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Cord blood cell therapy for ischemic stroke

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Title:
Cord blood cell therapy for ischemic stroke
Physical Description:
x, 133 leaves : ill. , ; 29 cm.
Language:
English
Creator:
Vendrame, Martina
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla.
Publication Date:

Subjects

Subjects / Keywords:
Cord Blood Stem Cell Transplantation   ( mesh )
Fetal Blood   ( mesh )
Infarction, Middle Cerebral Artery -- prevention & control   ( mesh )
Cerebrovascular Accident -- prevention & control   ( mesh )
Inflammation   ( mesh )
Spleen   ( mesh )
Stem Cells   ( mesh )
Hematopoietic Stem Cells   ( mesh )
MCAO
neuroprotection
neuroinflammation
spleen
stem cells
Dissertations, Academic -- Pathology and Laboratory Medicine -- Doctoral -- USF   ( lcsh )
Dissertations, Academic -- Pathology and Laboratory Medicine -- Doctoral -- USF   ( mesh )
Genre:
government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Summary:
ABSTRACT: Infusion of the "mononuclear fraction" of human cord blood cells (HUCBC), which is composed of immature blood cells and hematopoietic progenitors, is known to reduce neurobehavioral deficits in rats subject to middle cerebral artery occlusion (MCAO). When MCAO rats are infused with 10⁶ cells 24 hours after the induction of the stroke, their motor function improves. To extend these findings, we first examined the behavioral recovery of MCAO rats in the presence of increasing doses of HUCBC. The recovery in behavioral performance seen with measurements of spontaneous activity and motor deficits, depended on the amount of cells delivered, with 10⁶ HUCBC being the threshold for significant behavioral recovery. Measurements of the ischemic volume revealed an inverse relationship between HUCBC dose and damage volume, which reached significance at the higher HUCBC doses (10⁷ and 3-5x10⁷ cells). Moreover, investigation of the distribution of the intravenously injected cells showed that HUCBC were localized to the injured brain hemisphere and the spleen. Given these findings, we hypothesized that there may be a role of HUCBC in the modulation of the peripheral or brain-localized immune response that is normally evoked after stroke. Results on the effect of HUCBC infusion on splenocytes indicated that HUCBC treatment prevented the alterations in splenocyte type (CD8+ depletion) and function (T-cell suppression) induced by stroke. In particular, examination of cytokine production from splenocyte cultures of HUCBC-treated MCAO rats revealed increased production of IL-10 and decreased production of IFNgamma relative to MCAO rats. Microglia (immunostained with a CD11b antibody) and B cells (identified with the B220 cell marker) that were increased after MCAO were dramatically decreased after HUCBC treatment. Proinflammatory cytokines such as TNF-alpha, IL-1beta and IL-2 were upregulated after MCAO surgery and their expression was abrogated after HUCBC infusion. All these findings indicate that the action of HUCBC may be specifically related to the modulation of the stroke-induced inflammatory response, providing a possible mechanism by which cord blood cells have been repeatedly reported to promote functional recovery from ischemic injury.
Thesis:
Thesis (Ph.D.)--University of South Florida, 2004.
Bibliography:
Includes bibliographical references.
Statement of Responsibility:
by Martina Vendrame.
General Note:
Includes vita.

Record Information

Source Institution:
University of South Florida Library
Holding Location:
University of South Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 001670430
oclc - 56564337
usfldc doi - E14-SFE0000462
usfldc handle - e14.462
System ID:
SFS0025154:00001


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ABSTRACT: Infusion of the "mononuclear fraction" of human cord blood cells (HUCBC), which is composed of immature blood cells and hematopoietic progenitors, is known to reduce neurobehavioral deficits in rats subject to middle cerebral artery occlusion (MCAO). When MCAO rats are infused with 10 cells 24 hours after the induction of the stroke, their motor function improves. To extend these findings, we first examined the behavioral recovery of MCAO rats in the presence of increasing doses of HUCBC. The recovery in behavioral performance seen with measurements of spontaneous activity and motor deficits, depended on the amount of cells delivered, with 10 HUCBC being the threshold for significant behavioral recovery. Measurements of the ischemic volume revealed an inverse relationship between HUCBC dose and damage volume, which reached significance at the higher HUCBC doses (10 and 3-5x10 cells). Moreover, investigation of the distribution of the intravenously injected cells showed that HUCBC were localized to the injured brain hemisphere and the spleen. Given these findings, we hypothesized that there may be a role of HUCBC in the modulation of the peripheral or brain-localized immune response that is normally evoked after stroke. Results on the effect of HUCBC infusion on splenocytes indicated that HUCBC treatment prevented the alterations in splenocyte type (CD8+ depletion) and function (T-cell suppression) induced by stroke. In particular, examination of cytokine production from splenocyte cultures of HUCBC-treated MCAO rats revealed increased production of IL-10 and decreased production of IFNgamma relative to MCAO rats. Microglia (immunostained with a CD11b antibody) and B cells (identified with the B220 cell marker) that were increased after MCAO were dramatically decreased after HUCBC treatment. Proinflammatory cytokines such as TNF-alpha, IL-1beta and IL-2 were upregulated after MCAO surgery and their expression was abrogated after HUCBC infusion. All these findings indicate that the action of HUCBC may be specifically related to the modulation of the stroke-induced inflammatory response, providing a possible mechanism by which cord blood cells have been repeatedly reported to promote functional recovery from ischemic injury.
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C or d B l ood C e l l T he r a py f o r I s c he m i c S t r oke by M a r t i na V e ndr a m e A di s s e r t a t i on s ubm i t t e d i n pa r t i a l f u l f i l l m e nt of t he r e qui r e m e nt s f or t he de g r e e of D oc t or of P hi l os ophy D e pa r t m e nt of P a t hol ogy C ol l e ge of M e di c i ne U ni ve r s i t y of S ou t h F l o r i da C o M a j or P r of e s s or : A l i s on E W i l l i ng P h. D C o M a j or P r of e s s or : P a t r i c i a K r uk, P h. D K e i t h R P e nnypa c ke r P h D P a ul R S a nbe r g P h. D D S c J ua n S a nc he z R a m os M D P h D D a t e of A ppr ova l : J ul y 15, 2004 K e yw or ds : M C A O s t e m c e l l s ne ur oi n f l a m m a t i on ne ur op r ot e c t i on, s pl e e n. C opyr i ght 2004 M a r t i na V e ndr a m e

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i T A B L E O F C O N T E N T S L I S T O F T A B L E S vi L I S T O F F I G U R E S vi i A B S T R A C T i x C H A P T E R O N E I N T R O D U C T I O N 1 1. 1 E p i de m i ol ogy o f S t r oke 1 1. 2 P a t hol ogy of S t r oke 2 1. 3 T he G oa l of S t r oke T he r a py 4 1. 3. 1 T hr om bol yt i c T he r a py: T i s s ue P l a s m i noge n A c t i v a t or ( t P A ) 4 1. 3. 2 N e ur opr ot e c t i ve S t r a t e gi e s 5 1. 3. 3 A nt i I nf l a m m a t o r y S t r a t e gi e s 6 1. 3. 4 C l i ni c a l T r i a l s on S t r oke 8 1. 4 T he R ode nt M i ddl e C e r e br a l A r t e r y O c c l us i o n M ode l of S t r oke 9 1. 5 H e m a t ot he r a py F or R e pa i r of I s c he m i c B r a i n 12 1. 5. 1 C e l l T he r a py f o r S t r oke 12 1. 5. 2 H um a n E m br yoni c T e r a t oc a r c i nom a C e l l s L i ne 13 1. 5. 3 B one M a r r ow C e l l s 14 1. 5. 4 C or d B l ood C e l l s f or B r a i n R e pa i r 17 1. 6 C ur r e nt C l i ni c a l U s e of C or d B l ood C e l l T he r a py 19 1. 7 W ha t I s C or d B l ood? 20 1. 8 C onc l us i on. 22 C H A P T E R T W O G E N E R A L M A T H E R I A L S A N D M E T H O D S 24 2. 1 T he M i ddl e C e r e br a l A r t e r y O c c l us i on R a t M ode l 24

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i i 2. 1. 1 A ni m a l H ous i ng a nd M a i nt e na nc e 24 2. 1. 2 S ur gi c a l P r oc e dur e 24 2. 1. 3 P os t S ur gi c a l P ha r m a c ol ogi c a l a n d P hys i c a l S upp or t 25 2. 2 C or d B l ood C e l l s T r a ns pl a nt a t i on 26 2. 2. 1 P r e pa r a t i on of C r yopr e s e r ve d C e l l s 26 2. 2. 2 I nt r a F e m or a l T r a ns pl a nt a t i on 27 2. 2. 3 I nt r a P e ni l e V e i n T r a ns pl a nt a t i on 27 2. 3 A ni m a l S a c r i f i c e a nd P r e pa r a t i on of T i s s ue s 28 C H A P T E R T H R E E D O S E E F F E C T O F C O R D B L O O D I N F U S I O N O N F U N C T I O N A L A N D H I S T O L O G I C A L R E C O V E R Y O F R A T S S U B J E C T E D T O M I D D L E C E R E B R A L A R T E R Y O C C L U S I O N 29 3. 1 I nt r oduc t i on 29 3. 2 M a t e r i a l s a nd M e t hods 31 3. 2. 1 B e ha vi or T e s t s 31 3. 2. 1 1 M e a s ur e m e nt of S pon t a ne ous A c t i vi t y 32 3. 2. 1 2 S t e p T e s t 33 3. 2. 1 3 E l e va t e d B ody S w i ng T e s t 33 3. 2. 2 B r a i n H i s t ol ogi c a l A s s e s s m e nt 34 3. 2. 2 1 N i s s l T hi oni ne S t a i ni ng 34 3. 2. 2 2 M e a s ur e m e nt of I nf a r c t V ol um e 34 3. 2. 3 T r a c ki ng T he I nt r a v e nous T r a ns pl a nt e d C e l l s 35 3. 2. 3 1 P ol ym e r a s e C ha i n R e a c t i on 35 3. 2. 3 1. 1 D N A E xt r a c t i on 35 3. 2. 3 1. 2 P C R P r ot oc ol f or N e s t e d P r i m e r s 36 3. 2. 3 1. 3 P ol ya c r yl a m i de G e l P r e pa r a t i on a nd S i l ve r S t a i ni ng 37 3. 2. 3 2 I m m unohi s t oc he m i s t r y T e c hni que s 37 3. 2. 3 2. 1 Q ue nc hi ng of A ut of l uor e s c e nc e 37 3. 2. 3 2. 2 I m m unohi s t oc he m i s t r y P r ot oc ol 38 3. 2. 4 S t a t i s t i c a l A na l ys i s 38 3. 3 R e s ul t s 38 3. 3. 1 I nf us i on of C or d B l ood C e l l s R e duc e s T he S t r oke I nduc e H ype r a c t i vi t y 38 3. 3. 1 1 D a yt i m e S pont a ne ous A c t i vi t y T e s t 38 3. 3. 1 2 N i ght T i m e S pont a ne ous A c t i vi t y T e s t 42

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i i i 3. 3. 2 I nf us i on o f H i gh D os e s of C or d B l ood C e l l s I nduc e s a C om pl e t e R e c ove r y of A s ym m e t r y D e f i c i t s M e a s ur e d w i t h T he S t e p T e s t 46 3. 3. 3 C or d B l ood I nf us i on R e duc e s L ong T e r m D e f i c i t s i n T he E l e va t e d B ody S w i ng T e s t 46 3. 3. 4 C or d B l ood I nf us i on I nduc e s H i s t ol ogi c a l B e ne f i t i n B r a i ns of R a t s S ub j e c t t o M C A O 49 3. 3. 5 C or d B l ood I nf us i on I nduc e s R e duc t i on of I nf a r c t V ol um e i n M C A O B r a i ns 49 3. 3. 6 C or d B l ood C e l l s A r e D e t e c t e d i n S pl e e n a nd R i gh t B r a i n H e m i s phe r w i t h N e s t e d P C R f o r H um a n P r i m e r s 53 3. 3. 7 I m m unoc he m i c a l L a be l i ng O f H um a n C e l l s w i t h A n A nt i body A ga i ns t H um a n N uc l e i C onf i r m s T he P C R R e s ul t s 56 3. 4 D i s c us s i on 56 C H A P T E R F O U R E X A M I N A T I O N O F T H E E F F E C T O F H U C B C I N F U S I O N O N S T R O K E I N D U C E D A L T E R A T I O N S O F S P L E N O C Y T E S T Y P E A N D F U N C T I O N 4. 1 I nt r oduc t i on 61 4. 2 M a t e r i a l s a nd M e t hods 64 4. 2. 1 2, 3, 5 T r i phe nyl t e t r a z ol i um C hl or i de ( T T C ) S t a i ni n g a nd I nf a r c t V ol um e Q ua nt i f i c a t i on 65 4. 2. 2 D e t e c t i on of R N A E xp r e s s i on of C yt oki ne G e ne s 65 4. 2. 2 1 R N A E x t r a c t i on 65 4. 2. 2 2 R N A s e P r ot e c t i on A s s a y 66 4. 2. 3 S pl e noc yt e I s ol a t i on a nd C ul t ur e s 67 4. 2. 4 P r ol i f e r a t i on A s s a y 68 4. 2. 5 I m m unophe not ypi ng of S pl e e n C e l l s 68 4. 3. 6 C yt oki ne E nz ym e L i nke d I m m unoa ds or be nt A s s a ys 69

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i v 4. 3. 6 1 Q ua nt i f i c a t i on of S a m pl e P r ot e i n A m ount s 69 4. 3. 6 2 E nz ym e L i nke d I m m unoa ds or be nt A s s a ys 69 4. 3 R e s ul t s 70 4. 3. 1 C or d B l ood R e s c ue s S t r oke I nduc e d S pl e e n W e i ght R e duc t i on W hi c h C or r e l a t e s W i t h B r a i n I nf a r c t V o l um e s 70 4. 3. 2 E f f e c t o f C or d B l ood on P he not ype of I s c he m i c R a t D e r i ve d S pl e noc yt e s 73 4. 3. 3 E f f e c t of C or d B l ood on P r oduc t i on of P r o a nd A nt i I nf l a m m a t o r y C yt oki ne s i n T he S p l e e n 76 4. 3. 4 C or d B l ood D e pr e s s e s T he S pl e ni c T C e l l P r ol i f e r a t i on i n S t r oke d R a t s E nha nc i ng T he P r oduc t i on of I l 10 a nd B l oc ki ng T he R e l e a s e of I F N ga m m a 76 4. 4 D i s c us s i on 79 C H A P T E R F I V E M O D U L A T I O N O F S T R O K E I N D U C E D B R A I N I N F L A M M A T O R Y E V E N T S B Y C O R D B L O O D I N F U S I O N 84 5. 1 I nt r oduc t i on 84 5. 2 M a t e r i a l s a nd M e t hods 88 5. 2. 1 S e pa r a t i on o f C e l l s by P e r c ol l G r a di e nt 90 5. 2. 2 I m m unol a be l i ng P r ot oc ol A nd P r e pa r a t i on of S a m pl e s f o r F l ow C yt om e t r y A na l ys i s 90 5. 2. 3 A c q ui s i t i on a nd A na l ys i s of D a t a 92 5. 3 R e s ul t s 92 5. 3. 1 H U C B C I nf us i on R e duc e s T he S t r oke I ndu c e d U pr e gul a t i on o f C D 45/ C D 11b P os i t i ve a nd B 220 P os i t i ve C e l l s W i t hi n T he B r a i n 92 5. 3. 2 H U C B C I nf us i on D oe s N ot A l t e r T he P r e s e nc e O f C D 45/ C D 3 P os i t i ve C e l l s W i t hi n T he I s c he m i c B r a i n. 98 5. 3. 3 T he P e r c e nt a ge of N e ut r ophi l s I s N ot A l t e r e d 48 H our s a f t e r S t r oke S ur ge r y 98 5. 3. 4 M e s s e nge r R N A E xpr e s s i on O f P r o I n f l a m m a t or y C yt oki ne s I s R e gul a t e d by T he I nj e c t i on of C or d B l ood C e l l s 98

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v 5. 3. 5 H U C B C I nf us i on D e pr e s s e s T he E xp r e s s i on of P r o I nf l a m m a t or y C yt oki ne s T ha t A r e U p R e gul a t e d W i t hi n T he I s c he m i c B r a i n. 102 5. 4 D i s c us s i on 107 C H A P T E R S I X C O N C L U D I N G R E M A R K S 111 6. 1 N e ur obe ha vi or a l a nd N e ur opa t hol ogi c a l be ne f i t s i nduc e d by H U C B C i nf us i on i n t he M C A O a ni m a l m ode l of s t r oke 113 6. 2 W ha t i s t he m e c ha ni s m by w hi c h H U C B C e xe r t t he i r be ne f i c i a l e f f e c t s ? 115 6. 3 S um m a r y a nd C onc l us i on 118 R E F E R E N C E S 120 A B O U T T H E A U T H O R E nd P a ge

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vi L I S T O F T A B L E S T a bl e 3. 1 O ve r a l l A N O V A f or D a yt i m e S pont a ne ous A c t i vi t y: p va l ue F r a t i o de gr e e s of f r e e dom 40 T a bl e 3. 2 P os t hoc S t a t i s t i c a l C om pa r i s on f or D a yt i m e S pont a ne ous A c t i vi t y 41 T a bl e 3. 3 O v e r a l l A N O V A f or N i gh t t i m e S pont a ne ous A c t i vi t y: p va l ue F r a t i o, de gr e e s of f r e e dom 45

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vi i L I S T O F F I G U R E S F i gur e 1. 1 S c he m a o f t he M C A O pr oc e dur e 11 F i gur e 1. 2 F l ow c yt om e t r y a na l ys i s on t he c or d bl ood m ononuc l e a r f r a c t i on 23 F i gur e 3. 1 D a yt i m e s pont a ne ous a c t i vi t y 43 F i gur e 3. 2 O ve r a l l D a yt i m e s pont a ne ous a c t i vi t y 44 F i gur e 3. 3 S t e p t e s t 47 F i gur e 3. 4 E l e va t e d B ody S w i ng T e s t ( E B S T ) 48 F i gur e 3. 5 N i s s l T hi oni ne s t a i ni ng of M C A O r a t br a i n 50 F i gur e 3. 6 H U C B C t r e a t m e nt r e duc e s M C A O br a i n i nj ur y 51 F i gur e 3. 7 R e pr e s e nt a t i ve N i s s l T hi oni ne s t a i ni ng of b r a i n c or ona l 52 F i gur e 3. 8 D os e e f f e c t o f H U C B C r e l e a ve d by qu a nt i f i c a t i on of t he i s c he m i c vol um e 54 F i gur e 3. 9 P C R l oc a l i z a t i on o f i nt r a ve nous H U C B C i nt o va r i ous 55 F i gur e 3. 10 I m m unohi s t oc he m i s t r y c onf i r m s P C R l oc a l i z a t i on of H U C B C 57 F i gur e 4. 1 C or d bl ood r e s c ue s s pl e e n w e i ght r e duc t i on i nduc e d by s t r oke 72 F i gur e 4. 2 R e duc t i o n of M C A O i nduc e d i s c he m i c vol um e by H U C B C i nf us i on. 73 F i gur e 4. 3 I nve r s e c or r e l a t i on be t w e e n s pl e e n w e i g ht a nd % i s c he m i c br a i n vol um e 75 F i gur e 4. 4 S t r oke i nduc e d c ha nge s i n s pl e e n C D 8+ c e l l s r e ve r s e d by H U C B C 76 F i gur e 4. 5 R N A s e pr ot e c t i on a s s a y f or S pl e e n T N F a l pha I L 6 a nd I L 1b. 78

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vi i i F i gur e 4. 6 R N A s e pr ot e c t i on a s s a y f or S pl e e n I F N ga m m a I L 10 a nd I L 2. 79 F i gur e 4. 7 P r ol i f e r a t i on a s s a y on s pl e noc yt e s 81 F i gur e 4. 8 I F N ga m m a & I L 10 E L I S A on s upe r n a t a nt s f r om s pl e noc yt e s c ul t ur e s 82 F i gur e 5. 1 S c he m a of pe r c ol l g r a di e nt br a i n c e l l i s ol a t i on pr oc e dur e 93 F i gur e 5. 2 F l ow c yt om e t r y of br a i n m i c r ogl i a c e l l s f ol l ow i ng M C A O 94 F i gur e 5. 3 F l ow c yt om e t r y de t e c t i on o f br a i n T c e l l s f ol l ow i ng M C A O 95 F i g ur e 5. 4 F l ow c yt om e t r y de t e c t i on o f br a i n B c e l l s 96 F i gur e 5. 5 F l ow c yt om e t r y de t e c t i on o f br a i n ne ut r ophi l s a f t e r M C A O 97 F i gur e 5. 6 H U C B C e f f e c t on i nf l a m m a t or y c e l l m i gr a t i on i nt o t he M C A O r a t br a i n 98 F i gur e 5. 7 R N A s e pr ot e c t i on a s s a y f or br a i n T N F a l pha a nd I L 1 be t a m R N A l e ve l s 100 F i gur e 5. 8 R N A s e pr ot e c t i on a s s a y f or br a i n T N F be t a a nd I L 2 m R N A l e ve l s 101 F i gur e 5. 9 R N A s e pr ot e c t i on a s s a y f or br a i n I L 6 I L 10 a nd I L 4 m R N A l e ve l s 102 F i gur e 5. 10 B r a i n T N F a l pha a nd I L 1 p r ot e i n l e ve l s f ol l ow i ng M C A O 104 F i gur e 5. 11 B r a i n I L 2, I L 6 a nd I L 10 pr o t e i n l e v e l s f ol l ow i ng M C A O 105

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i x C or d B l ood C e l l T he r a py F or I s c he m i c S t r oke M a r t i na V e ndr a m e A B S T R A C T I nf us i on of t he m ononuc l e a r f r a c t i on o f hum a n c or d bl ood c e l l s ( H U C B C ) w hi c h i s c om pos e d of i m m a t ur e bl ood c e l l s a nd he m a t opoi e t i c pr oge ni t or s i s know n t o r e duc e ne ur obe ha vi or a l de f i c i t s i n r a t s s ubj e c t t o m i ddl e c e r e br a l a r t e r y oc c l us i on ( M C A O ) W he n M C A O r a t s a r e i nf us e d w i t h 10 6 c e l l s 2 4 ho ur s a f t e r t he i nduc t i on o f t he s t r oke t he i r m ot or f unc t i on i m pr ove s T o e xt e nd t he s e f i n di ngs w e f i r s t e xa m i ne d t he be ha vi or a l r e c ove r y o f M C A O r a t s i n t he pr e s e nc e of i nc r e a s i ng dos e s of H U C B C T he r e c ove r y i n be ha vi or a l pe r f o r m a nc e s e e n w i t h m e a s ur e m e nt s of s pont a ne ous a c t i vi t y a nd m ot or de f i c i t s de pe nde d on t he a m ount o f c e l l s de l i ve r e d, w i t h 10 6 H U C B C be i ng t he t hr e s hol d f or s i gni f i c a nt be ha vi or a l r e c ove r y. M e a s ur e m e nt s of t he i s c he m i c vol um e r e ve a l e d a n i nve r s e r e l a t i ons hi p be t w e e n H U C B C dos e a nd da m a ge vol um e w hi c h r e a c he d s i gni f i c a nc e a t t he hi ghe r H U C B C dos e s ( 10 7 a nd 3 5x10 7 c e l l s ) M or e ove r i nve s t i ga t i on of t he di s t r i but i on o f t he i nt r a ve nous l y i nj e c t e d c e l l s s how e d t ha t H U C B C w e r e l oc a l i z e d t o t he i nj ur e d b r a i n he m i s phe r e a nd t he s p l e e n. G i ve n t he s e f i ndi ngs w e hypot he s i z e d t ha t t he r e m a y be a r ol e of H U C B C i n t he m odul a t i on of t he pe r i phe r a l or br a i n l oc a l i z e d i m m une r e s pons e t ha t i s nor m a l l y e voke d a f t e r s t r oke R e s ul t s on t he e f f e c t of H U C B C i nf us i on on s pl e n oc yt e s i ndi c a t e d t ha t H U C B C t r e a t m e nt pr e ve nt e d t he a l t e r a t i ons i n s pl e noc yt e t y pe ( C D 8+ de pl e t i on) a nd f unc t i on ( T c e l l s uppr e s s i on) i nduc e d by s t r oke I n pa r t i c ul a r e xa m i na t i on of c yt oki ne pr oduc t i on

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x f r om s pl e noc yt e c ul t ur e s o f H U C B C t r e a t e d M C A O r a t s r e ve a l e d i nc r e a s e d p r oduc t i on of I L 10 a nd de c r e a s e d pr oduc t i on o f I F N ga m m a r e l a t i ve t o M C A O r a t s M i c r ogl i a ( i m m unos t a i ne d w i t h a C D 11b a nt i body) a nd B c e l l s ( i de nt i f i e d w i t h t he B 220 c e l l m a r ke r ) t ha t w e r e i nc r e a s e d a f t e r M C A O w e r e dr a m a t i c a l l y de c r e a s e d a f t e r H U C B C t r e a t m e nt P r oi n f l a m m a t or y c yt oki ne s s uc h a s T N F a l pha I L 1be t a a nd I L 2 w e r e upr e gul a t e d a f t e r M C A O s ur ge r y a nd t he i r e xpr e s s i on w a s a br oga t e d a f t e r H U C B C i nf us i on. A l l t he s e f i nd i ngs i ndi c a t e t ha t t he a c t i on of H U C B C m a y be s pe c i f i c a l l y r e l a t e d t o t he m odul a t i on o f t he s t r oke i nduc e d i nf l a m m a t or y r e s pons e pr o vi di n g a pos s i bl e m e c ha ni s m by w hi c h c or d bl ood c e l l s ha ve be e n r e pe a t e dl y r e por t e d t o p r om ot e f unc t i ona l r e c ove r y f r om i s c he m i c i nj ur y

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1 C H A P T E R O N E I N T R O D U C T I O N 1. 1 E p i d e m i ol ogy of s t r ok e A l t hough t he w or l dw i de i nc i de nc e of s t r oke i n u nk now n, i n t he U ni t e d S t a t e s i t i s w e l l doc um e nt e d t ha t s t r oke a f f e c t s a ppr oxi m a t e l y 700 000 A m e r i c a ns pe r ye a r ( H D S S 2004 U pda t e ) O f t he s e onl y one t hi r d a c hi e ve s f ul l r e c ove r y, one t h i r d s uc c um bs w i t hi n t he f ol l ow i ng 12 m ont hs a nd t he r e m a i ni ng t hi r d a r e a f f e c t e d by pe r m a ne nt di s a bi l i t i e s ( H D S S 2004 U pda t e ) A l t hough, s t r oke a f f e c t s a l l a ge gr oups t he r i s k i nc r e a s e s dr a m a t i c a l l y w i t h a gi ng [ m o r e t ha n 90 % of a l l s t r o ke s oc c ur r i ng i n pe opl e of a ge 55 a nd ol de r ] ( H D S S 2004 U pda t e ) T he r i s k f a c t or s a s s oc i a t e d w i t h s t r oke i nc l ude : hype r t e ns i on, t oba c c o us e di a be t e s h i gh a l c ohol i nt a ke poor d i e t e xc e s s i ve w e i ght f a m i l y hi s t or y of c a r d i ova s c ul a r di s e a s e a nd pr e vi ous e pi s ode s of s t r oke or t r a ns i e nt i s c he m i c a t t a c ks ( T I A s ) ( G r e e n G P 2004 ) S t r oke i s a l r e a dy t he t hi r d l e a di ng c a us e of de a t h a nd t he l e a di ng c a us e of di s a bi l i t y, but a s t he A m e r i c a n popul a t i on m e di a n a ge i s e xpe c t e d t o i nc r e a s e ove r t he ne xt t w o de c a de s s t r oke l oom s a n e ve r i nc r e a s i ng t h r e a t f o r t he A m e r i c a n s oc i e t y.

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2 1. 2 P at h ol ogy of S t r ok e T he t e r m s t r oke ge ne r a l l y r e f e r s t o a ny c a r di ova s c ul a r a c c i de nt t ha t oc c ur s i n t he c e nt r a l ne r vous s ys t e m ( C N S ) T hi s gr oup of va s c ul a r pa t hol ogi e s of t he br a i n i nc l ud e s he m or r ha gi c a c c i de nt s ( due t o t he r up t ur e o f a b l o od ve s s e l ) a s w e l l a s di s or de r s t ha t f ol l ow t he oc c l us i on o f a b l ood ve s s e l i e i s c he m i c i nj ur i e s T he s e l a t t e r i s c he m i c s t r oke s e nc om pa s s t he m a j or i t y o f s t r oke s ( m or e t ha n 80% ) ( H D S S 2004 U pda t e ) A bout 50 pe r c e nt o f i s c he m i c s t r oke s a r e c a us e d b y c e r e br a l t h r om bos i s w hi c h i s c a us e d by t he f o r m a t i on o f a b l ood c l ot w i t hi n c e r e br a l a r t e r i e s t ha t a r e da m a ge d by a r t e r i os c l e r os i s T hr om bos i s c a n a f f e c t l a r ge ve s s e l s s uc h a s t he c a r ot i d, m i ddl e c e r e br a l or ba s i l a r a r t e r i e s a s w e l l a s s m a l l ve s s e l s ( l e nt i c ul os t r i a t e ba s i l a r pe ne t r a t i ng m e dul l a r y) c a us i ng a t ype of t h r om bot i c s t r oke know n a s a l a c una r s t r oke T he ot he r 50 pe r c e nt o f i s c he m i c s t r oke s i s c a us e d by c e r e br a l e m bol i s m a t ype of i s c he m i c s t r oke t ha t oc c ur s a s a r e s ul t o f bl oc ka ge of c i r c ul a t i on t o a por t i on of t he br a i n pa r e nc hym a by a n e m bol us T he e m bol us c a n be c om pos e d of a va r i e t y o f s ubs t a nc e s ( s uc h a s f a t or a i r ) but m or e f r e que nt l y i s de r i ve d f r om a bl ood c l ot m a i nl y or i gi na t i ng f r om t he he a r t o r t he w a l l s of t he c a r ot i d a r t e r i e s T he e m bol us m ove s f r e e l y t hr ough t he bl ood s t r e a m unt i l i t l odge s w i t hi n a n a r t e r y t oo s m a l l t o a l l ow i t s pa s s a ge bl oc ki ng t he bl ood be hi nd i t f r o m pa s s i ng t hr ough W i t hi n s e c onds t o m i nut e s of t he l os s of pe r f us i on t o a n a r e a o f br a i n pa r e nc hym a a n i s c he m i c c a s c a d e i s t r i gge r e d, t he r e s ul t of w hi c h i s i r r e ve r s i bl e da m a ge or i nf a r c t i on of t he a r e a l a c ki ng bl ood f l ow ( c or e ) a r ound w hi c h t he r e i s a n a r e a o f pot e nt i a l l y r e ve r s i bl e i nj ur y, c a l l e d t he pe num br a A t t he c e l l ul a r l e ve l t he e l e c t r ophys i ol ogi c a l pr ope r t i e s o f t he ne ur ons s ubj e c t t o i s c he m i a a r e a l t e r e d, a s a c ons e que nc e of t he s udde n de p l e t i on of

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3 A T P i n r e s pons e t o hypoxi a a nd t he c ons e que nt f a i l ur e of c e l l m e m br a ne i on t r a ns por t s ys t e m s ( D i a r r a S he l don e t a l 1999; S he l don a nd C hur c h 2004) T he r e s ul t i ng i nt r a c e l l ul a r f l ow of c a l c i um a nd ot he r c a t i ons c a u s e s t he r e l e a s e of s e ve r a l ne ur ot r a ns m i t t e r s s uc h a s l a r ge a m oun t s of g l ut a m a t e w hi c h a c t i va t e s N m e t hyl D a s pa r t a t e ( N M D A ) a nd ot he r e xc i t a t or y r e c e pt or s ( L i u, G r os s m a n e t a l 2001) T he s e c e l l s t he n be c om e de pol a r i z e d, c a us i ng a ddi t i ona l c a l c i um i nf l ux a ddi t i ona l r e l e a s e of gl ut a m a t e a nd l oc a l a m pl i f i c a t i on of t he i ni t i a l i s c he m i c i ns ul t T hi s e no r m ous c a l c i um i nf l ux l e a ds t o ne u r on c e l l de a t h by a c t i va t i ng va r i ous de gr a da t i ve e nz ym e s i nc l udi ng c a l pa i n a nd m e t a l l opr ot e a s e s w hi c h de gr a de l a m i ni n a nd ot he r c om pone nt s of t he br a i n m a t r i x ( F ukuda F i ni e t a l 2004) A num be r of t oxi c c om pounds a r e ge ne r a t e d by t hi s pr oc e s s i nc l udi ng f r e e oxyge n r a di c a l s a nd ni t r i c oxi de w hi c h t he m s e l ve s i nduc e a ddi t i ona l ne ur ona l da m a ge ( L i u G r os s m a n e t a l 2001) W i t hi n hour s t o da ys a f t e r a s t r oke a n i nf l a m m a t or y r e a c t i on i s e voke d w hi c h pr ovoke s t he r e l e a s e of pr o i n f l a m m a t or y c yt oki ne s s uc h a s t um or ne c r os i s f a c t or a l pha ( T N F a l ph a ) w i t hi n t he i s c he m i c br a i n ( B ut t i ni A ppe l e t a l 1996) A l t hough i ni t i a l l y pr ot e c t i ve t hi s i nf l a m m a t or y r e a c t i on i f c hr oni c a l l y pr e s e nt e xt e nds t he da m a ge t o t he s ur r oundi ng t i s s ue ( de l Z oppo, B e c ke r e t a l 2001) W i t hout t he r a pe ut i c i nt e r ve nt i on, t he pe num br a w i l l be c ons um e d by t h i s c a s c a de of m ul t i pl e i ns ul t s a nd i ne vi t a bl y l os e i t s f unc t i ona l i t y c om pl e t e l y.

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4 1. 3 T h e goal of s t r ok e t h e r ap y T he c e nt r a l goa l of s t r oke t he r a py i s t o s a l va ge t he pe num br a l r e gi on T h i s c a n be obt a i ne d by 1) r e duc i ng t he dur a t i on of i s c he m i a i e r e i ns t a t i ng t he bl ood f l ow t o t he c om pr om i s e d a r e a ; or 2) l i m i t i ng t he s e ve r i t y o f c e l l i nj ur y a s s e e n w i t h s t r a t e gi e s ba s e d on ne ur ona l pr o t e c t i on. T he i s c he m i c c a s c a de of f e r s m ul t i pl e s t e ps a t w hi c h s uc h s t r a t e gi e s c a n be a ppl i e d a nd m a ny i nt e r ve nt i ons a i m e d a t s t oppi ng t hi s c a s c a de of e ve nt s a r e be i ng i nve s t i ga t e d. A ke y f a c t or i s t he t i m i ng of r e s t o r i ng c e r e br a l bl ood f l ow gi ve n t ha t c l i ni c a l s t udi e s ha ve c onc l ude d t ha t r e pe r f us i o n m us t oc c ur w i t hi n 3 hour s i n or de r t o s a l va ge t he pe num br a ( N I N D S 1995) 1. 3. 1 T h r om b ol yt i c T h e r ap y: T i s s u e P l as m i n oge n A c t i vat or ( t P A ) T hr om bol yt i c s w or k by b r e a ki ng up t he c l ot r e s pons i bl e f or obs t r uc t i ng t he bl ood f l ow a l l ow i ng bl ood t o r e f l ow t h r ough t he oc c l ude d ve s s e l I n 1995, t he t i s s ue P l a s m onoge n A c t i va t or ( t P A ) S t udy c onduc t e d by N a t i ona l I ns t i t ut e of N e u r ol ogi c a l D i s or de r s a nd S t r oke ( D e pa r t m e nt of H e a l t h a nd H um a n S e r vi c e s a t t he N a t i ona l I ns t i t ut e o f H e a l t h, B e t he s da M A ) publ i s he d t he r e s ul t s of t w o r a ndo m i z e d c l i ni c a l t r i a l s on 600 s t r oke pa t i e nt s w hi c h c onc l ude d t ha t t he r e w a s a n i m p r o ve m e nt by t hr om bo l yt i c t P A us e i n pa t i e nt s w he n t r e a t e d w i t hi n 90 m i nut e s o f s t r oke ons e t ( N I N D S 1995) T he U S F ood a nd D r ug A dm i ni s t r a t i on ( F D A ) a ppr ove d t P A a s a t r e a t m e nt f o r s t r oke i n 1996 w i t h i t s e m pl oym e nt be i ng r e s t r i c t e d t o a 3 hour s t i m e w i n dow f r om s t r oke ons e t T h i s t r e a t m e nt r e m a i ns t he onl y e f f e c t i ve t he r a pe ut i c opt i on f o r s t r oke A r e c e nt s t udy publ i s he d i n t he L a nc e t ( H a c ke D onna n e t a l 2004) s how s t he r e s ul t s of a n e xt e ns i ve a na l ys i s of a bout 3, 000 s t r oke pa t i e nt s w ho w e r e r a ndom i z e d f o r t r e a t m e nt w i t h t P A or a pl a c e bo, i n s i x

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5 c ont r ol l e d c l i ni c a l t r i a l s : t he f i ndi ngs de m ons t r a t e t ha t t P A t r e a t m e nt w i t hi n 3 hou r s of s t r oke ons e t i nduc e d s i gni f i c a nt r e c ove r y M or e o ve r t he s t udy s ugge s t s t ha t t P A gi ve n up t o 4 hour s a f t e r t he s t r oke ons e t m a y be o f be ne f i t but t he a ut hor s poi nt out t ha t t he r e i s a de c r e a s i ng e f f e c t i ve ne s s w i t h a l m os t no be ne f i t a t a l l w he n t P A t r e a t m e nt i s done a t 6 hour s pos t s t r oke ons e t W hi l e t he r e i s s t r ong e v i de nc e t ha t e a r l y t r e a t m e nt w i t h t P A c a n i m pr ove a s t r oke pa t i e nt 's pos s i bi l i t y of c om pl e t e r e c ove r y, on l y a n e s t i m a t e d 2 t o 5 pe r c e nt of a l l e l i gi bl e s t r oke pa t i e n t s r e c e i ve t hr om bol yt i c s t r e a t m e nt 1. 3. 2 N e u r op r ot e c t i ve S t r a t e gi e s N e ur opr ot e c t i ve t he r a pi e s a r e t r e a t m e nt s i nt e nde d t o bl oc k t he c e l l ul a r b i oc he m i c a l a nd m e t a bol i c pr oc e s s e s of ne ur ona l de a t h dur i ng or f o l l ow i ng t he i s c he m i c i ns ul t T he y i nc l ude a n a r r a y of pha r m a c ol ogi c i nt e r ve nt i ons s uc h a s a nt a goni s t s of gl ut a m a t e r e c e pt or s c a l c i um c ha nne l bl oc ke r s a nt i e de m a a g e nt s a nd ne ur ot r ophi c f a c t or s S t udi e s on s t r oke a ni m a l m ode l s ha ve s how n t ha t t he s e s ubs t a nc e s ha ve a be ne f i c i a l e f f e c t a s s e e n by e i t he r be ha vi or a l m e a s ur e s a nd/ or i n r e duc t i on of i nf a r c t vol um e of t he s t r oke br a i n. F o r i ns t a nc e N m e t hyl D a s pa r t a t e ( N M D A ) r e c e pt or a nt a goni s t s ( s uc h a s C ona nt oki n G a nd t he c om pound S M 18400) a r e k now n t o r e duc e i n f a r c t vol u m e i n s t r oke d r a t s ( O ht a ni T a na ka e t a l 2000; W i l l i a m s L i ng e t a l 2002) C a l c i um c ha nne l s bl oc ke r s s uc h a s ni m odi pi ne ( K or e nkov P a hnke e t a l 2000 ) ha ve a l s o be e n e xa m i ne d i n r ode nt m ode l s of c e r e br a l i s c he m i a a nd s how n t o p os s e s s ne ur opr ot e c t i ve pr ope r t i e s S om e e vi de nc e a l s o s ugge s t s t ha t m a gne s i um s ul f a t e ( M gS 2 O H 3 ) m a y i nh i bi t i s c he m i c da m a ge by bl oc ki ng c a l c i um c ha nne l s I nt r a ve nous de l i ve r y of 5% M gS 2 O H 3 ( 90 m g/ kg )

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6 up t o 6 hour s pos t s t r oke i s pr ove n t o i m pr ove ne u r ol ogi c a l out c om e a nd r e duc e br a i n i nf a r c t vol um e i n r a t s ( Y a ng, L i e t a l 2000) M or e r e c e nt l y, ne ur o t r ophi c f a c t or s ha ve a l s o be e n i nt r oduc e d i nt o t he e xpe r i m e nt a l s e t t i ng w i t h ve r y p r om i s i ng r e s ul t s I n s t r oke d r a t s i nt r a ve nous i nf us i on of br a i n de r i ve d ne ur ot r ophi c f a c t or ( B D N F ) ( s t a r t i ng a t 30 m i nut e s a f t e r M C A O s ur ge r y a nd p r ol onge d f or 2 hour s ) i nduc e d a r e duc t i on i n i n f a r c t vol u m e vi s i bl e a t 5 hour s pos t s t r oke ( B e r ge r S c ha bi t z e t a l 2004) W he n a ppl i e d i n s yne r gy w i t h hypot he r m i a t he s a m e t r e a t m e nt c a n dr a m a t i c a l l y i nhi bi t t he r e l e a s e of gl ut a m a t e i n t he c or pus s t r i at um ( B e r ge r S c ha bi t z e t a l 2004 ) R e c e nt l y, vi r a l ve c t or m e di a t e d ge ne t r a ns f e r of ne ur ot r ophi c f a c t or s ha s be e n c ons i de r e d a s a ne w t he r a pe ut i c a l t e r na t i ve f o r i s c he m i c br a i n i nj ur y I nt r a ve nt r i c ul a r ge ne de l i ve r y o f gl i a l c e l l l i ne de r i ve d ne ur ot r ophi c f a c t or ( G D N F ) ne r ve gr ow t h f a c t or ( N G F ) B D N F i ns ul i n l i ke g r ow t h f a c t or 1 ( I G F 1) or va s c ul a r e ndot h e l i a l gr ow t h f a c t or ( V E G F ) up t o 6 hou r s a f t e r t he i s c he m i c i ns ul t bl oc ke d ne ur ona l de a t h i nduc e d by oc c l us i on of t he bi l a t e r a l c a r ot i d a r t e r i e s i n ge r bi l s ( S hi r a kur a I noue e t a l 2004) 1. 3. 3 A n t i i n f l a m m at or y S t r at e gi e s T he i de a of bl oc ki ng t he i nf l a m m a t o r y r e a c t i on i nd uc e d by i s c he m i a i n t he br a i n ha s be e n t he ba s i s of s e ve r a l e xpe r i m e nt a l t he r a pe ut i c a ppr o a c he s f or s t r oke A nt i i n f l a m m a t or y a ge nt s w e r e f i r s t i nt r oduc e d i n t hi s a r e na f o r t he i r a nt i c oa gul a nt pr ope r t i e s A ge nt s l i ke a s pi r i n, i bupr o f e n a nd s e r i ne pr ot e a s e i nhi bi t or s s uc h a s t he ur i na r y t r yps i n i nh i bi t or ( U T I ) ha ve be e n s how n t o a m e l i o r a t e be ha vi or a l d e f i c i t s i n r ode nt m ode l s of i s c he m i c i nj ur y a nd c onc om i t a nt l y r e duc e i n f a r c t vol um e ( C ol e P a t e l e t a l 1993; Y a no A nr a ku e t

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7 a l 2003; B e r ge r X i a e t a l 2004) G r e a t s uc c e s s i n t he e xpe r i m e nt a l s e t t i ng ha s be e n obt a i ne d w i t h t he e m pl oy m e nt of bl oc ke r s of t he i nf i l t r a t i on of i nf l a m m a t or y c e l l s t o t he br a i n p a r e nc hym a F o r e xa m pl e i nt r a ve nous a ppl i c a t i on of i nt e r f e r on be t a ( I F N be t a ) a c yt oki ne w i t h a nt i i n f l a m m a t or y pr ope r t i e s a t t e nu a t e d t he de ve l opm e nt of br a i n i nf a r c t i on by 70% i n s t r oke d r a t s ( V e l dhui s D e r ks e n e t a l 2003) T h i s e f f e c t w a s obt a i ne d w he n I F N be t a i nf us i on w a s be gun 6 hou r s a f t e r ons e t o f s t r oke s ym pt om s I m m unohi s t oc he m i s t r y a t 24 hour s a f t e r s t r oke ons e t s how e d t ha t I F N be t a a l m os t c om pl e t e l y bl oc ke d t he m i g r a t i on o f i nf l a m m a t or y c e l l s s uc h a s ne ut r ophi l s a nd m onoc yt e s i nt o t he C N S pa r e nc hym a O t he r s t udi e s ha ve a ppl i e d a l t e r na t i ve m ol e c ul a r s t r a t e gi e s t o pr e ve nt t he i n f i l t r a t i on of i nf l a m m a t o r y c e l l s s uc h a s pha r m a c ol ogi c a l l y bl oc ki ng a dhe s i on m ol e c ul e s F or e xa m pl e t he dy a d C D 18 I nt e r C e l l ul a r A dhe s i on M ol e c ul e 1 ( I C A M 1) a t t r a c t e d a l ot of a t t e nt i on w he n a nt i bodi e s bl oc ki ng t he i r i nt e r a c t i on i nduc e d ne ur ol ogi c a l i m pr ove m e nt a nd r e duc e d i nf a r c t s i z e i n s t r oke d r ode nt s ( Z ha ng, C hopp e t a l 1994; H a r t l S c hur e r e t a l 19 96) T he pot e nt i a l a ppl i c a t i on of t he s e s t r a t e gi e s ha s be e n c onf i r m e d by s e ve r a l e xpe r i m e nt a l s t udi e s but c l i ni c a l t r i a l s ba s e d on s uc h s t r a t e gi e s h a ve yi e l de d l e s s e nt hus i a s m I n 1 995 t he E n l i m om a b A c ut e S t r oke t r i a l c onc l ude d t ha t i nt r a ve nous a dm i ni s t r a t i on of t he m ur i ne a nt i hum a n I C A M 1 a nt i body di d not i nduc e a ny be ne f i t i n s t r oke pa t i e nt s ( D e G r a ba 1998) D e s pi t e s e ve r a l l ogi s t i c a nd pr a gm a t i c r e a s ons t ha t c oul d e xpl a i n s uc h f a i l ur e t he r a pe ut i c a ppr oa c he s ba s e d on s i m i l a r s t r a t e gi e s s t i l l r e m a i n c onf i ne d t o t he e xpe r i m e nt a l s e t t i ng.

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8 1. 3. 4 C l i n i c al t r i al s on S t r ok e A s of 2004, t he r e a r e 38 s t r oke t r i a l s on goi ng w or l dw i de ( A H A 2004) S om e o f t he s e a r e f oc us e d on i nt r a a r t e r i a l r e va s c ul a r i z a t i on s t r a t e gi e s ba s e d on t he i de a t ha t i t i s e f f i c a c i ous t o r e s t or e t he bl ood f l ow e i t he r be f or e t he ve s s e l ha s be e n f ul l y obs t r uc t e d or a f t e r t he s t r oke O ne of t he s e t r i a l s i s t he C a r ot i d a nd V e r t e br a l A r t e r y T r a ns l um i na l A ngi opl a s t y S t udy ( C A V A T A S ) t ha t de t e r m i ne s t he be ne f i t s a nd r i s ks of pe r c ut a ne ous t r a ns l um i na l a ngi opl a s t y of t he c a r ot i d a nd ve r t e br a l a r t e r i e s i n pa t i e nt s w i t h s ym pt om a t i c a nd a s ym pt om a t i c c e r e br ova s c ul a r di s e a s e O t he r t r i a l s f oc us on t he c ont r ol of phys i ol ogi c a l pa r a m e t e r s t ha t a r e no r m a l l y a l t e r e d a f t e r a n i s c he m i c i ns ul t T he C ont r ol l i ng H ype r t e ns i on a nd H ypot e ns i on I m m e d i a t e l y P os t s t r oke ( C H H I P S ) T r i a l i nvol ve s a l m os t 2, 000 pa t i e nt s a c r os s t he U ni t e d K i ngdom a nd a i m s a t t he c ont r ol l i ng bl ood pr e s s ur e w i t h de pr e s s or t he r a py w i t hi n 12 h our s pos t s t r oke O t he r on goi ng t r i a l s a ppl y ne ur opr ot e c t i ve s t r a t e gi e s s uc h a s i nt r a ve nou s de l i ve r y of m a gne s i um s ul f a t e a f t e r t he de m ont r a t i on t ha t m a gne s i um s ul f a t e i s s a f e a n d w e l l t ol e r a t e d i n s t r oke pa t i e nt s T he I nt r a ve nous M a gne s i um E f f i c a c y i n S t r oke T r i a l ( I M A G E S ) t r i a l i s c oor di na t e d by t he U ni ve r s i t y of C a l i f o r ni a L os A nge l e s a nd i s de s i gne d t o t e s t t he e f f i c a c y of M gS 2 O H 3 gi ve n a t 12 hou r s of s t r oke ons e t F i na l l y t he r e a r e o n goi ng s t r oke t r i a l s t ha t t e s t t he e f f i c a c y of vi t a m i ns t r e a t m e nt i n pa t i e nt s w i t h r e c u r r e nt T r a ns i e nt i s c he m i c A t t a c ks ( T I A ) T he V i t a m i ns t o pr e ve nt S t r oke ( V I T A T O P S ) t r i a l a dm i ni s t e r s vi t a m i n s uppl e m e nt s of B 12, B 6 a nd f ol a t e t o pa t i e nt s w ho ha d a T I A w i t h i n t he l a s t 7 m ont hs

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9 1. 4 T h e r od e n t m i d d l e c e r e b r al ar t e r y oc c l u s i on m o d e l of s t r ok e S e ve r a l a ni m a l m ode l s of s t r oke ha ve be e n de ve l ope d i n or de r t o t e s t ne w t he r a pe ut i c opt i ons f or t r e a t m e nt o f s e que l a e of b r a i n i s c he m i a A m ong a l l t he s e m ode l s t he m i ddl e c e r e br a l a r t e r y oc c l us i on ( M C A O ) t ha t w e us e i n o ur l a bor a t or y i s t he m os t c om m onl y e m pl oye d. O t he r s t r oke m ode l s of c e r e br a l i s c he m i a i nc l ude bi l a t e r a l c a r ot i d oc c l us i on ( E kl of a nd S i e s j o 1972 ) i nt r a c r a ni a l c om pr e s s i on t ha t pr odu c e s i s c he m i a a nd i nc r e a s e d i nt r a c r a ni a l pr e s s ur e ( L j unggr e n R a t c he s on e t a l 1974) uni l a t e r a l c a r ot i d oc c l us i on pl us hypoxi a ( N a ka j i m a I s hi da e t a l 1999) c om p r e s s i on of t he ne c k w i t h a c uf f ( S i e m kow i c z a nd H a ns e n 1978) c om m on c a r ot i d a r t e r y o r i n t e r na l c a r ot i d a r t e r y oc c l us i on w i t h t hr om bot i c bl ood c l ot ( O ve r ga a r d, S e r e ghy e t a l 1 992) a s w e l l a s f ou r ve s s e l oc c l us i on ( P ul s i ne l l i a nd B r i e r l e y 1979) A l l t he s e s t r oke m ode l s do not pr ovi de a m ode l of f oc a l i s c he m i a i e a n i s c he m i c l e s i on l i m i t e d t o s pe c i f i c br a i n a r e a s a s t he M C A O m ode l doe s a nd a s i t oc c ur s i n t he ne ur opa t hol ogy of c l i ni c a l s t r oke T he r e f or e t he o c c l us i on of t he M C A r e m a i ns t he m ode l of c hoi c e t ha t m os t c l os e l y m i m i c k s t he r e a l s c e na r i o of br a i n i s c he m i c e ve nt s A ddi t i ona l l y, t he M C A i s a l s o t he ve s s e l m os t c om m onl y oc c l ude d i n e m bol i c s t r oke s ( S ym on, D or s c h e t a l 1975) T he M C A O m ode l i nvol ve s t he s e l e c t i ve oc c l us i on of t he r i ght or l e f t M C A D ur i ng t he l a s t t hr e e de c a de s i t ha s be e n s ubj e c t e d t o s e ve r a l v a r i a t i ons i n or de r t o opt i m i z e t e c hni c a l a s pe c t s out c om e r e pr oduc i bi l i t y a nd r e l i a bi l i t y ( T a m ur a G r a ha m e t a l 1981; B e l a ye v, A l ons o e t a l 1996 ; Z ha ng C hopp e t a l 1997 ) M os t of t he s e s t udi e s a r e done on r ode nt s s uc h a s m i c e a nd r a t s a l t hough e a r l y s t udi e s e m pl oye d f e l i ne s a nd pr i m a t e s be c a us e t he s e c oul d be t t e r m i m i c t he c l i n i c a l ne ur ol ogi c a l

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10 s yndr om e s f ol l ow i ng s t r oke i n m e n. H ow e ve r i s s ue s r e l a t e d t o ha ndl i ng c os t s a nd a ni m a l e t hi c a l c onc e r ns i nduc e d a s hi f t t o r ode nt m ode l s ( G a r c i a L os s i ns ky e t a l 1978 ) T he f i r s t M C A oc c l us i on pr oc e dur e s f r om t he 70 s i nvol ve d t he l i ga t i on a nd s e ve r a nc e of t he M C A a f t e r f r ont ot e m por a l c r a ni ot om y ( R ob i ns on, S hoe m a ke r e t a l 1975 ; R obi ns on 1979) T hi s pr oc e dur e w a s a bl e t o i nduc e a l e s i on l i m i t e d t o t he r a t c e r e b r a l c or t e x w i t h a m i ni m a l va r i a t i on i n t he l e s i on s i z e b e t w e e n a ni m a l s H ow e ve r t he l e s i on a r e a di d not a ppe a r t o c ons i s t e nt l y r e s e m bl e t he i s c he m i c a r e a da m a ge d i n hum a n M C A s t r oke ( R obi ns on 1979) T a m u r a e t a l ( T a m ur a G r a ha m e t a l 1981) a nd l a t e r L onga e t a l ( L onga W e i ns t e i n e t a l 1989) de s c r i be d a t e c hni q ue i n w hi c h a s ut ur e w a s i ns e r t e d i nt r a l um i na l l y f r om t he bi f u r c a t i on of t he c om m on c a r ot i d a r t e r y. S i nc e t he l e ngt h of t he s ut ur e c or r e s ponds t o t he di s t a nc e f r om i t s i ns e r t i o n t o t he ba s e of t he M C A ( a bout 20m m de pe ndi ng on t he s i z e o f t he a ni m a l ) t he t i p of t he s ut ur e w oul d r e a c h t he ba s e of t he M C A a nd oc c l ude i t t he or e t i c a l l y A s c he m a t i c dr a w i ng o f t hi s p r oc e dur e i s i l l us t r a t e d i n F i gur e 1 1. T hi s p r oc e dur e c oul d be p e r f or m e d w i t hou t c r a ni ot om y w i t h a s i m pl e ne c k i nc i s i on a nd bl unt di s s e c t i on of t he c o m m on c a r ot i d a r t e r y. M or e ove r on s ubs e que nt pa t hol ogi c a l e xa m i na t i on t he c e r e br a l l e s i on r e s e m bl e d m or e c l os e l y t ha t s e e n i n hum a n M C A s t r oke ( T a m u r a G r a ha m e t a l 1981) F ur t he r m o r e t hi s pr oc e dur e w a s r e ve r s i bl e a l l ow i ng t he s ut ur e t o be r e m ove d a t a l a t e r t i m e ( L onga W e i ns t e i n e t a l 1989) T hi s opt i on ha s i nde e d a l l ow e d ot he r i nve s t i ga t or s t o m odi f y t hi s pr oc e dur e a nd pe r f o r m a t r a ns i e nt M C A O : r e m ova l of t he s ut ur e a f t e r a c e r t a i n t i m e a l l ow s a r e pe r f us i on o f t he i s c he m i c a r e a m i m i c ki ng w ha t c a n ha ppe n i n a n e m bol i c or t hr om bo e m bol i c oc c l us i on, w he n s p ont a ne ous r e m ova l of t he e m bo l us a l l ow s bl ood t o r e f l ow t hr ough t he

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12 i s c he m i c a r e a T hi s va r i a t i on of pe r f o r m i ng a t r a ns i e nt oc c l us i on ha s be e n a dopt e d by s e ve r a l i nve s t i ga t or s ( L i C hopp e t a l 2000 ; C he n, S a nbe r g e t a l 2 001) O t he r va r i a t i ons of t hi s M C A O pr oc e dur e a r e f or t he m os t pa r t t e c hni c a l m a i nl y i nvol vi ng di f f e r e nt poi nt s of i ns e r t i on of t he s ut ur e a nd i nnova t i ons i n s ur gi c a l di s s e c t i on t e c hni que s I n t he pr oc e dur e de s c r i be d by B e l a ye v e t a l ( B e l a ye v, A l ons o e t a l 1996 ) t he s ut ur e i s i ns e r t e d t hr ough t he p r oxi m a l e xt e r na l c a r ot i d a r t e r y t he n i nt o t he i nt e r na l c a r ot i d a r t e r y a nd f i na l l y i nt o t he ba s e of t he M C A T h i s pr oc e dur e i nc l ude s t he i s ol a t i on a nd c oa gul a t i on of t he br a nc he s of t he e xt e r na l c a r ot i d a r t e r y a nd l i ga t i on of t he pt e r ygopa l a t i ne a r t e r y T h i s t e c hni que ha s be e n us e d s uc c e s s f ul l y i n s e ve r a l r e po r t s ( W i l l i ng, J i a ng e t a l 2002; B e l a ye v, K hout or ova e t a l 2003) a nd i t i s t he t e c hni que c ur r e nt l y us e d i n our l a bor a t or y S pe c i f i c a l l y, w e e m pl oye d t he pe r m a ne nt oc c l us i on m ode l i e w i t hout r e m ova l of t he s ut ur e a f t e r p l a c e m e nt ( W i l l i ng, L i xi a n e t a l 2003; W i l l i ng, V e ndr a m e e t a l 2003) 1. 5 H e m at ot h e r ap y f or r e p ai r o f i s c h e m i c b r ai n 1. 5. 1 C e l l t h e r ap y f or s t r ok e C e l l t r a ns pl a nt a t i on ha s r e c e nt l y e m e r ge d a s a t he r a pe ut i c a l t e r na t i ve f or ne ur ode ge ne r a t i ve di s e a s e s i nc l udi ng s t r oke T h i s f i e l d ha s r e c e i ve d i nc r e a s i ng a t t e nt i on a f t e r t he r e vol ut i ona r y di s c ove r y t ha t t he br a i n a l s o pos s e s s e s a r e ge ne r a t i ve c a pa c i t y, t hr ough t he p r e s e nc e of ne ur a l s t e m c e l l s pr e s e nt e ve n i n t he a dul t hum a n b r a i n ( R e ynol ds a nd W e i s s 1992; S ong, S t e ve ns e t a l 20 02) N e ur a l s t e m c e l l s w hi c h a r e a bl e t o s e l f r e ne w a nd c om m i t t o ne ur ona l a s t r oc yt i c o r ol i gode ndr ogl i a l l i ne a ge s a r e

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13 pr i m a r l y de r i ve d f r om hum a n e m br yos ( R e ubi nof f I t s yks on e t a l 2001) H ow e ve r t he i de a of ha r ve s t i ng t he s e c e l l s f r om s uc h s our c e s ha s ge ne r a t e d e t hi c a l c onc e r ns T w o a l t e r na t i ve c e l l s our c e s a r e r e pr e s e nt e d by t he hum a n e m br yoni c t e r a t oc a r c i nom a c e l l s l i ne s w hi c h c a n be di f f e r e nt i a t e d i n v i t r o i nt o pos t m i t ot i c ne ur ons or by s t e m c e l l s i s ol a t e d f r om he m a t opoi e t i c t i s s ue s ( s uc h a s a dul t bone m a r r ow a nd c or d bl ood ) w hi c h de m ons t r a t e t he c a pa c i t y t o a t t a i n ne ur ona l a nd gl i a l pr ope r t i e s i n v i t r o but not de r i ve d f r om e m b r yoni c s our c e s 1. 5. 2 H u m an e m b r yon i c t e r at oc ar c i n om a c e l l s l i n e s T he hum a n e m br yona l c a r c i nom a c e l l l i ne ( N T e r a 2/ D 1 or N T 2 c e l l s ) a r e c a pa bl e of di f f e r e nt i a t i ng i nt o pos t m i t ot i c ne ur on l i ke c e l l s ( h N T or N T 2N c e l l s ) a f t e r e xpos ur e t o r e t i noi c a c i d ( P l e a s ur e a nd L e e 1993 ) S t udi e s of N T 2N c e l l s t r a ns pl a nt e d i nt o t he C N S of n on di s e a s e d r ode nt s s how e d t ha t N T 2N c e l l s s ur vi ve d, i n t e gr a t e d i nt o t he c e r e br a l m i c r oe nvi r om e nt a nd de ve l oppe d m ol e c ul a r a nd s t r uc t ur a l c ha r a c t e r i s t i c of ne ur ons ( T r oj a now s ki M a nt i one e t a l 1993) T he s e c e l l s c a n e ngr a f t a nd s ur vi ve up t o one ye a r i n r ode nt b r a i ns w i t hout r e ve r t i ng t o a t um o r i ge ni c s t a t e ( T r oj a now s ki M a nt i one e t a l 1993) A ddi t i ona l l y N T 2N c e l l s ha ve be e n t r a ns p l a nt e d i nt o t he s t r i a t um o f i s c he m i c r a t s s how i ng gr a f t i ng a nd s ur vi va l a t 6 m ont hs pos t t r a ns pl a nt i n a ddi t i on t o s i gni f i c a nt a m e l i or a t i on of f unc t i ona l s t r oke i nduc e d de f i c i t s ( B or l onga n, T a j i m a e t a l 1998 ) B a s e d on t he s e a ni m a l s t udi e s a s t r oke pha s e I c l i ni c a l t r i a l e m pl oyi ng N T 2N c e l l s w a s s t a r t e d i n 2000 a t t he U ni ve r s i t y o f P i t t s bur gh ( P A ) ( K ond z i ol ka W e c hs l e r e t a l 2000) T hi s t r i a l i nc l ude d 12 pa t i e nt s ( a ge d 44 t o 75 ye a r s ) w i t h ba s a l ga ngl i a i nf a r c t a nd f i xe d m ot or de f i c i t s w i t h a s t r oke c l i ni c a l l y s t a bl e f or a t l e a s t 2 m ont hs ( t ha t oc c ur r e d 6 m ont hs t o 6

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14 ye a r s pr e vi ous l y) I m a gi ng e va l ua t i ons s h ow e d no a dve r s e c e l l r e l a t e d e f f e c t s o f t he a l l ogr a f t ( be i ng t he s e c e l l s de r i ve d f r om a hum a n c e l l l i ne a nd t r a ns pl a nt e d i nt o un r e l a t e d hum a n r e c i pi e nt s ) N e ur ol ogi c a l s c or e s s how e d i m pr ove m e nt i n s i x of t he t w e l ve pa t i e nt s a nd pos i t r on e m i s s i on t om ogr a ph y s c a ns a t 6 m ont hs c on f i r m e d t he pr e s e nc e of vi a bl e c e l l s ( K ondz i ol ka W e c hs l e r e t a l 2000 ) A ut ops y f i ndi ngs i n t he br a i n of a pha s e I c l i ni c a l s t r oke t r i a l pa t i e nt i m pl a nt e d w i t h hum a n N T 2N c e l l s ( w ho di e d o f c a us e s unr e l a t e d t o t he s t r oke ) f ound t he t r a ns pl a nt e d c e l l s a dj a c e nt t o t he i n f a r c t a r e a a t 27 m ont hs a f t e r s ur ge r y w i t h no e vi de nc e o f de ve l op m e nt of ne opl a s m ( N e l s on, K ondz i ol ka e t a l 2002 ) T he s e f i ndi ngs i ndi c a t e d t ha t i m pl a nt e d N T 2N c e l l s c oul d s ur vi ve f or a t i m e gr e a t e r t ha n 2 ye a r s i n t he hum a n b r a i n w i t hout a d ve r s e e f f e c t s s uppor t i ng t he i de a t ha t t he r a pi e s ba s e d on i m pl a nt a t i on of hu m a n e m br yo na l c a r c i nom a c e l l l i ne s c a n be f e a s i bl e a nd non de l e t e r i ous 1. 5. 3 B on e m ar r ow c e l l s H e m a t opoi e t i c s t e m c e l l s ( H S C ) de r i ve d f r o m bon e m a r r ow w e r e f i r s t i de nt i f i e d f or t he i r a bi l i t y t o r e c ons t i t ut e bl ood l i ne a ge s a nd t he y w e r e s ubs e que nt l y s how n t o di f f e r e nt i a t e i nt o ot he r c e l l t ype s i nc l udi ng ne ur oe c t ode r m i c c e l l s s uc h a s ne ur ons a nd gl i a l c e l l s D e s pi t e s om e s ke pt i c i s m r e ga r di ng t he c a pa c i t y of t he s e c e l l s t o ge ne r a t e c e l l s of s uc h di s s i m i l a r l i ne a ge a n e ve r i nc r e a s i ng body o f e vi d e nc e s ugge s t s t ha t de ve l opm e nt of ne ur ons a nd a s t r oc yt e s f r om bone m a r r ow de r i ve d H S C s i s a bona f i de phe nom e non. I n a ddi t i on t o he m a t opoi e t i c s t e m c e l l s bone m a r r ow c ont a i ns non he m a t opoi e t i c pr e c ur s or s c e l l s w hi c h a r e a l s o r e f e r r e d t o a s m e s e nc hym a l c e l l s or bone m a r r ow s t r om a l s t e m c e l l s ( B M S C ) w hi c h ha ve a l s o be e n s how n t o di f f e r e nt i a t e i nt o gl i a a nd ne ur ons i n v i t r o

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15 S e ve r a l i nde pe nde nt r e por t s ha ve c onf i r m e d t he ob s e r va t i on t ha t s ys t e m i c a l l y i nf us e d B M S C s c a n r e popul a t e a num be r of non he m a t opo i e t i c t i s s ue s ( P e r e i r a O 'H a r a e t a l 1998) T o a s s e s s w he t he r B M S C s c oul d a l s o r e c ons t i t ut e C N S t i s s ue s bone m a r r ow c e l l s ge ne t i c a l l y m a r ke d w i t h a r e t r ovi r a l t a g w e r e i nj e c t e d i nt r a ve nous l y i n s ubl e t ha l l y i r r a di a t e d W B B 6F 1yJ K i t W / K i t W v m i c e ( c ons i de r e d a s good r e c i pi e nt s f o r bone m a r r ow t r a ns pl a nt be c a us e t he y pos s e s s ge ne t i c a l l y de f e c t i ve he m a t opoi e t i c s ys t e m ) ( E gl i t i s a nd M e z e y 1997) W e e ks a f t e r t he i nj e c t i o n, t a gge d c e l l s w e r e obs e r ve d i n t he br a i n w he r e t he y w e r e w i de l y d i s t r i but e d i n t he c or t e x, hi ppoc a m pus t ha l a m us c e r e be l l um a nd br a i n s t e m T he s e c e l l s e xpr e s s e d e i t he r m i c r ogl i a l m a r ke r s ( F 4/ 80 ) or G F A P s ugge s t i ng t ha t p r e c ur s or s r e s i de nt i n t he b one m a r r ow w e r e a bl e t o r e c ons t i t ut e C N S gl i a l popul a t i ons ( E gl i t i s a nd M e z e y 1997) O t he r s ha ve c onf i r m e d t hi s obs e r va t i on ( K ope n, P r oc kop e t a l 1999; N a ka no, M i gi t a e t a l 2001) a nd f ur t he r de m ons t r a t e d t ha t i nf us e d bone m a r r ow de r i ve d c e l l s c a n a l s o de ve l o p ne ur a l t r a i t s a f t e r gr a f t i ng i nt o t he br a i n, a s s e e n w i t h t he e xpr e s s i on of N e u N ( B r a z e l t on, R os s i e t a l 2000; M e z e y, C ha ndr os s e t a l 2000) N S E ( M e z e y, C ha ndr os s e t a l 2000 ) c l a s s I I I be t a t ubul i n a nd t he 200kD ne ur of i l a m e nt ( B r a z e l t on, R os s i e t a l 2 000) B M S C s ha ve a l s o be e n t r a ns pl a nt e d i nt r a c e r e br a l l y. W he n i nj e c t e d i nt o t he s t r i a t um o f a l bi no r a t b r a i n, B M S C c e l l s e ngr a f t e d a nd m i g r a t e d f r om t he t r a ns pl a nt s i t e t o di f f e r e nt C N S a r e a s a l ong know n pa t hw a ys of ne u r a l s t e m c e l l m i gr a t i on ( A z i z i S t oke s e t a l 1998) i nd i c a t i ng t ha t t he s e c e l l s c a n r e s pond t o l o c a l c ue s M or e ove r a f t e r t he e ngr a f t m e nt t he s e c e l l s l os t m a r ke r s t ypi c a l of c ul t ur e d B M S C s a nd de ve l ope d phe not ype s s i m i l a r t o a s t r oc yt e s

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16 I n l i ght of t he s e r e s ul t s i n non di s e a s e d a ni m a l s b one m a r r ow de r i ve d c e l l s ha ve be e n s ubs e que nt l y s t udi e d i n a ni m a l m ode l s of ne ur ode ge ne r a t i ve di s e a s e w i t h t he i de a t ha t t he s e c e l l s m i ght a l s o be c a pa bl e of r e c ons t i t ut i ng da m a ge d t i s s ue s a nd c ons e que nt l y r e s t or i ng br a i n f unc t i on. I n r ode nt m ode l s of s t r ok e t r a ns pl a nt a t i on o f B M S C s e i t he r i nt r a s t r i a t a l l y or i nt r a va s c ul a r l y f ound t ha t t he B M S C s hom e d t o t he i s c he m i c bounda r y z one a nd bor e ne ur ona l a nd gl i a l a nt i ge ns ( L i C hopp e t a l 2000; C he n, L i e t a l 2001 ; L i C he n e t a l 2001 ) T he s a m e i nve r s t i ga t or s ha ve r e por t e d t ha t B M S C s c a n i nduc e t he i n s i t u e xpr e s s i on of va s c ul a r e ndot he l i a l gr ow t h f a c t or ( V E G F ) a nd i t s r e c e pt or t he r e by pr om ot i ng ne o va s c ul a r i z a t i on p r oc e s s e s i n t he pe num br a l r e gi on ( C he n, Z ha ng e t a l 2003) N ot a l l t he i nve s t i ga t or s be l i e ve t ha t a r e a l ne ur ona l / gl i a l di f f e r e nt i a t i on of t he s e c e l l s i s pos s i bl e E ve n a c c e pt i ng t he i de a t ha t B M c e l l s t r u l y de ve l op a ne ur ona l / gl i a l t r a i t i n v i v o t he f u nc t i ona l i t y o f t he s e c e l l s a s ne ur ons or gl i a ha s not ye t be e n pr ove n. O ne e m e r gi ng i de a i s t ha t bone m a r r ow de r i ve d c e l l s m a y i nduc e t he di f f e r e nt i a t i on of r e s i de nt t ot i pot e nt s t e m c e l l s i nt o f unc t i ona l c e l l s t ha t r e c ons t i t ut e da m a ge d ne ur ons A n i nc r e a s e i n e ndoge nous ne ur a l s t e m c e l l pr o l i f e r a t i on w i t hi n t he s ub ve nt r i c ul a r z one a nd t he hi ppoc a m pus ha s be e n obs e r ve d a f t e r B M S C s t r a ns pl a nt a t i on ( M a hm ood, L u e t a l 2001) A l t e r na t i ve l y, t he obs e r ve d f unc t i ona l r e c ov e r y m a y be a r e s ul t of t he i nc r e a s e d e xpr e s s i on of ne ur ot r ophi c / g r ow t h f a c t or s T hi s ph e nom e non c oul d be f unc t i on o f e i t he r t he e ndoge nous pr oduc t i on of gr ow t h f a c t or s i nduc e d by B M S C s or by de l i ve r y of t he s e f a c t or s f r om t he t r a ns pl a nt e d c e l l s ( C he n, K a t a k ow s ki e t a l 2002; M a hm ood, L u e t a l 2004) I n v i t r o s t udi e s e m pl oyi ng T B I c ondi t i o ne d B M S C s c ul t ur e s ha ve s how n a t i m e de pe nde nt i nc r e a s e i n B D N F N G F V E G F a nd he pa t oc yt e gr ow t h f a c t or ( H G F )

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17 i ndi c a t i ng a r e s pons i ve pr oduc t i on of t he s e gr ow t h f a c t or s by t he B M S C s ( C he n, K a t a kow s ki e t a l 2002) R e c e nt r e po r t s s ugge s t a ddi t i ona l m e c ha ni s m s t ha t t r a ns pl a nt e d B M S C s m a y unde r go. F or i ns t a nc e t he e xp r e s s i on of t r a ns pl a nt e d s t e m c e l l s by ne ur a l phe not ype m a y be e xpl a i ne d by t he i r f us i on w i t h e ndoge nous c e l l s ( T e r a da H a m a z a ki e t a l 2002) T hi s hypo t he s i s know n a s t he f us i on t he or y i s s uppor t e d by i n v i t r o c o c ul t ur e e xpe r i m e nt s s how i ng e m br yoni c s t e m c e l l s a r e a bl e t o f us e w i t h bone m a r r ow c e l l s or ne ur a l s t e m c e l l s ( T e r a da H a m a z a ki e t a l 2002; Y i ng, N i c hol s e t a l 2002) C e l l f us i on m a y i nde e d ha ve a be ne f i c i a l f unc t i on f or i ns t a nc e t r a ns pl a nt e d c e l l s m a y pr ovi de l a c ki ng ge ne t i c m a t e r i a l t o a c e l l ot he r w i s e unde r g oi ng ne c r os i s H ow e ve r t he i s s ue t ha t ha s ye t t o be r e s ol ve d i s w he t he r e xpr e s s i on of a di pl oi d nuc l e a r m a t e r i a l m a y on t he c ont r a r y l e a d t o t um or oge ne t i c pr oc e s s e s 1. 5. 4 C or d B l ood C e l l s f or B r ai n R e p ai r A c c um ul a t i ng e vi de nc e s uppor t s t he i de a t ha t s i m i l a r t o bone m a r r ow c e l l s c or d bl ood c e l l s unde r go a phe not ypi c c onve r s i on i nt o ne ur on a l a nd gl i a l l i ne a ge s w he n e xpos e d t o de f i ne d c ul t ur e c ondi t i ons ( S a nc he z R a m os S ong e t a l 2001; B i c kne s e G oodw i n e t a l 2002) H um a n um bi l i c a l c o r d bl ood c e l l s ( H U C B C ) t r e a t e d w i t h r e t i noi c a c i d ( R A ) a nd ne r ve gr ow t h f a c t or ( N G F ) e xp r e s s e d m ol e c ul a r m a r ke r s s uc h a s M us a s hi 1 ( a s pe c i f i c m a r ke r f o r ne ur a l s t e m c e l l s ) a nd be t a t ubul i n I I I ( s t r uc t ur a l pr ot e i ns c ha r a c t e r i s t i c o f ne ur ons ) ( S a nc he z R a m os S ong e t a l 2001) A l s o t r e a t m e nt o f H U C B C w i t h f i b r obl a s t gr ow t h f a c t or ( F G F ) a nd e pi de r m a l g r ow t h f a c t o r ( E G F ) i nduc e s t he e xpr e s s i on of ne ur a l a nd gl i a l m a r ke r s a s s e e n by W e s t e r n i m m unobl ot s a nd i m m unoc yt oc he m i c a l de t e c t i on

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18 of bot h be t a t ubul i n I I I a nd gl i a l f i b r i l l a r y a c i di c pr ot e i n ( G F A P ) ( B i c kne s e G oodw i n e t a l 2002) T he s e f i ndi ngs l e d t o t he i de a t ha t c or d bl ood c e l l s c oul d m i m i c t he be ne f i c i a l e f f e c t s s e e n a f t e r bone m a r r ow c e l l t r a ns pl a nt a t i on i n a ni m a l m ode l s of ne u r ode ge ne r a t i ve di s e a s e s T he r e f or e c or d bl ood c e l l s ha ve be e n us e d i n m ode l s o f s t r oke a nd t r a um a t i c br a i n i nj ur y s ubs e que nt l y de m ons t r a t i ng t ha t c or d bl ood c oul d a l s o r e s c ue m ot or de f i c i t s i nduc e d i n t he s e m ode l s T he m os t a dva nc e d w or k w i t h c or d bl ood a s a t r e a t m e nt f o r ne ur ode ge ne r a t i on ha s be e n c onduc t e d on s t r oke a ni m a l m ode l s T he f i r s t r e por t of be ne f i t f r om H U C B C t r a ns pl a nt a t i on i n a n a ni m a l m ode l of s t r oke oc c ur r e d i n 2001, w he n H U C B C w a s i nf us e d i n t he M C A O m ode l a nd s i gni f i c a nt f unc t i ona l r e c ove r y w a s obs e r ve d ( C he n, S a nbe r g e t a l 2001) T r e a t m e nt a t 24 hour s a f t e r M C A O w i t h 3x10 6 H U C B C i nj e c t e d i nt o t a i l ve i n w a s r e po r t e d t o i m p r ove f unc t i ona l r e c ove r y, a s e vi de nc e d by t he r ot a r od t e s t a nd M odi f i e d N e ur ol ogi c a l S e ve r i t y S c or e ( m N S S ) ( C he n, S a nbe r g e t a l 2001) I n t he s a m e s t udy, t r e a t m e nt a t 7 da ys a f t e r M C A O w i t h t he s a m e a m ount of c e l l s s i gni f i c a nt l y i m pr ove d f unc t i on onl y on t he m N S S A not he r s t udy c onc l ude d t ha t t he be ha vi or a l r e c ove r y i nduc e d i n M C A O r a t s by 10 6 H U C B C w he n de l i ve r e d i nt r a ve nous l y 24 hour s pos t s t r oke w a s c om pa r a bl e i f not be t t e r t ha n t he r e c ove r y obs e r ve d a f t e r i nt r a c e r e br a l t r a ns pl a nt a t i on of 2 50 000 c e l l s ( W i l l i ng, L i xi a n e t a l 2003) I nve s t i ga t or s s how e d a s i m i l a r r e c ove r y be t w e e n t he t w o gr oups i n s pont a ne ous a c t i vi t y a nd t he pa s s i ve a voi da nc e t a s k, w hi l e i n t he s t e p t e s t s i gni f i c a nt i m p r ove m e nt s w e r e f ound onl y a f t e r i n t r a ve nous de l i ve r y o f t he H U C B C

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19 1. 6 C u r r e n t C l i n i c al U s e of C or d B l ood C e l l T h e r a p y C or d bl ood ha s be e n us e d e xt e ns i ve l y a s a n a l t e r na t i ve s our c e of he m a t opoi e t i c s t e m c e l l s i n a l l oge ne i c s t e m c e l l t r a ns pl a nt a t i on f or t he t r e a t m e nt of a c qui r e d a nd ge ne t i c di s e a s e s T he f i r s t r e l a t e d hum a n um bi l i c a l c o r d b l ood ( H U C B ) t r a ns pl a nt w a s pe r f or m e d i n t he s e ve nt i e s i n a c hi l d w i t h a c ut e l ym phobl a s t i c l e uke m i a ( E nde a nd E nde 19 72) a nd s i nc e t he n, t hous a nds of H U C B t r a ns pl a nt s ha ve be e n pe r f or m e d w o r l dw i de w i t h t he m a j or i t y pe r f or m e d a s t he r a py f or ha e m a t ol ogi c a l m a l i gna n c i e s ( L e w i s 2002) F r om t he s e e a r l y e xpe r i e nc e s s om e c l e a r a dva nt a ge s of us i ng H U C B c e l l s ha ve be c om e e vi de nt F i r s t t he H U C B c e l l s a r e r e a di l y a va i l a bl e C ont r a r y t o bone m a r r ow c e l l s w hos e ha r ve s t r e qui r e s a bone m a r r ow punc t u r e c o r d bl o od c e l l s c a n be obt a i ne d f r om t he um bi l i c a l c or d o f a ny he a l t hy ne ona t e I m por t a nt l y H U C B C c a n be e a s i l y c r yopr e s e r ve d a nd s t or e d i n c or d bl ood c e l l ba nks unt i l ne e de d f o r t he t r a ns pl a nt T he s e c r yopr e s e r ve d c or d bl ood c e l l s ha ve be e n r e por t e d t o s how no d i f f e r e nc e i n e ngr a f t m e nt c a pa bi l i t i e s w he n ba nke d up t o 3 ye a r s ( G oodw i n G r unz i nge r e t a l 2003 ) S e c ondl y, t he r e i s a l ow e r i nc i de nc e of gr a f t ve r s us hos t di s e a s e ( G V H D ) w i t h H U C B t r a ns pl a nt s c om pa r e d t o bone m a r r ow t r a ns pl a nt s T hi s ha s a l l ow e d t he us a ge of c or d bl ood c e l l t r a ns pl a nt s f r om un r e l a t e d donor s w i t h a m i ni m a l i nc i de nc e o f i m m une r e a c t i ons T hi s a dva nt a ge i s p r oba bl y r e l a t e d t o t he i m m unol ogi c i m m a t ur i t y t ha t c or d bl ood c e l l s ha ve be e n s how n t o pos s e s s ( L e w i s 20 02) T hi r dl y t he r i s k of vi r a l t r a ns m i s s i on w i t h H U C B t r a ns pl a nt i s m i ni m a l B one m a r r ow t r a ns pl a nt c a n be j e opa r di z e d by t he t r a ns m i s s i on of pa t hoge ns f r om t he dono r t o t he hos t t ha t c a n f a t a l l y i n f e c t t he r e c i pi e nt O ne of t h os e i s t he E ps t e i n B a r r v i r us ( E B V ) w hi c h i s r e s pons i bl e f or t he ons e t of l ym phopr ol i f e r a t i ve di s or de r s W hi l e bone m a r r ow

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20 t r a ns pl a nt a r e ge ne r a l l y a c c om pa ni e d by a s i gni f i c a nt i nc i de nc e of E B V t r a ns m i s s i on, a f t e r c or d bl ood t r a ns pl a nt s t he i nc i de nc e of E B V a s s oc i a t e d l ym phopr ol i f e r a t i ve di s or de r s i s l ow ( B a r ke r M a r t i n e t a l 2001) 1. 7 Wh at i s c or d b l ood ? C or d B l ood i s t he bl ood c ont a i ne d i n t he pl a c e nt a a nd um bi l i c a l c or d t ha t i s no r m a l l y di s c a r de d a f t e r de l i ve r y of t he ne ona t e C or d bl ood c a n be c ol l e c t e d a f t e r c l a m pi ng a nd c ut t i ng t he um bi l i c a l c or d, i m m e di a t e l y a f t e r t he bi r t h of t he ba by T he a c t ua l pa r t o f t he c or d bl ood t ha t i s us e d f o r c e l l t r a ns pl a nt i s t he m ononuc l e a r f r a c t i on a f r a c t i on of t he bl ood c ont a i ni ng m a i nl y m ononuc l e a r c e l l s t ha t i s obt a i ne d by c e l l g r a di e nt s e pa r a t i on t e c hni que s C o r d bl ood ba nks c ol l e c t t he um bi l i c a l c or d bl ood i s ol a t e t he m ononuc l e a r f r a c t i on a nd c r yopr e s e r ve i t C r yop r e s e r va t i on of t he s e c e l l s a l l ow s f or l ong t e r m s t or a ge pe r m i t t i ng t he i r us e f or f ut u r e t r a ns pl a nt s T he m ononuc l e a r f r a c t i on i s a he t e r oge ne ous p opul a t i on of c e l l s t ha t a r e di s t i ngui s he d f r om e a c h ot he r by t he e xpr e s s i on of di f f e r e nt c e l l s ur f a c e a nt i ge ns T he s t e m c e l l s ubs e t i s r e pr e s e nt e d by c e l l s be a r i ng t he C D 34 m ol e c ul e a c om m on m a r ke r f or hum a n he m a t opoi e t i c s t e m c e l l s L i t e r a t ur e r e por t s t ha t t hi s f r a c t i on c ons t i t ut e s l e s s t ha n 1 pe r c e nt o f t he e nt i r e m ononuc l e a r f r a c t i on ( P r a nke F a i l a c e e t a l 2001) T h i s m e a ns f o r i ns t a nc e t ha t i n a n H U C B C t r a ns pl a nt of 100, 000 c e l l s a ppr oxi m a t e l y 1000 t o 2 000 c e l l s w i l l be he m a t opoi e t i c s t e m c e l l s S o m e i nve s t i ga t or s ha ve i de nt i f i e d s om e s ubc l a s s e s of c e l l s w i t hi n t he C D 34 s t e m c e l l f r a c t i on O ne s ubt ype p f C D 34 pos i t i ve c e l l s be a r t he c K I T t r a ns m e m br a ne t yr os i ne ki na s e gr ow t h f a c t or r e c e pt or ( C D 117) w ho s e l i ga nd i s t he s t e m

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21 c e l l f a c t or ( S C F ) ( L a ve r A bboud e t a l 1995) T he C D 117 S C F i nt e r a c t i on i s t hought t o be r e s pons i bl e f or t he a dhe s i on of s t e m c e l l s t o t he m i c r oe nvi r onm e nt ( L a ve r A bboud e t a l 1995) A not he r s ubt ype o f t he C D 34 pos i t i ve c e l l s i s r e pr e s e nt e d by c e l l s be a r i ng C D 34 a nd C D 133. T he s e c e l l s ha ve be e n s how n t o e xpr e s s t he V E G F a nd ot he r a ngi oge ni c f a c t or s s ugge s t i ng a r o l e of t he s e c e l l s i n t he r e gul a t i on of he m a t opoi e s i s a nd t he ge ne r a t i on of ne w c a pi l l a r i e s a t t he i nj u r y s i t e ( P om yj e Z i vny e t a l 2003) A t hi r d s ubs e t of C D 34 c e l l s i s one t ha t doe s not be a r t he C D 38 m a r ke r T hi s f r a c t i on, C D 34 pos i t i ve but C D 38 ne ga t i ve i s c ons i de r e d a m or e i m m a t ur e f r a c t i on a nd ha s be e n c or r e l a t e d s t r i c t l y w i t h t he r e ge ne r a t i ve c a pa c i t y of s t e m c e l l s ( E nc a bo, M a t e u e t a l 2003) I n a ddi t i on t o t he C D 34 pos i t i ve s t e m c e l l popul a t i on, l ym phoc yt e s m onoc yt e s a nd a s m a l l f r a c t i on of c e l l s be a r i ng a he t e r oge ne ous a r r a y of s ur f a c e m a r ke r s m a ke up t he r e m a i ni ng c e l l s w i t hi n t he m ononuc l e a r f r a c t i on ( P r a nke F a i l a c e e t a l 2001) T he l ym phoc yt e s a r e r e por t e d t o c ons t i t ut e f r om 30 up t o 45 pe r c e nt of t he e nt i r e f r a c t i on ( P r a nke F a i l a c e e t a l 2001; N a ga m ur a I noue M or i e t a l 2004) T hi s va r i a bi l i t y i s pr oba bl y due t o di f f e r e nt m e t hodol ogi e s o f s e pa r a t i on of t he m ononuc l e a r f r a c t i on. A l t e r na t i ve l y, i t m a y a t t r i but a bl e t o t he va r i a bi l i t y be t w e e n di f f e r e nt ba bi e s b l ood a nd t he i m m une s t a t us of t he m ot he r A bout ha l f of t h e l ym phoc yt e s a r e r e por t e d t o be T c e l l s a nd t he ot he r ha l f B c e l l s T he T c e l l s a r e a p pr oxi m a t e l y 80% C D 4 pos i t i ve w i t h t he r e m a i ni ng 20% be i ng C D 8 pos i t i ve T c e l l s E xpa ns i on of s pe c i f i c T c e l l s ubs e t s c a n be done i n v i t r o w i t h t he us e of s pe c i f i c c yt oki ne s s uc h a s i nt e r l e uki n 7 or i nt e r l e uki n 2 or t he S C F ( S a nc he z A l f a ni e t a l 2003) T he f r a c t i on of m onoc yt e s i s r e por t e d i n t he l i t e r a t ur e a s be i ng qui t e va r i a bl e s om e w he r e be t w e e n 10 t o 30 pe r c e nt o f t he w hol e m ononuc l e a r f r a c t i on ( P r a nke F a i l a c e e t a l 2001; S or g, A ndr e s e t a l 2001) T he s e c e l l s

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22 e xpr e s s a nt i ge ns c ha r a c t e r i s t i c of m onoc yt e s s uc h a s C D 14, C D 19, C D 11b a nd C D 26L ( S or g, A ndr e s e t a l 2001 ) T he r e m a i ni ng c e l l s o f t he m ononuc l e a r f r a c t i on c ons i s t s of va r i ous c e l l t ype s ; i nc l udi ng: de ndr i t i c c e l l s w hi c h do not e xpr e s s a ny s pe c i f i c m a r ke r but c a n be m o r phol ogi c a l l y di s t i ngui s he d f r om ot h e r m e s e nc hym a l de r i ve d c e l l s a s w e l l a s a C D 31 pos i t i ve popul a t i on ( a l s o c a l l e d P E C A M 1) w hi c h i s ge ne r a l l y e xpr e s s e d by m a t ur e he m a t opoi e t i c c e l l s ( P r a nke F a i l a c e e t a l 2 001) F i gur e 1. 2 i l l us t r a t e s a t ypi c a l f l ow c yt om e t r y a na l ys i s of t he c om pos i t i on o f c e l l s i n t he m ononuc l e a r f r a c t i on of c o r d bl ood a s t ypi c a l l y e m pl oye d i n ou r l a bor a t o r y. T he s t e m c e l l s ubs e t ( C D 34+ c e l l s ) a c c oun t e d f or l e s s t ha n 1 % of t he e nt i r e f r a c t i on C e l l s t ha t a r e C D 11b pos i t i ve ( s uc h a s m onoc yt e s ) r e pr e s e nt e d a bout 36% o f t he f r a c t i on a nd C D 4 pos i t i ve T c e l l s a r e a l m os t 50% B c e l l s ( a s s t a i ne d f or t he B c e l l m a r ke r B 220) oc c upy a f r a c t i on of a bout 16 % 1. 8 C on c l u s i on T he w or k e nc l os e d i n t hi s di s s e r t a t i on i s b r oke do w n i n t hr e e m a i n c ha pt e r s F i r s t w e de t e r m i ne d w he t he r t he r e w a s r e l a t i ons hi p be t w e e n num be r o f c or d bl ood c e l l s i n f us e d a nd be ha vi or a l / hi s t ol ogi c a l out c om e s ( C ha pt e r T hr e e ) S e c ondl y, w e f oc us e d on t he i nve s t i ga t i on of t he pos s i bl e e f f e c t of c or d bl ood i n t r a ve nous i nj e c t i on on t he pe r i phe r a l i m m une s ys t e m pa r t i c ul a r l y i n t he s pl e e n ( C ha pt e r F our ) F i na l l y, w e e xpl or e d t he r ol e of c or d bl ood c e l l s on t he r e gul a t i on of t he i nf l a m m a t or y e ve nt s t ha t f ol l ow i s c he m i a i n t he br a i n ( C ha pt e r F i ve )

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24 C H A P T E R T WO G E N E R A L M A T E R I A L S A N D M E T H O D S 2. 1 T h e M i d d l e C e r e b r al A r t e r y O c c l u s i on R a t M o d e l 2. 1. 1 A n i m al h o u s i n g an d m ai n t e n an c e A dul t S pr a gue D a w l e y r a t s ( a ve r a ge body w e i ght r a nge : 175. 0 25 0 g ) w e r e us e d i n a l l t he s e e xpe r i m e nt s A ni m a l s w e r e hous e d i n t he V i va r i um f a c i l i t y of t he C ol l e ge o f M e di c i ne of t he U ni ve r s i t y o f S ou t h F l o r i da T he y w e r e a l l ow e d t o a c c l i m a t e f or 7 da ys be f or e unde r goi ng a ny e xpe r i m e nt a l p r oc e dur e a nd ke pt unde r 12 hour s da r k/ l i ght c ondi t i ons i n a t e m pe r a t u r e c ont r ol l e d r oom w i t h w a t e r a nd c how ad l i bi dum V i va r i um pe r s onne l r e pl e ni s he d w a t e r a nd c how da i l y a nd r o ut i ne l y pe r f o r m e d c a ge c l e a ni ng. 2. 1. 2 S u r gi c al p r oc e d u r e A l l s ur gi c a l pr oc e dur e s w e r e pe r f or m e d i n s ur gi c a l r oom s of t he V i va r i um f a c i l i t y of t he C ol l e ge of M e di c i ne ( U ni ve r s i t y o f S out h F l o r i da ) a c c or di ng t o t he gui de l i ne s of t he N a t i ona l I ns t i t ut e of H e a l t h a s a ppr ove d by t he U n i ve r s i t y of S out h F l or i da I ns t i t ut i ona l A ni m a l C a r e a nd U s e C om m i t t e e T he r a t s w e r e a n e s t he t i z e d w i t h I s of l uor a ne m i xe d w i t h 2 t o 5 pe r c e nt of ox yge n. I nduc t i on o f t he a ne s t he s i a w a s pe r f or m e d de l i ve r i ng t he ga s m i xt ur e a t t he f l ow r a t e of 5 l i t e r s pe r m i nut e W he n t he a ni m a l w a s i n de e p s l e e p ( a s de t e r m i ne d by non r e s pons i ve ne s s t o t oe pi nc h) t he a ne s t he s i a w a s de c r e a s e d t o t he f l ow

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25 of 2 l i t e r s pe r m i nut e a nd m a i nt a i ne d a s s uc h f or t h e e nt i r e s ur gi c a l p r oc e dur e T he ne c k a r e a w a s s ha ve d a nd c ove r e d w i t h B e t a di ne s ol ut i o n ( A bbot t ) W i t h t he us e of bl unt s ur gi c a l t e c hni que s c ut a ne ous a nd s ubc ut a ne ous t i s s ue s a nd t he s ubm a ndi bul a r s a l i va r y gl a nd w e r e di s s e c t e d f r e e t o a l l ow t he e xpos ur e o f t he r i ght c om m on c a r ot i d a r t e r y T hi s a r t e r y w a s c a r e f ul l y di s s e c t e d f r e e of t he t e nt h c r a ni a l ne r ve T he i nt e r na l a nd e xt e r na l c a r ot i d a r t e r i e s w e r e e xpos e d a nd t he i r s m a l l c ol l a t e r a l ve s s e l s c a ut e r i z e d. T he e xt e r na l c a r ot i d w a s t he n l i ga t e d p r oxi m a l l y a nd di s t a l l y a n d s ubs e que nt l y s e ve r e d w i t h a s ha r p i ns t r um e nt T he p t e r ygopa l a t i ne a r t e r y w a s t he n i s ol a t e d a nd t i e d o f f w i t h a 5 0 s ut ur e T he e m bol us us e d t o i nduc e t he ve s s e l oc c l us i on w a s a c om m e r c i a l l y a va i l a bl e nyl on l i ne of t he di a m e t e r of 4 um t ha t w a s c ut t o a l e ngt h of 40 m m T he e m bol us w a s i ns e r t e d t hr ough t he e xt e r na l c a r ot i d a r t e r y i nt o t he i nt e r na l c a r ot i d t o t he o r i gi n o f t he m i ddl e c e r e br a l a r t e r y ( a ppr oxi m a t e l y 25 m m f r om poi nt o f e nt r y) T he di a g r a m i n F i gur e 1. 1 ( C ha pt e r 1) s how s a pi c t o r i a l r e pr e s e nt a t i on. A f t e r w a r ds t he e m bol us w a s t i e d i n pe r m a ne nt l y a nd a l l t he t i s s ue s r e pos i t i one d i n t he i r c or r e c t a na t om i c a l l oc a t i on. T he ne c k i nc i s i on w a s c l os e d w i t h 3 0 s ut ur e O nc e a l l t e c hni c a l pr oc e dur e s w e r e c onc l ude d, t he a ne s t he s i a w a s s t oppe d, t he a ni m a l w a s pl a c e d i n a c l e a n c a ge on a w a r m i ng pa d a nd obs e r ve d unt i l a w a ke ni ng. 2. 1. 3 P os t s u r gi c al p h ar m ac ol ogi c al a n d p h ys i c al s u p p or t I m m e di a t e l y a f t e r t he s ur gi c a l p r oc e dur e a l l t he r a t s r e c e i ve d a n i nj e c t i on w i t h K e t of e n ( 0. 4 m l i nt r a m us c ul a r l y) a nd B a yt r i l ( 0 1 m l s ubc ut a ne ous l y) a s a c ont r ol o f pos t s ur gi c a l pa i n a nd i nf e c t i ous r e a c t i ons T he s e i nj e c t i ons w e r e r e pe a t e d f or f our da ys a f t e r t he

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26 s ur gi c a l pr oc e dur e A l l a ni m a l s w e r e unde r t he c on t i nuous obs e r va t i on of t he V i va r i um s t a f f 2. 2 C or d B l ood C e l l s T r an s p l an t at i o n 2. 2. 1 P r e p ar at i on o f C r yop r e s e r ve d C e l l s C or d bl ood c e l l s w e r e dona t e d f r om a c or d bl ood c e l l ba nk ( S a ne r on C C E L T he r a pe ut i c s I nc T a m pa F L ) T he m ononuc l e a r f r a c t i on w a s s e pa r a t e d by F i c ol l gr a di e nt by t he c e l l ba nk a nd pr e s e r ve d i nt o H a nks B a l a nc e d S a l t S ol ut i on ( H B S S ) w i t h 5 pe r c e nt D M S O i n a l i qui d ni t r oge n c ont a i ne r T he m ononuc l e a r c e l l s ha r ve s t e d f r om a s i ngl e de l i ve r y w e r e pl a c e d i nt o f i ve 1 m l c r yopr ot e c t i ve vi a l s a t a c o nc e nt r a t i on of 10, 000 000 c e l l s pe r m l A t t he t i m e of us e e nough vi a l s a s ne e de d w e r e r a pi dl y t ha w e d a t 37C i n a w a t e r ba t h a nd ge nt l y t r a ns f e r r e d i nt o a 15 m l c e nt r i f uge t ub e c ont a i ni ng e i t he r 10 m l o f I s ol yt e S pH 7. 4 ( B B r a un/ M c G a w P ha r m a c e ut i c a l s ) or H B S S ( G i bc o) T he c e l l s w e r e t he n c e nt r i f uge d a t t he s pe e d of 600g f o r 15 m i nut e s T he s upe r na t a nt w a s di s c a r de d a nd r e pl e ni s he d w i t h f r e s h s ol ut i on, a nd r e s pun a ga i n. T hi s pr oc e s s w a s r e pe a t e d t hr e e t i m e s V i a bi l i t y of t he c e l l s a nd de t e r m i na t i on of t he num be r of c e l l s w e r e pe r f or m e d by t he t r ypa n bl ue dye e xc l u s i on m e t hod. B r i e f l y, 90 l of t r ypa n bl ue dy e di l ut e d a t a c onc e nt r a t i on of 4 pe r c e nt w a s c om bi ne d w i t h 10 l of c e l l s us pe ns i on. T he c e l l s i n f i ve qua dr a nt s of t he he m oc yt om e t e r w e r e c ount e d a nd t he r e s ul t s a ve r a ge d. C e l l num be r w a s a dj us t e d a c c or di ng t o t he de s i de r a t e d c onc e nt r a t i on a nd a vol um e of 500 l of c e l l s us pe n s i on w a s us e d i n e a c h t r a ns pl a nt

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27 2. 2. 2 I n t r a f e m or al t r an s p l an t at i on O n t he da y of t he t r a ns pl a nt t he a ni m a l s w e r e a ne s t he t i z e d w i t h i s of l uor a ne a s de s c r i be d a bove f or t he M C A O s ur ge r y. T he s upe r f i c i a l a r e a of t he r i ght l e g a nd t hi gh w e r e s ha ve d a nd B e t a di ne s ol ut i on a ppl i e d. C ut a ne ous a nd s ubc ut a ne ous t i s s ue s w e r e c a r e f ul l y di s s e c t e d us i ng bl unt t e c hni que s a nd t he f e m or a l v e i n w a s i s ol a t e d. A H a m i l t on s yr i nge w i t h a 26 ga uge ne e dl e w a s e m pl oye d f or t he t r a ns pl a nt O nc e t he ne e dl e w a s i ns e r t e d i nt o t he l um e n of t he f e m o r a l ve i n, t he ve i n w a s a r ound t he ne e dl e w a s l i ga t e d. T he c e l l s w e r e de l i ve r e d i n a vol um e o f 500 l ove r a 5 m i n ut e pe r i od. A f t e r t he ne e dl e w a s w i t hdr a w n, t he l i ga t i on w a s t i ght e ne d f ur t he r a nd t he i nc i s i on c l os e d us i ng a 3 0 s i l k s ut ur e A f t e r w a r ds t he a ne s t he s i a w a s s t oppe d, t he a ni m a l w a s pl a c e d i n i t s c a ge a nd obs e r ve d unt i l a w a ke ni ng. T he r a t s t ha t d i d not r e c e i ve a c e l l t r a ns pl a nt r e c e i ve d 500 l t he H U C B C f r e e r e s us pe ns i on s ol ut i on ( I s ol yt e S pH 7. 4) us e d i n t he a ni m a l s r e c e i vi ng c e l l s 2. 2. 3 I n t r a p e n i l e ve i n t r an s p l an t at i o n S i m i l a r t o t he p r oc e dur e out l i ne d a bove t he a ni m a l s w e r e r e a ne s t he t i z e d w i t h i s of l uor a ne T he pe ni s of t he a ni m a l w a s c a r e f ul l y e xpos e d a nd t he dor s a l ve i n i de nt i f i e d. B e t a di ne s ol ut i on w a s a ppl i e d t o t he w hol e e xpos e d s ur f a c e o f t he ge ni t a l s A 1c c s yr i nge w i t h a 31 ga uge ne e dl e w a s e m pl oye d f or t he c e l l t r a ns pl a nt O nc e t he ne e dl e w a s i ns e r t e d i nt o t he l um e n of t he do r s a l ve i n, t he c e l l s w e r e de l i ve r e d i n a vol um e o f 500 l ove r a 5 m i nut e pe r i od A f t e r t he ne e dl e w a s w i t h dr a w n, pr e s s ur e w a s a ppl i e d on t he ve i n f or a ppr oxi m a t e l y 20 s e c onds T he a ne s t he s i a w a s s t oppe d, t he a ni m a l w a s pl a c e d i n i t s c a ge a nd obs e r ve d unt i l r e ga i ni ng c ons c i ous ne s s A s f or t he f e m o r a l t r a ns pl a nt s t he

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28 r a t s t ha t di d no t r e c e i ve H U C B C w e r e i nj e c t e d w i t h 500 l t he H U C B C f r e e r e s us pe ns i on s ol ut i on ( I s ol yt e S pH 7 4 or H B S S ) 2. 3 A n i m al s ac r i f i c e an d p r e p ar at i on of t i s s u e s A l l s a c r i f i c e pr oc e dur e s w e r e pe r f or m e d i n t he V i va r i um f a c i l i t y of t he C ol l e ge of M e di c i ne ( U ni ve r s i t y of S out h F l or i da ) a c c or di ng t o t he gui de l i ne s of t he N a t i ona l I ns t i t ut e of H e a l t h a ppr ove d by t he U ni ve r s i t y o f S out h F l or i da I ns t i t ut i ona l A ni m a l C a r e a nd U s e C om m i t t e e A l l r a t s w e r e a ne s t he t i z e d w i t h pe nt oba r bi t a l ( 150 m g/ kg i nt r a pe r i t one a l l y) W he n a ni m a l s w e r e non r e s po ns i ve t o t oe pi nc h, a 22 ga uge ne e dl e w a s i ns e r t e d i nt o t he l e f t ve nt r i c l e t he a ur i c ol a o f t he r i ght a t r i um w a s pe r f or a t e d, a nd a ni m a l s w e r e pe r f us e d w i t h 0. 1 M phos pha t e buf f e r ( P B ) pH 7. 4, f ol l ow e d by 4% pa r a f or m a l de hyde i n P B ( P F A ) A f t e r w a r d, br a i n a nd ot h e r o r ga ns ( he a r t l ungs l i ve r ki dne y, s pl e e n, t hym us ) w e r e ha r ve s t e d a nd pl a c e d i n 10 f ol d vol um e of 4% P F A f or 24 hour s A t t he e nd of t he pos t f i x pe r i od, or ga ns w e r e pl a c e d i n 20% s uc r os e i n 0 1 M P B f or 24 hour s a nd f i na l l y c r yopr e s e r ve d a t 80C W he n pe r f us i on of t he a ni m a l s w a s not ne c e s s a r y ( s uc h a s i n e xpe r i m e nt s c onduc t e d i n C ha pt e r 4 a nd 5) a ni m a l s w e r e a ne s t he t i z e d w i t h I s of l uor a ne a nd de c a pi t a t e d us i ng a s t a nda r d gui l l ot i ne

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29 C H A P T E R T H R E E D O S E E F F E C T O F C O R D B L O O D I N F U S I O N O N F U N C T I O N A L A N D H I S T O L O G I C A L R E C O V E R Y O F R A T S S U B JE C T E D T O M I D D L E C E R E B R A L A R T E R Y O C C L U S I O N 3. 1 I n t r od u c t i on S e ve r a l s t udi e s ha ve r e por t e d t ha t i nf us i on o f hum a n um bi l i c a l c or d bl ood c e l l s ( H U C B C ) a f t e r t he i nduc t i on of br a i n i s c he m i a c a n i m pr ove be ha vi o r a l out c om e ( C he n, S a nbe r g e t a l 2001; W i l l i ng, L i xi a n e t a l 2003; W i l l i ng, V e nd r a m e e t a l 2003) T he s e m i na l f i ndi ngs of C he n e t a l ( C he n, S a nbe r g e t a l 2001) de m ons t r a t e d t ha t t r e a t m e nt by i nt r a ve nous de l i ve r y o f H U C B C 24 hour s a f t e r M C A O i n r a t s c oul d i m pr ove f unc t i ona l r e c ove r y, a s e vi de nc e d by i m p r ove m e nt s on t he r ot a r od t e s t a nd M odi f i e d N e ur ol ogi c a l S e ve r i t y S c or e ( m N S S ) S ubs e que nt s t udi e s ha ve s how n t ha t t he be ha vi or a l r e c ove r y i nduc e d by i nt r a ve nous H U C B C t r e a t m e nt of M C A O r a t s i s c om pa r a bl e i f not be t t e r t ha n t he r e c ove r y obs e r ve d a f t e r i nt r a c e r e br a l t r a ns pl a nt a t i on ( W i l l i ng L i xi a n e t a l 2003) T he s e obs e r va t i ons ha ve r a i s e d gr e a t ho pe a nd i nt e r e s t i n t he a r e a o f he m a t ol ogi c a l s t e m c e l l t r a ns pl a nt a t i on t he r a py f or s t r oke H ow e ve r s e ve r a l i m por t a nt que s t i ons r e ga r di ng H U C B C t he r a pe ut i c pot e nt i a l r e m a i n una ns w e r e d: 1) w ha t i s t he be s t dos e f or t he s e c e l l s ? 2) W ha t i s t he t he r a pe ut i c t i m e w i ndow f or e m pl oym e nt o f t he s e c e l l s pos t s t r oke ( hour s da ys w e e ks ) ? 3) i s i t s ol e l y t he s t e m c e l l popu l a t i on m e di a t i ng

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30 r e c ove r y a nd 4 ) w ha t i s t he t he r a pe ut i c m e c ha ni s m ( ne ur on r e pl a c e m e nt or ne ur on s pa r i ng) ? I n t hi s s t udy, w e e xa m i ne d t he H U C B C t he r a pe ut i c dos e i n s t r oke a s a ns w e r i ng t hi s que s t i on pr ovi de s a n i m por t a nt pl a t f o r m f r om w hi c h t he r e m a i ni ng que s t i ons m a y be a ddr e s s e d. T he s t udi e s by C he n e t a l ( C he n, S a nbe r g e t a l 2001) a nd W i l l i ng e t a l ( W i l l i ng, L i xi a n e t a l 2003) ha ve e m pl oye d H U C B C dos e s a r ound 10 6 c e l l s a nd s how n a m e l i or a t i on of s t r oke i nduc e d f unc t i ona l de f i c i t s S t udi e s us i ng H U C B C i n ot he r ne ur ode ge ne r a t i ve di s e a s e s m ode l s ha ve s how n f unc t i ona l r e c ove r y r e qui r e d f r om r e l a t i ve l y f e w ( 10, 000 ) up t o m e ga dos e s ( s uc h a s 5x10 7 c e l l s ) of H U C B C ( C he n a nd E nde 2000; G a r buz ova D a vi s W i l l i ng e t a l 2003) G i ve n t he w i de dos e r a nge i n w hi c h H U C B C ha d be e n r e por t e d t o be t he r a pe ut i c w e s ought t o i nve s t i ga t e w he t he r t he r e w a s a n opt i m a l dos e a t w hi c h H U C B C s how e d e f f i c a c y i n t he M C A O m ode l I n pa r t i c ul a r w e w a nt e d t o de t e r m i ne w he t he r i m pr ove m e nt i n be ha vi or a l ou t c om e m e a s ur e s w oul d s how a H U C B C dos e de pe nde nt e f f e c t A ddi t i ona l l y, w e m e a s ur e d t he i nf a r c t vol u m e t o e va l ua t e qua nt i t a t i ve l y w he t he r H U C B C c oul d r e duc e t he a m ount of t i s s ue da m a ge pr e s e nt a f t e r M C A O a s t he r e a r e no c ons i s t e nt r e por t s i n t he l i t e r a t ur e on w he t he r c o r d bl ood t r e a t m e nt c oul d a m e l i or a t e b r a i n s t r uc t ur a l a nd hi s t ol ogi c a l a l t e r a t i ons i nduc e d by M C A O ; t hi s m a y be i m por t a nt f e a t ur e s w he n e va l ua t i ng t he i m pa c t o f c or d bl ood c e l l i nf u s i on i n t hi s di s e a s e m ode l A l t hough i nt r a c e r e br a l t r a ns pl a n t a t i on i s c om m onl y us e d t o de l i ve r c e l l s f or ne ur ona l c e l l t r a ns pl a nt a t i on, t he i nt r a ve nous r out e ha s a l s o be e n de m ons t r a t e d t o be e f f e c t i ve w he n he m a t opoi e t i c c e l l s ( bone m a r r ow or bl ood d e r i ve d c e l l s ) a r e us e d, a s t he s e ha ve be e n de t e c t e d i n t he b r a i n pa r e nc hym a a nd s how n t o i nduc e be ha vi or a l r e c ove r y i n

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31 a ni m a l m ode l s of ne u r ode ge ne r a t i ve di s or de r s ( G a r buz ova D a vi s W i l l i ng e t a l 2003; M a hm ood, L u e t a l 2004) A ddi t i ona l l y, s om e i nv e s t i ga t or s ha ve de m ons t r a t e d t ha t c or d bl oo d c e l l s a nd ot he r t ype s of c e l l s c a n be c he m ot a c t i c a l l y a t t r a c t e d t o t he s i t e of i nj ur y ( A boody, B r ow n e t a l 2000; C he n, S a nbe r g e t a l 2001) S i nc e H U C B C w e r e e xc l us i v e l y e m pl oye d t hr ough t he i nt r a ve nous r out e i n t he c u r r e nt s t udy, w e w onde r e d w he t he r t he s e c e l l s c oul d s pe c i f i c a l l y r e a c h a nd s u r vi ve i n t he i nj ur y he m i s phe r e 4 w e e ks pos t i nf us i on. M ove ove r gi ve n t he i nt r a ve nous de l i ve r y r out e a nd t he pos s i bl e r e l e va nc e t o t he i r t he r a pe ut i c e f f i c a c y w e pr of i l e t he H U C B C di s t r i but i on i n ot he r or ga n s ys t e m s 3. 2 M at e r i al s an d M e t h od s A ni m a l s w e r e di vi de d i nt o s e ve n e xpe r i m e nt a l gr o ups : s ha m s ur ge r y ( n= 4) M C A O onl y ( n= 13) r a t s i nf us e d w i t h 10 4 ( n= 6) 10 5 ( n= 6 ) 10 6 ( n= 6) 10 7 ( n= 6) o r 3 5 x10 7 ( n= 4) H U C B C 24 hour s a f t e r M C A O A f t e r t he t r a ns pl a nt a l l a ni m a l s ( i nc l udi ng a ni m a l s t ha t ha d not r e c e i ve d a c e l l t r a ns pl a nt ) w e r e i nj e c t e d w i t h c yc l os por i n ( C S A ) i n t he a m ount o f 10 m g pe r ki l ogr a m s o f w e i ght of t he a ni m a l S i nc e c yc l os por i n ha s be e n s how n t o be ne ur opr ot e c t i ve i n a ni m a l m ode l s of s t r oke ( B o r l o nga n, S t a hl e t a l 1999) i t i s c r i t i c a l t o c ont r ol f o r t hi s i n a ny t r a ns pl a nt s t udy t ha t a dm i n i s t e r s t hi s dr ug. T he r e f o r e i n our s t udy, a l l a ni m a l s r e ga r dl e s s of t he t r e a t m e nt ( H U C B C or m e di a ) w e r e i nj e c t e d w i t h c yc l os por i ne U s i ng t hi s de s i gn e ns ur e s t ha t a ny c ha nge s e e n i n t he c e l l t r a ns pl a nt e d a ni m a l s r e l a t i ve t o non t r a ns pl a nt e d a ni m a l s c a nnot be e xpl a i ne d by c yc l os por i n a dm i ni s t r a t i on a l one C yc l os por i ne w a s di l ut e d i n c om m e r c i a l l y a va i l a bl e ol i ve oi l a t a c onc e nt r a t i on of 1: 1 T he i nj e c t i on w a s r e pe a t e d d a i l y f or t he du r a t i on of t hi s s t udy. I n

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32 or de r t o pr ot e c t t he i m m unoc om pr o m i s e d a ni m a l s f r om oppo r t uni s t i c i nf e c t i ons a l l a ni m a l s w e r e hous e d i n a ve nt e d hood a nd t he i r d r i nki ng w a t e r w a s r e pl a c e d w i t h a c i di f i e d w a t e r 3. 2. 1 B e h avi or t e s t s T he ba t t e r y o f t e s t s c ons i s t e d of m e a s ur e m e nt s of s pont a ne ous a c t i vi t y, e l e va t e d body s w i ng t e s t a nd s t e p t e s t a nd w e r e pe r f o r m e d i n a l l a ni m a l s be f or e M C A O a nd a t 2 a nd a t 4 w e e ks a f t e r M C A O 3. 2. 1 1 M e as u r e m e n t o f S p on t a n e ou s ac t i vi t y S pont a ne ous a c t i vi t y w a s m e a s ur e d w i t h t he a ut o m a t e d V e r s a M a x S ys t e m ( A c c us c a n I ns t r um e nt s C ol um bus O H U S A ) F or t he da yl i ght s pont a ne ous a c t i vi t y t e s t r a t s w e r e pl a c e d f or 1 hour i n a P l e xi gl a s box ( 35x20x30 c m ) w i t h be ddi ng ; t he box w a s s ur r ounde d by t w o l e ve l s of i n f r a r e d be a m s T hi r t e e n l oc om ot or pa r a m e t e r s i nc l ud i ng hor i z ont a l a c t i vi t y ( H A ) m ove m e nt num be r ( M V N ) m ove m e nt t i m e ( M V T ) t ot a l di s t a nc e ( T D ) ve r t i c a l a c t i vi t y ( V A ) ve r t i c a l m ov e m e nt s ( V M N ) s t e r e ot ypy num be r ( S N ) s t e r e ot ypy c ount s ( S C ) s t e r e ot ypy t i m e ( S T ) c l oc kw i s e r ot a t i on ( C R ) c ount e r c l oc kw i s e r ot a t i on ( C C R ) c e nt e r di s t a nc e ( C D ) a nd c e nt e r t i m e ( C T ) w e r e e va l ua t e d. A c t i v i t y w a s r e c or de d e ve r y 5 m i nut e s f or a t ot a l t i m e o f 1 hour T he ni ght t i m e s pont a ne ous a c t i vi t y t e s t w a s c onduc t e d w i t h t he s a m e pr o c e dur e s e xc e pt t ha t t he a c t i vi t y w a s m e a s ur e d f or 12 hou r s t h r ough t h e da r k pha s e of t he l i ght c yc l e a nd r e c or de d e ve r y hour

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33 3. 2. 1 2 S t e p t e s t T hi s t e s t w a s pe r f or m e d on a ha r d f l a t s ur f a c e w he r e t he r a t s w e r e pl a c e d f o r a f e w m i nut e s be f or e t he a c t ua l t e s t i n o r de r t o l e t t he m h a bi t ua t e t o t he t e s t i ng a r e a T he r a t s w e r e he l d t i ght l y i m m obi l i z i ng a l l f our pa w s e xc e pt e i t he r t he r i ght or l e f t f or e l i m b W i t h t he f r e e f or e l i m b pl a c e d on t he s ur f a c e t he y w e r e dr a gge d f or a l e ngt h of 1 m e t e r t ow a r ds t h e di r e c t i on o f t he pl a c e d f or e l i m b T he num be r of s t e ps m a de w a s c ount e d. T he s a m e pr oc e dur e w a s pe r f or m e d w i t h t he ot he r f or e l i m b d r a ggi ng t he a ni m a l f or t he s a m e di s t a nc e i n t he oppos i t e di r e c t i on T he d i f f e r e nc e be t w e e n num be r of s t e ps pe r f or m e d w i t h t he r i ght pa w a nd num be r o f s t e ps pe r f o r m e d w i t h t he l e f t pa w w a s c a l c ul a t e d. 3. 2. 1 3 E l e vat e d B od y S w i n g t e s t F or t hi s t e s t t he a ni m a l w a s pl a c e d on a P l e xi gl a s s c a ge w i t h f r e s h be ddi ng a nd a l l ow e d t o a c c l i m a t e f or a bout 5 m i nut e s be f or e s t a r t i ng t h e t e s t R a t s w e r e he l d t i ght l y by t he t a i l a t a ppr oxi m a t e l y 10 c m a bove t he s ur f a c e of t he t e s t i ng a r e a W he n r a t s a r e he l d a s de s c r i be d, t he y s pont a ne ous l y l i f t t he i r body t ow a r d t he r i gh t or t he l e f t of t he i r m i dl i ne body a xi s T he f r e que nc y of l e f t o r r i ght s w i ngs w a s c ount e d ove r 20 c ons e c ut i ve t r i a l s B e c a us e i t ha s be e n obs e r ve d t ha t p r i or t o unde r go i ng s t r oke s om e r a t s a l r e a dy de m ons t r a t e a n a s ym m e t r y, s w i ngi ng m or e t ow a r d s one s pe c i f i c s i de w e c a l c ul a t e d t he e l e va t e d body s w i ng t e s t bi a s by t a ki ng t he a bs ol ut e va l ue of t he di f f e r e nc e be t w e e n t he num be r of s w i ngs on t he i m pa i r e d s i de m i nus 10 s i nc e a nonbi a s e d a ni m a l s houl d t ur n t o bot h s i de s e qua l l y. T he r e f o r e a r a t t ha t pr e s e nt s n o bi a s w i l l s c or e 0 a nd a n i nc r e a s i ng s c or e r e pr e s e nt s gr e a t e r l a t e r a l m ove m e nt w i t h a m a xi m um s c or e be i ng t ha t of 1 0.

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34 3. 2. 2 B r ai n h i s t ol ogi c al as s e s s m e n t F our w e e ks a f t e r t he t r a ns pl a nt a ni m a l s w e r e s a c r i f i c e d a nd br a i n w e r e ha r ve s t e d a s de s c r i be d i n c ha pt e r 2. 3. 2. 2 1 N i s s l T h i on i n e s t ai n i n g C r yopr e s e r ve d br a i ns w e r e c ut w i t h a c r yos t a t i n 2 0um t hi c k s e c t i ons A l l s e c t i ons w e r e pr e s e r ve d a t 80C unt i l us e T he N i s s l T hi oni ne dye w a s obt a i ne d by c om bi ni ng 625m l of T hi on i ne w i t h 500 m l o f a s ol ut i on pr e pa r e d by a ddi ng 18 m l of 6% g l a c i a l a c e t i c s ol ut i on w i t h 100m l of 1N N a O H T he s t a i ni ng w a s pe r f or m e d a s f ol l ow s C r yop r e s e r ve d s l i de s w e r e a l l ow e d t o r e a c h r oom t e m pe r a t u r e a nd w e r e s ubs e que nt l y s oa ke d i n 100% e t ha nol f or 10 m i nut e s T hi s s t e p w a s f o l l ow e d by t he i m m e r s i on of t he s l i de s i n a de c r e a s i ng e t ha nol gr a di e nt : 95% e t ha nol f o r 2 m i nut e s 70% e t ha nol f or 2 m i nut e s 50% e t ha nol f or 2 m i nut e s A f t e r w a r ds t he s l i de s w e r e i m m e r s e d i n di s t i l l e d w a t e r f o r 1 m i nut e a nd t he N i s s l T hi on i n dye w a s t he n a ppl i e d f or 50 s e c onds T he s l i de s w e r e i m m e di a t e l y r e s oa ke d i n w a t e r f o r 2 m i nut e s a nd t he n de hydr a t e d t h r ough a n i nc r e a s i ng e t ha nol gr a di e nt ( 50 % e t ha nol f or 3 m i nu t e s 70% e t ha nol f or 3 m i nut e s a nd 95% e t ha nol f or 3 m i nut e s a nd 100% e t ha nol f or 7 m i nut e s ) F i na l l y s l i de s w e r e l e f t i n xyl e ne f or 15 m i nut e s S l i de s w e r e t he n c ove r s pl i ppe d w i t h P e r m ount ( P r oS c i T e c h) 3. 2. 2 2 M e as u r e m e n t o f I n f ar c t vol u m e T he a r e a s of c e r e b r a l i nf a r c t i on w e r e de l i ne a t e d a t s i x pr e s e l e c t e d c or ona l s e c t i ons a t a di s t a nc e of 1 m m a pa r t be gi nni ng 1. 7 m m a nt e r i or t o br e gm a t h r ough 3 3 m m pos t e r i or t o br e gm a T he a r e a of i s c he m i c da m a ge a nd t he t ot a l a r e a of t he c ont r a l a t e r a l he m i s phe r e w e r e qua nt i f i e d us i ng a n i m a ge a na l yz e r ( I m a ge P r o P l us ) T he t ot a l vol um e o f i s c he m i c t i s s ue a nd t ha t of t he c ont r a l a t e r a l he m i s phe r e w e r e c a l c ul a t e d a s f ol l ow s : t ot a l

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35 vol um e = s um ( a r e a of pr e de f i ne d c or ona l s e c t i ons ( m m ) ) 1 6 x ( i nt e r s e c t i on di s t a nc e ( m m ) ) w he r e e a c h m e a s ur e d a r e a o f t he s i x c or ona l s e c t i ons w a s m ul t i pl i e d by t he di s t a nc e t o t he s ubs e que nt s e c t i on, a nd t he t ot a l vo l um e w a s obt a i ne d by c a l c ul a t i ng t he s um of t he s e m e a s ur e s S i nc e a t one m ont h a f t e r M C A O t he i nj ur e d b r a i n he m i s phe r e ha s t ypi c a l l y unde r gone a s hr i nka ge pr oc e s s t ha t oc c ur s t o a va r i a bl e de g r e e w e f ound i t ne c e s s a r y t o nor m a l i z e t he vol um e o f da m a ge f or t he de c r e a s e d s i z e of t h e he m i s phe r e I n or de r t o do s o, w e c a l c ul a t e d t he vol um e of i s c he m i c da m a ge a s pe r c e nt of vol um e of c ont r a l a t e r a l ( i nt a c t ) he m i s phe r e T he a m ount of d a m a ge w a s qua nt i f i e d by t a ki ng i nt o a c c ount t he obs e r va bl e da m a ge ( t he ne c r ot i c c or e a nd t he bys t a nde r w hi t e t i s s ue a s s t a i ne d w i t h N i s s l T hi oni ne ) i n a ddi t i on t o t he l a c una r s pa c e s 3. 2. 3 T r ac k i n g t h e i n t r ave n ou s t r an s p l an t e d c e l l s T he s a m e a ni m a l s e m pl oye d i n t he be ha vi or a l a nd hi s t opa t hol ogi c a l s t udi e s unde r w e nt pol ym e r a s e c ha i n r e a c t i on ( P C R ) a na l ys i s f or hum a n ne s t e d pr i m e r s a nd i m m unohi s t oc he m i s t r y a na l ys i s A ni m a l s w e r e t a ke n f r om a l l s e ve n e xpe r i m e nt a l c ondi t i ons a s de s c r i be d pr e vi ous l y. A l l a ni m a l s w e r e t r a ns pl a nt e d i nt r a ve nous l y 24 hour s a f t e r t he s t r oke s ur ge r y a nd s a c r i f i c e d 4 w e e ks a f t e r t h e c e l l t r a ns pl a nt a c c or di ng t o t he m e t hods de s c r i be d i n C ha pt e r 2 H a l f of t he a ni m a l s pe r gr oup unde r w e nt P C R a na l ys i s t he ot he r ha l f unde r w e nt i m m unohi s t oc he m i c a l a na l ys i s 3. 2. 3 1 P ol ym e r as e C h ai n R e ac t i o n 3. 2. 3 1. 1 D N A e xt r ac t i on B r a i ns a n d ot he r or ga ns ( he a r t l ungs l i ve r ki dne y s pl e e n, t hy m us bone m a r r ow ) w e r e s a m pl e d. A f r a gm e nt o f a ppr oxi m a t e l y 50 m 3 f o r e a c h s a m pl e d or ga n w a s

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36 s us pe nde d i n 500 l o f l ys i s buf f e r ( 400 g/ m l of P r ot e i na s e K i n 20m M T r i s C L 5m M E D T A 400m M N a C L 1% S D S ) l e f t f or 12 hou r s i n a w a t e r ba t h a t 55 C A n e qua l vol um e of P he nol ( I nvi t r oge n) w a s a dde d t o t he s us pe ns i on, t he e nt i r e m i xt ur e w a s t he n vor t e xe d a nd s pun f o r 3 t i m e s a t 1500g f or 10 m i n ut e s T he a que ous pha s e w a s c ol l e c t e d a nd r e s us pe nde d i n P he nol t hr e e t i m e s a nd s pun a s i n t he pr e vi ous s t e p. T he s a m e pr oc e s s w a s r e pe a t e d onc e w i t h C hl or of o r m ( G i bc o ) D N A w a s s ubs e que nt l y pr e c i pi t a t e d by a ddi ng a n e qua l vol um e of I s opr opa nol ( S i gm a ) A f t e r c e nt r i f ugi ng t he t ube f o r 5 m i nut e s a t 1200g, a D N A pe l l e t w a s obt a i ne d T h i s w a s t he n w a s he d i n 70% e t ha nol a nd dr i e d a t r oom t e m pe r a t ur e f or 15 m i nut e s T he pe l l e t w a s t he n di s s ol ve d i n 100 l o f T r i s E D T A ( T E ) ( pH 8. 0 ) a t 37 C ove r n i ght F o r e a c h s a m pl e t he D N A obt a i ne d w a s qua nt i f i e d us i ng t he S m a r t S pe c s pe c t r a phot om e t e r s e t a t t he w a ve l e ngt h of 260 n m 3. 2. 3 1. 2 P C R p r ot oc ol f or n e s t e d p r i m e r s P C R w a s r un f or t he hum a n gl yc e r ol 3 phos pha t e de hydr oge na s e ge ne us i ng t he f ol l ow i ng pr i m e r s ( s e ns e : G G C T G G G A C T C A T G G A G A T a nt i s e ns e : C G G G T A A G T C G T T G A G A A A G ) a t a c on c e nt r a t i on of 500nM f or 30 c yc l e s E a c h c yc l e w a s r un a s f ol l ow s : 95C f or 30 s e c 67C f o r 30 s e c 72C f or 30 s e c A s e c ond P C R w a s r un f or ne s t e d pr i m e r s ( s e ns e : T C T T G G A G A G C T G T G G T G T T G a nt i s e ns e : G T T A C C T G A A A G G A C T G C ) a t a c onc e nt r a t i on of 500 nM f o r 30 c yc l e s E a c h c yc l e w a s r un a s f ol l ow s : 95C f or 2 m i n 95C f or 3 0 s e c 60C f o r 30 s e c 72C f o r 30 s e c A pos i t i ve c ont r ol of hum a n D N A ( 60pg) a nd a non t e m pl a t e s a m pl e w e r e r un w i t h e a c h r e a c t i on.

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37 3. 2. 3 1. 3 P ol yac r yl am i d e ge l p r e p ar at i on an d S i l ve r s t ai n i n g D N A pr oduc t s w e r e r un on 3 5% pol ya c r yl a m i de g e l s E a c h ge l w a s pr e pa r e d by a ddi ng 100 l of a m m oni um pe r s ul f a t e ( A P S B i o r a d) a nd 5 l of t e t r a m e t hyl e t hyl e ne di a m i ne ( T E M E D B i or a d) t o a s ol ut i on c om pos e d of 2m l o f a c r yl a m i de ( B i or a d) 2m l of T B E ( T r i s 27g r B or i c A c i d 13 75gr a nd 10m l E D T A ) a nd 6 m l of w a t e r G e l s w e r e pr e pa r e d us i ng t he B i or a d ge l m ount i ng c a s s e t t e s a f t e r 15 m i nut e s of s ol i di f i c a t i on S a m pl e s w e r e r un i n t he a m ount o f 5 l a dde d t o 2 l of l oa di ng buf f e r A 100 ba s e pa i r l a dde r ( N e w E ngl a nd B i ol a bs ) w a s r un i n e a c h ge l A f t e r ge l s w e r e r un f or 20 m i nut e s a t 90 A m ps t he y w e r e s oa ke d i n 10 % e t ha nol f or 3 m i nut e s S i l ve r s t a i ni ng w a s pe r f o r m e d by i m m e r s i ng t he ge l s i n 1% n i t r i c a c i d f or 3 m i nut e s A f t e r r i ns i ng w i t h di s t i l l e d w a t e r t he ge l s w e r e s oa ke d i n 0. 1% s i l ve r ni t r a t e f o r 10 m i nu t e s i n t he da r k T he ge l s w e r e t he n t r a ns f e r r e d t o t he de ve l opi ng s ol ut i on ( 6g s odi um c a r bona t e 120 l f or m a l de hyde i n 100m l of w a t e r ) un t i l t he ba nds a ppe a r e d. A f t e r s oa ki ng t he ge l s i n 1 0 % a c e t i c a c i d f or 5 m i nut e s t he ge l s w e r e l e f t i n 1 % gl yc e r ol f o r 2 ho ur s F i na l l y, ge l s w e r e dr i e d be t w e e n c e l l opha ne f i l m s a nd s c a nne d. 3. 2. 3 2 I m m u n oh i s t oc h e m i s t r y T e c h n i q u e s 3. 2. 3 2. 1 Q u e n c h i n g of A u t of l u or e s c e n c e T he s e c t i ons w e r e r i ns e d 3 t i m e s f or 10 m i n w i t h p hos pha t e buf f e r e d s a l i ne ( P B S ) a nd a n a ut of l uor e s c e nc e que nc hi ng s ol ut i on ( ki ndl y pr ovi de d by D r A nn S t e e l e A l l C hi l d r e ns H os pi t a l D e pa r t m e nt of P a t hol ogy S t P e t e r s bur g F l or i da ) w a s a ppl i e d f or 10 s e c onds a t r oom t e m pe r a t ur e a t a di l ut i on o f 1: 100 f r om t he gi ve n s t oc k c onc e nt r a t i on.

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38 3. 2. 3 2. 2 I m u n oh i s t oc h e m i s t r y p r ot oc ol P r i m a r y m ous e m onoc l ona l a nt i body a ga i ns t hum a n nuc l e i ( H uN u, C he m i c on, I nc ) w a s a ppl i e d ove r ni ght a t a 1: 50 di l ut i on S l i de s w e r e t he n r i ns e d 3 t i m e s f or 10 m i n w i t h P B S f ol l ow e d by a t w o hour i nc uba t i on i n a 1: 5 00 di l ut i on of f l uor e s c e i n i s ot hi oc ya na t e ( F I T C ) c onj uga t e d goa t a nt i m ous e s e c onda r y a nt i body ( A l e xa 488, M ol e c ul a r P r obe s ) C ont r ol s l i de s w e r e s t a i ne d w i t h t he s a m e pr ot oc ol w i t h t he om i s s i on of t he pr i m a r y a nt i body. S l i de s w e r e w a s he d f i ve t i m e s i n P B S a nd D A P I s t a i ni ng ( M ol e c ul a r P r obe s ) w a s pe r f or m e d t o vi s ua l i z e nuc l e a t e d c e l l s F i na l l y, s l i de s w e r e c ove r s l i ppe d w i t h V e c t a s hi e l d a nd pr e s e r ve d i n t he da r k a t 4C 3. 2. 4 S t at i s t i c al A n al ys i s T he be ha vi or a l t e s t da t a w e r e a na l yz e d us i ng a na l y s i s of va r i a nc e ( A N O V A ) w i t h r e pe a t e d m e a s ur e s A hom oge ne i t y of va r i a nc e w a s a l s o a ppl i e d i f t h i s t e s t w a s s i gni f i c a nt ( p< 0. 05) t he K r us ka l l W a l l i s H T e s t w a s e m pl oye d f or pos t hoc c om pa r i s ons S t a t i s t i c a l a na l ys i s of t he br a i n i nf a r c t m e a s ur e m e nt s w a s pe r f or m e d w i t h A N O V A 3. 3 R e s u l t s 3. 3. 1 I n f u s i on o f c or d b l ood c e l l s r e d u c e s t h e s t r ok e i n d u c e d h yp e r ac t i vi t y. 3. 3. 1 1 D ayt i m e s p on t a n e ou s ac t i vi t y t e s t F or e l e ve n of t he t h i r t e e n a na l yz e d pa r a m e t e r s t he ove r a l l A N O V A r e ve a l e d s i gni f i c a nt di f f e r e nc e s be t w e e n t he gr oups ( p< 0. 0001 t o p< 0 0 4) M or e ove r on ni ne of t he t hi r t e e n va r i a bl e s t he r e w e r e s i gni f i c a nt i nt e r a c t i ons be t w e e n gr oups a nd l e ngt h of t i m e pos t

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39 t r a ns pl a nt gr oups a nd obs e r va t i ona l pe r i od a nd g r oups l e ngt h of t i m e pos t t r a ns pl a nt a nd obs e r va t i ona l pe r i od ( p< 0 001 t o p< 0 03) ( T a bl e 3. 1) F or t he pa r a m e t e r s C C R H A M V T S C V M N di f f e r e nc e s be t w e e n t he dos e gr oups w e r e e xa m i ne d a t 2 a nd 4 w e e ks ( T a bl e 3. 2) O n C C R a t 2 w e e ks t h e a ni m a l s t r e a t e d w i t h 10 5 10 6 a nd 3 5x10 7 c e l l s w e r e s i gni f i c a nt l y l e s s a c t i ve t ha n t he m e di a t r e a t e d c ont r ol s ( p= 0. 03, p= 0. 005 p= 0. 04 r e s pe c t i ve l y) A t 4 w e e ks t he 10 7 H U C B C gr oup a l s o be c a m e s i gni f i c a nt l y l e s s hype r a c t i ve t ha n t he s t r oke on l y r a t s ( p= 0. 03) a nd t he 10 6 a nd 3 5x10 7 gr oups r e m a i ne d s i gni f i c a nt l y l e s s a c t i ve ( p= 0. 008 a nd p= 0. 02 r e s pe c t i ve l y) S i m i l a r l y, on H A t he r e w a s a s i gni f i c a nt r e duc t i on of m ove m e nt i n t he 10 5 a nd 10 6 a nd 3 5x10 7 gr oups c om pa r e d t o t he non r e c i pi e nt M C A O r a t s a t 2 w e e ks ( p= 0. 005 f or a l l t hr e e gr oups ) a nd a t 4 w e e ks t he 10 6 a nd 10 7 dos e gr oup s w e r e s i gni f i c a nt l y l e s s a c t i ve t ha n t he M C A O onl y a ni m a l s ( bot h p= 0. 05 ) O n M V T a t 2 w e e ks pos t t r a ns pl a nt t he a ni m a l s i n t he 10 4 10 5 a nd 10 6 gr oups w e r e s i gni f i c a nt l y l e s s a c t i ve t ha n t he c ont r ol s ( p= 0. 009 p= 0. 043, p= 0 02) A t 4 w e e ks t he onl y g r oup t ha t w a s s i gni f i c a nt l y l e s s a c t i ve w a s t he 10 7 gr oup ( p= 0. 05 ) O n S C t he r e w a s a r e duc t i on i n hype r a c t i vi t y onl y a t 2 w e e ks f or t he gr oup 10 6 ( p= 0. 04 ) O n V M N t he r e w e r e no di f f e r e nc e s be t w e e n t he t r e a t e d gr oups a nd t he c ont r ol s but t he r e w a s a s i gni f i c a nt r e duc t i on o f t he a c t i vi t y be t w e e n t he 10 4 dos e gr oup a nd t he 3 5x10 7 gr oup ( p= 0. 010 ) O n C R T D a nd V A t he r e w e r e no i nt e r a c t i ons s o w e e xa m i ne d t he di f f e r e nc e s be t w e e n gr oups r e ga r dl e s s of t he t i m e pos t t r a ns pl a nt or t he obs e r va t i ona l pe r i od ( T a bl e 3. 2 ) O n C R r a t s t ha t ha d be e n t r e a t e d w i t h 10 5 a nd 10 6 c e l l s w e r e l e s s a c t i ve t ha n t he s t r oke onl y c ont r ol s ( p= 0 046 a nd p= 0. 005 r e s pe c t i ve l y) I n a ddi t i on, a ni m a l s t ha t r e c e i ve d 10 6 c e l l s w e r e s i gni f i c a nt l y l e s s a c t i ve t ha n a ni m a l s i n t he

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40 T a bl e 3. 1 O ve r a l l A N O V A f or D a yt i m e S pont a ne ous A c t i vi t y: p va l ue F r a t i o, de gr e e s of f r e e dom V a r i a b l e G r o u p G r o u p x t i m e p o s t i n f u s i o n G r o u p x o b s e r v a t i o n a l p e r i o d G r o u p x t i m e p o s t i n f u s i o n x o b s e r v a t i o n a l p e r i o d C o u n t e r c l o c k w i s e r e v ( C C R ) 0 0 0 3 4 0 5 6 3 8 0 0 9 0 2 0 3 5 6 3 8 < 0 0 0 0 1 3 2 7 6 6 4 1 8 0 0 0 7 1 5 3 6 6 4 1 8 C e n t e r t i m e ( C T ) 0 1 2 1 8 2 6 3 8 0 1 9 8 1 5 1 6 3 8 < 0 0 0 0 1 2 2 0 6 6 4 1 8 < 0 0 0 0 1 2 7 7 6 6 4 1 8 C e n t e r d i s t a n c e ( C D ) 0 0 7 7 2 0 8 6 3 8 0 7 4 3 0 5 8 6 3 8 0 0 2 5 1 4 0 6 6 4 1 8 0 9 7 4 0 6 7 6 6 4 1 8 C l o c k w i s e r o t a t i o n ( C R ) 0 0 0 2 4 2 1 6 3 8 0 9 6 5 0 2 2 6 3 8 < 0 0 0 0 1 2 1 1 6 6 4 1 8 0 1 0 7 1 2 4 6 6 4 1 8 H o r i z o n t a l a c t i v i t y ( H A ) 0 0 0 2 4 3 2 6 3 8 0 1 2 7 1 7 9 6 3 8 0 1 5 0 1 1 9 6 6 4 1 8 0 0 3 3 1 3 8 6 6 4 1 8 M o v e m e n t n u m b e r ( M V N ) 0 1 7 1 1 6 0 6 3 8 0 0 0 1 4 3 5 6 3 8 < 0 0 0 0 1 2 8 5 6 6 4 1 8 < 0 0 0 0 1 3 0 0 6 6 4 1 8 M o v e m e n t t i m e ( M V T ) < 0 0 0 0 1 4 6 9 4 6 3 8 < 0 0 0 0 1 2 0 5 8 6 3 8 < 0 0 0 0 1 2 9 4 9 6 6 4 1 8 < 0 0 0 0 1 2 2 2 3 6 6 4 1 8 S t e r e o t y p y n u m b e r ( S N ) 0 0 4 2 4 8 6 3 8 0 0 0 0 1 6 1 5 6 3 8 < 0 0 0 0 1 1 8 8 6 6 4 1 8 < 0 0 0 0 1 2 0 7 6 6 4 1 8 S t e r e o t y p y c o u n t s ( S C ) 0 0 1 5 3 0 5 6 3 8 0 0 6 3 2 2 0 6 3 8 0 0 0 1 1 7 1 6 6 4 1 8 < 0 0 0 0 1 2 4 2 6 6 4 1 8 T o t a l d i s t a n c e ( T D ) 0 0 1 4 3 0 8 6 3 8 0 3 2 8 1 1 9 6 3 8 0 0 0 1 1 6 9 6 6 4 1 8 0 8 7 3 0 7 9 6 6 4 1 8 V e r t i c a l a c t i v i t y ( V A ) 0 0 2 7 2 7 0 6 3 8 0 5 9 8 0 7 6 6 3 8 < 0 0 0 0 1 2 1 9 6 6 4 1 8 0 2 3 3 1 1 3 6 6 4 1 8 V e r t i c a l m o v e m e n t s ( V M N ) < 0 0 0 0 1 8 5 8 6 3 8 0 1 2 7 1 7 9 6 3 8 < 0 0 0 0 1 2 6 8 6 6 4 1 8 < 0 0 0 0 1 4 9 4 6 6 4 1 8 S t e r e o t y p y t i m e ( S T ) 0 0 3 0 2 6 3 6 3 8 0 0 0 0 5 5 2 4 6 3 8 0 0 0 1 1 6 9 6 6 4 1 8 < 0 0 0 0 1 3 0 1 6 6 4 1 8

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41 T a bl e 3. 2 P o s t hoc S t a t i s t i c a l C om pa r i s on f o r D a yt i m e S pon t a ne ous A c t i vi t y. V a r i a b l e T i m e o f a n a l y s i s S i g n i f i c a n c e ( p a n d 2 b e t w e e n t h e m e d i a t r e a t e d a n d H U C B C t r e a t e d g r o u p s C o u n t e r c l o c k w i s e r e v ( C C R ) 2 w e e k s 1 0 5 ( p = 0 0 3 1 2 = 4 4 3 ) 1 0 6 ( p = 0 0 0 5 1 2 = 7 8 7 ) 3 5 x 1 0 7 ( p = 0 0 4 1 2 = 4 1 5 ) 4 w e e k s 1 0 6 ( p = 0 0 0 8 1 2 = 6 9 2 ) 1 0 7 ( p = 0 0 3 1 2 = 4 4 3 ) 3 5 x 1 0 7 ( p = 0 0 2 1 2 = 5 1 2 ) C l o c k w i s e r o t a t i o n ( C R ) 2 a n d 4 w e e k s 1 0 5 ( p = 0 0 4 6 1 2 = 3 9 7 ) 1 0 6 ( p = 0 0 0 5 1 2 = 7 8 1 ) H o r i z o n t a l a c t i v i t y ( H A ) 2 w e e k s 1 0 5 ( p = 0 0 5 1 2 = 3 7 2 ) 1 0 6 ( p = 0 0 5 1 2 = 3 7 2 ) 3 5 x 1 0 7 ( p = 0 0 5 1 2 = 3 8 6 ) 4 w e e k s 1 0 7 ( p = 0 0 5 1 2 = 3 7 2 ) 1 0 6 ( p = 0 0 5 1 2 = 3 7 2 ) M o v e m e n t t i m e ( M V T ) 2 w e e k s 1 0 4 ( p = 0 0 0 9 1 2 = 6 6 9 ) 1 0 5 ( p = 0 0 4 3 1 2 = 4 0 6 ) 1 0 6 ( p = 0 0 2 0 1 2 = 5 4 0 ) 4 w e e k s 1 0 7 ( p = 0 0 5 1 2 = 3 7 2 ) S t e r e o t y p y c o u n t s ( S C ) 2 w e e k s 1 0 6 ( p = 0 0 4 1 2 = 4 0 6 ) 4 w e e k s n s T o t a l d i s t a n c e ( T D ) 2 a n d 4 w e e k s 1 0 4 ( p = 0 0 4 1 2 = 3 9 1 ) 1 0 5 ( p = 0 0 4 1 2 = 4 0 3 ) 1 0 6 ( p = 0 0 0 1 1 2 = 9 7 6 ) 1 0 7 ( p = 0 0 4 6 1 2 = 3 9 7 ) 3 5 x 1 0 7 ( p = 0 0 5 1 2 = 3 7 1 ) V e r t i c a l a c t i v i t y ( V A ) 2 a n d 4 w e e k s 1 0 5 ( p = 0 0 0 2 1 2 = 9 2 7 ) V e r t i c a l m o v e m e n t s ( V M N ) 2 w e e k s n s 4 w e e k s b e t w e e n 1 0 4 a n d 3 5 x 1 0 7 ( p = 0 0 1 0 1 2 = 6 5 4 )

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42 10 4 dos e g r oup ( p= 0 056) S i m i l a r l y, on T D w he r e a s i gni f i c a nt a c t i vi t y r e duc t i on w a s pr e s e nt i n a l l dos e g r oups ( 10 4 f o r p= 0. 04 10 5 f or p= 0. 04, 10 6 f or p= 0 0018, 10 7 f or p= 0. 046 a nd 3 5x10 7 f or p= 0. 05) t he r e w a s m or e a c t i vi t y r e duc t i on i n t he 10 6 g r oup c om pa r e d t o t he 10 4 g r oup ( p= 0. 05) O n V A w he r e t he 10 5 gr oup w a s l e s s a c t i ve t ha n M C A O onl y ( p= 0. 002) t he gr oup 10 6 w a s l e s s a c t i ve t ha n t he 10 4 gr oup a nd t he 10 5 gr oup ( p= 0 02 a nd p= 0 05) I n F i gu r e 3. 1 w e pr e s e nt t he t ot a l a c t i vi t y a t 2 a nd 4 w e e ks ( e xpr e s s e d a s pe r c e nt of ba s e l i ne ) f o r t w o r e pr e s e nt a t i ve pa r a m e t e r s ( C C R a nd H A ) F i gur e 3. 2 pr e s e nt s t he a c t i vi t y of a l l r a t s m e a s ur e d w i t h t he s a m e t w o pa r a m e t e r s but i l l us t r a t e d a l ong t he e nt i r e obs e r va t i ona l pe r i od of t he t e s t ( 60 m i nut e s ) 3. 3. 1 2 N i gh t t i m e s p on t an e ou s ac t i vi t y t e s t N i ght t i m e s pont a ne ous a c t i vi t y w a s t e s t e d dur i ng t he ni ght a t i nt e r va l s of 1 hou r f o r a pe r i od of 13 hour s T he ove r a l l A N O V A r e ve a l e d s i gni f i c a nt di f f e r e nc e s be t w e e n t he gr oups f or e l e ve n of t he t hi r t e e n e xa m i ne d va r i a bl e s w i t h s i gni f i c a nc e s r a ngi ng f r om p< 0. 0001 t o p= 0. 05. D i f f e r e nc e s i n a c t i vi t y a m on g t he gr oups w e r e a l s o pr e s e nt a c r os s t i m e ( 2 a nd 4 w e e ks ) a nd a t t he d i f f e r e nt t i m e po i n t s ( 13 m e a s ur e s i n 13 hour t e s t ) f o r a l l t he m e a s ur e s e xc e pt f or C R M V N a nd S N ( p< 0. 0 001 t o p= 0. 0025 ) ( T a bl e 3 3) T he K r us ka l l W a l l i s H T e s t w a s e m pl oye d f or pos t hoc a na l ys i s A s i gni f i c a nt de c r e a s e i n a c t i v i t y of a ni m a l s i n t he t r e a t e d gr oups w a s f ound onl y i n H A ( dos e o f 10 5 f o r p= 0. 035)

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45 T a bl e 3. 3 O ve r a l l A N O V A f or N i ght t i m e S pont a ne ous A c t i vi t y: p va l ue F r a t i o de gr e e s of f r e e dom V a r i a b l e G r o u p G r o u p x t i m e p o s t t r a n s p l a n t G r o u p x o b s e r v a t i o n a l p e r i o d G r o u p x t i m e p o s t t r a n s p l a n t x o b s e r v a t i o n a l p e r i o d C o u n t e r c l o c k w i s e r e v ( C C R ) 0 0 5 8 2 2 5 6 3 8 0 0 0 8 3 4 1 6 3 8 < 0 0 0 0 1 4 4 5 7 2 < 0 0 0 0 1 4 6 0 7 2 C e n t e r t i m e ( C T ) 0 0 0 0 1 6 6 1 6 3 8 0 0 0 1 4 7 2 6 3 8 < 0 0 0 0 1 5 5 7 7 2 < 0 0 0 0 1 3 4 7 7 2 C e n t e r d i s t a n c e ( C D ) 0 0 2 9 2 6 7 6 3 8 0 0 1 4 3 0 9 6 3 8 < 0 0 0 0 1 3 5 3 7 2 < 0 0 0 0 1 3 4 8 7 2 C l o c k w i s e r o t a t i o n ( C R ) 0 0 5 3 2 3 0 6 3 8 0 2 6 4 1 3 3 6 3 8 0 0 2 4 1 3 9 7 2 4 5 6 0 2 5 1 1 1 1 7 2 4 5 6 H o r i z o n t a l a c t i v i t y ( H A ) 0 0 1 6 3 0 0 6 3 8 0 0 0 2 4 3 4 6 3 8 < 0 0 0 0 1 4 2 8 7 2 4 5 6 < 0 0 0 0 1 4 2 4 7 2 4 5 6 M o v e m e n t n u m b e r ( M V N ) 0 3 5 9 1 1 3 6 3 8 0 5 0 0 0 9 0 6 3 8 0 0 0 0 9 1 6 8 7 2 4 5 6 0 4 4 4 1 0 1 7 2 4 5 6 M o v e m e n t t i m e ( M V T ) 0 0 1 3 3 1 5 6 3 8 0 0 0 1 4 4 9 6 3 8 < 0 0 0 0 1 4 0 8 7 2 4 5 6 < 0 0 0 0 1 4 0 9 7 2 4 5 6 S t e r e o t y p y n u m b e r ( S N ) 0 4 8 5 1 4 4 6 3 8 0 7 1 7 1 0 2 6 3 8 < 0 0 0 0 1 4 4 2 7 2 4 5 6 0 3 8 8 1 0 3 7 2 4 5 6 S t e r e o t y p y c o u n t s ( S C ) 0 0 1 7 2 9 8 6 3 8 0 0 0 2 4 2 5 6 3 8 < 0 0 0 0 1 4 3 8 7 2 4 5 6 < 0 0 0 0 1 3 9 0 7 2 4 5 6 T o t a l d i s t a n c e ( T D ) 0 0 0 7 3 5 2 6 3 8 0 0 0 2 4 1 9 6 3 8 < 0 0 0 0 1 3 8 8 7 2 4 5 6 < 0 0 0 0 1 4 0 5 7 2 4 5 6 V e r t i c a l a c t i v i t y ( V A ) 0 2 2 1 1 4 5 6 3 8 0 1 3 4 1 7 5 6 3 8 < 0 0 0 0 1 1 9 8 7 2 4 5 6 0 0 0 2 1 5 9 7 2 4 5 6 V e r t i c a l m o v e m e n t s ( V M N ) 0 2 5 8 1 3 5 6 3 8 0 0 0 6 3 8 3 5 5 6 3 8 < 0 0 0 0 1 3 6 9 7 2 4 5 6 < 0 0 0 0 1 7 3 7 7 2 4 5 6 S t e r e o t y p y t i m e ( S T ) < 0 0 0 0 1 1 1 4 4 6 3 8 0 0 0 0 5 3 1 0 6 3 8 < 0 0 0 0 1 4 1 4 7 2 4 5 6 < 0 0 0 0 1 3 6 7 7 2 4 5 6

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46 3 3. 2 I n f u s i on of h i gh d os e s of c or d b l ood c e l l s i n d u c e s a c om p l e t e r e c ove r y of as ym m e t r y d e f i c i t s m e as u r e d w i t h t h e S t e p t e s t T he nor m a l r a t s t ook t he s a m e num be r of s t e ps w i t h t he r i gh t a nd t he l e f t pa w dur i ng a l l t e s t s A s s how n i n F i gu r e 3 3 t he gr oup o f r a t s t ha t w a s s t r oke d a nd di d not r e c e i ve a H U C B t r a ns pl a nt di s pl a ye d a n i nc r e a s e i n a s ym m e t r y ( e xpr e s s e d a s pe r c e nt of ba s e l i ne di f f e r e nc e be t w e e n r i ght a nd l e f t s t e ps ) of a bout 24 0% a nd 200% a t 2 a nd 4 w e e ks r e s pe c t i ve l y. G r oups t ha t r e c e i ve d t he h i ghe r dos e s of c e l l s s uc h a s 10 7 a nd 3 5x10 7 s how e d s i gni f i c a nt l y l e s s a s ym m e t r y t ha n t he non t r e a t e d gr oup ( p= 0. 01 a nd p= 0. 02 ) a t bot h 2 a nd 4 w e e ks I nt e r e s t i ngl y, a t 4 w e e ks bot h t he s e gr oups w e r e pe r f or m i ng a t pr e s t r oke ba s e l i ne l e ve l s A ddi t i ona l l y a t t hi s l a t e t i m e poi nt t he a ni m a l s of t he 10 5 gr oups a l s o pr e s e nt e d w i t h a s i gni f i c a nt r e duc t i on of a s ym m e t r y c om pa r e d t o t he a ni m a l s i n t he non t r e a t e d gr oup ( p< 0. 05 ) 3. 3. 3 C or d b l ood i n f u s i on r e d u c e s l on g t e r m d e f i c i t s i n t h e E l e vat e d B od y S w i n g T e s t P r i or t o M C A O a l l a ni m a l s ha d a bi a s of l e s s t ha n 2. A t 2 w e e ks pos t t r a ns pl a nt t he r e w e r e no s i gni f i c a nt di f f e r e nc e s be t w e e n dos e gr ou ps a nd s t r oke d a ni m a l s t ha t ha d not r e c e i ve d a c e l l i nf us i on. H ow e ve r a t 4 w e e ks t he bi a s of t he a ni m a l s t r e a t e d w i t h t he dos e of 10 6 a nd 3 5x10 7 w a s s i gni f i c a nt l y l e s s t ha n M C A O c ont r ol r a t s ( p= 0. 005 a nd p= 0. 04 r e s pe c t i ve l y) ( F i gur e 3. 4)

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49 3. 3. 4 C or d b l ood i n f u s i on i n d u c e s h i s t ol ogi c al b e n e f i t i n b r ai n s of r at s s u b j e c t t o M C A O I n t he i n f a r c t e d br a i n e xa m i ne d a t one m ont h a f t e r t he s t r oke f our d i s t i nc t phe nom e na w e r e a ppa r e nt F i r s t a t one m on t h a f t e r t he M C A O s ur ge r y t he i nj ur e d he m i s phe r e s ha d s hr unk. S e c ondl y, a s s how n i n F i gur e 3 5, t he r e w a s a l s o a l os s of t i s s ue ( obs e r va bl e a c r os s a l l br e gm a l e ve l s ) due t o t he l i que f a c t i on pr oc e s s t ha t f ol l ow s t he e de m a a nd ne c r ot i c e ve nt s t ypi c a l l y obs e r ve d w i t hi n t he f i r s t w e e k pos t s t r oke F our w e e ks a f t e r t he i s c he m i c i nj ur y m os t o f t he t i s s ue i nvol ve d i n t hi s pr oc e s s ha s be e n l os t a nd s ubs t i t ut e d by e m pt y s pa c e s ( ge ne r a l l y na m e d l a c una e ) T hi s f e a t ur e w a s m os t l y e vi de nt i n t he m os t c a uda l s e c t i ons e xa m i ne d. T hi r dl y w i t hi n t he s t r i a t um a nd c o r t e x t he r e w a s a n e xt e ns i ve l os s of c e l l s w i t h a c om pl e t e l os s of c yt oa r c hi t e c t ur e F ou r i n m os t of t he M C A O br a i ns i t w a s pos s i bl e t o i de nt i f y t he ne c r ot i c c or e a w e l l de f i ne d a r e a t ha t s t a i ne d m or e i ns e ns i t i ve l y w i t h t he N i s s l T hi oni ne dye ( r e pr e s e nt i ng t he a r e a of i r r e ve r s i bl e c e l l da m a ge ) I n t he b r a i ns of M C A O r a t s t ha t ha d r e c e i ve d a t l e a s t 10 6 c e l l s t he pr oc e s s of s hr i nka g e w a s e vi de nt t oge t he r w i t h t he va r i a bl e p r e s e nc e of t he ne c r ot i c c or e a nd s c a t t e r e d l a c una r s pa c e s O n t he c ont r a r y, t he l os s of c yt oa r c hi t e c t ur e i n s t r i a t um a nd c or t e x w a s l e s s dr a m a t i c a l m os t ne gl i gi bl e i n s om e br a i ns F i gur e 3. 6 s how s s om e of t he s e h i s t ol ogi c a l c ha r a c t e r i s t i c s i n de t a i l 3. 3. 5 C or d b l ood i n f u s i on i n d u c e s r e d u c t i on of i n f ar c t vol u m e i n M C A O b r ai n s T he i nf a r c t vol um e w a s qua nt i f i e d a s e xpl a i ne d i n M a t e r i a l a nd M e t hods s e c t i on a t s i x pr e de f i ne d c or ona l s e c t i ons F i gur e 3 7 p r e s e nt s a pi c t ur e of t he s i x c or ona l s e c t i ons

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53 ( f r om br e gm a 1. 7 t o b r e gm a 3. 3 ) s t a i ne d w i t h N i s s l T hi oni ne of t w o r e pr e s e nt a t i ve br a i ns t r e a t e d w i t h no c e l l s ( m e di a ) a nd w i t h 10 7 H U C B C I n a ddi t i on t o a r e duc t i on i n hi s t ol ogi c a l a nom a l i e s obs e r ve d w i t h H U C B C i nf us i on a f t e r M C A O t he r e w a s a l s o a r e duc t i on i n t he a m ount o f da m a ge o r i nf a r c t s i z e a s a c ons e que nc e of our t r e a t m e nt T he a dm i ni s t r a t i on of H U C B C a t t he dos e of 10 7 but n ot a t l ow e r dos e c oul d s i gni f i c a nt l y r e duc e t he vol um e s of i s c he m i c br a i n da m a ge ( F i g u r e 3. 8 ) I n f a r c t vol u m e i n t he s t r oke d gr oups w a s 33. 15 + 4. 29% of t he vol um e of c ont r a l a t e r a l he m i s phe r e w hi l e a ni m a l s of t he 10 7 dos e s s how e d i nf a r c t s i nvol vi ng onl y 11. 46 + 4. 13 % of t he he m i s phe r e A ni m a l s t ha t ha d r e c e i ve d a h i ghe r dos e of c e l l s w a s 20. 61 + 4. 16% of t he c ont r a l a t e r a l he m i s phe r e A s s how n i n F i gu r e 3 8, gr oups i nj e c t e d w i t h ot he r dos e s a l s o ha d a de c r e a s e d i nf a r c t vol um e de pe nde nt on t he a m ount of c e l l s i n f us e d. H ow e ve r di f f e r e nc e s be t w e e n t he i nf a r c t vol um e s of t he s e gr oups a nd t he c ont r ol s w e r e not s t a t i s t i c a l l y s i gni f i c a nt 3. 3. 6 C or d b l ood c e l l s ar e d e t e c t e d i n s p l e e n an d r i gh t b r ai n h e m i s p h e r e w i t h n e s t e d P C R f or h u m an p r i m e r s R i ght a nd l e f t br a i n he m i s phe r e s he a r t l ung, l i ve r s pl e e n, t hym us ki dne y a nd bone m a r r ow w e r e s a m pl e d. I n a ni m a l s o f t he l ow e s t dos e gr oups s uc h a s t he 10 4 a nd 10 5 c e l l gr oups no hum a n D N A w a s f ound i n a ny or ga n I n a ni m a l s i nj e c t e d w i t h 10 6 10 7 a nd 3 5x10 7 c e l l s hum a n D N A w a s f ound i n s pl e e n, r i gh t br a i n he m i s phe r e ( i nj ur e d ) a nd he a r t ( F i gur e 3 9 ) H um a n D N A w a s ne ve r de t e c t e d i n o t he r or ga ns s uc h a s t hym us l i ve r bone m a r r ow ki dne y a nd l ung

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56 3. 3. 7 I m m u n oc h e m i c al l a b e l i n g o f h u m an c e l l s w i t h a n an t i b od y agai n s t H u m an N u c l e i c on f i r m s t h e P C R r e s u l t s T he r e w a s e xt e ns i ve a ut of l uor e s c e nc e pr e s e n t i n s pl e e n, he a r t l i ve r a nd ki dne y. T he que nc hi ng s ol ut i on r e duc e d t he a ut o f l uor e s c e nc e i n a l l or ga ns e xa m i ne d e xc e pt i n l ung he a r t a nd t hym us w he r e t he que nc hi ng s ol ut i on de c r e a s e d t he i nt e r f e r i ng s i gna l onl y m i ni m a l l y. H um a n nuc l e i i m m unor e a c t i ve c e l l s w e r e de t e c t e d i n s pl e e n a nd r i ght b r a i n he m i s phe r e of a ni m a l s i nj e c t e d w i t h 10 6 10 7 a nd 3 5x10 7 c e l l dos e s ( F i gur e 3 10 ) H e r e t he i m m unopos i t i ve c e l l s s e e m e d t o be l oc a l i z e d w i t hi n bl ood ve s s e l s or a t t he pe r i va s c ul a r s pa c e F e w c e l l s w e r e de t e c t e d i n t he pa r e nc hym a of e i t he r s pl e e n or b r a i n. I n l i ve r a nd ki dne y no H U C B C w e r e f ound 3. 4 D i s c u s s i on T he r e duc t i on o f s t r oke i nduc e d hype r a c t i vi t y i s c ons i de r e d a s pe c i f i c a nd s e ns i t i ve m e a s ur e f or t he e va l ua t i ons of e xpe r i m e nt a l t r e a t m e nt s i n t he M C A O r a t s H e r e w e f ound t ha t H U C B C dos e s e qua l t o or gr e a t e r t ha n 10 6 w e r e a bl e t o i nduc e s i gni f i c a nt a c t i vi t y r e duc t i on w he n t hi s w a s m e a s ur e d a t 2 w e e ks D os e s hi ghe r t he n 10 6 s uc h a s 10 7 a nd 3 510 7 c oul d i nduc e a f ur t he r i m pr ove m e nt t ha t c oul d be a l s o o bs e r ve d a t 4 w e e ks A t l ow e r dos e s ( 10 4 a nd 10 5 ) a l t hough t he r e w a s a s i m i l a r t e nde nc y t hi s r e a c he d s i gni f i c a nc e onl y f or c e r t a i n m e a s ur e s W he n t he S t e p t e s t w a s pe r f o r m e d a f t e r t he e m pl o ym e nt of t h i s a r r a y o f H U C B C dos e s onl y t he hi ghe s t dos e s ha d a s i g ni f i c a nt e f f e c t D os e s of 10 7 a nd 3 5 10 7 w e r e a bl e t o r e ve r t t he m o t or a bi l i t y o f t he r a t s t o l e ve l s c om pa r a bl e t o nor m a l r a t s

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58 A l t hough t he s pont a ne ous a c t i vi t y t e s t a nd t he S t e p t e s t de m ons t r a t e d a n e f f e c t of H U C B C i nf us i on a t 2 w e e ks a f t e r t r a ns pl a nt a t i on, t he e l e va t e d body s w i ng t e s t di d not s how a ny s i gni f i c a nt i m pr ove m e nt a t t hi s e a r l y t i m e poi nt H ow e ve r a t 4 w e e ks a f t e r t r a ns pl a nt r a t s i nj e c t e d w i t h 10 6 a nd 3 5x10 7 c e l l s di s pl a ye d a s i gni f i c a nt l y de c r e a s e d bi a s c om pa r e d t o t he m e di a r e c e i vi ng r a t s C ur i ous l y, t he 10 7 dos e gr oup di d not de m ons t r a t e a ny s t a t i s t i c a l i m pr ove m e nt a s s how n by t he S pont a ne ous A c t i vi t y a nd S t e p T e s t f or t hi s gr oup. H ow e ve r r e vi e w of i ndi vi dua l pe r f o r m a nc e on t he E B S T s ugge s t e d t he pr e s e nc e of t w o out l i e r s ( > 2 5 S D f r o m m e a n) ; w he n t he s e w e r e e xc l ude d f r om t he a na l ys i s t he r e w a s s t a t i s t i c a l l y s i gni f i c a nt i m pr ove m e nt on t hi s t e s t a s w e l l ( p< 0. 031 ) W e c a n c onc l ude f r om t he s e obs e r va t i ons t ha t t he m i t i ga t i on of M C A O i nduc e d de f i c i t s de pe nds on t he a m ount o f H U C B C i nj e c t e d S pe c i f i c a l l y, w he n 10 6 o r hi ghe r a m ount s o f H U C B C a r e e m pl oye d a f t e r pe r m a ne nt M C A O t he r e i s s i gni f i c a nt r e c ove r y i n be ha vi or a l pe r f or m a nc e ; how e ve r a t t he hi ghe s t H U C B C dos e s e m pl oye d ( 10 7 a nd 3 5 x10 7 ) r e c ove r y of be ha vi o r a l f unc t i on s how s a pl a t e a u e f f e c t s how i ng no s i gni f i c a nt i m pr ove m e nt ove r t ha t of t he 10 7 dos e I t i s a l s o i m po r t a nt t o m e nt i on t ha t t he s e t hr e e d i f f e r e nt be ha vi or a l t e s t s ha ve not a l w a ys s how n i m pr ove m e nt s i n f unc t i on a t t he s a m e c e l l d os e l e ve l s F or i ns t a nc e t he a s ym m e t r y a na l y z e d w i t h t he S t e p t e s t s how s a n a m e l i or a t i on i n r a t s t r e a t e d w i t h 10 5 c e l l s w he n t hi s t he t e s t i s pe r f or m e d a t 4 w e e ks ; a t t he s a m e t i m e poi nt i n t he e l e va t e d body s w i ng t e s t t he gr oup of 10 5 doe s not pe r f or m be t t e r t he non t r e a t e d a ni m a l s S i m i l a r l y, t he 10 7 g r oup s how s a n opt i m a l pe r f o r m a nc e i n t he s t e p t e s t a t 2 a nd 4 w e e ks w hi l e a t bot h t he s e t i m e poi nt s t hi s g r oup f a i l s i n d i s pl a yi ng a t he r a pe ut i c a l be ne f i t w i t h

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59 t he e l e va t e d body s w i ng t e s t T hi s obs e r va t i on poi nt s out t he i m po r t a nc e of us i ng a n e nt i r e ba t t e r y o f t e s t s t o e va l ua t e t he e f f i c a c y o f a t r e a t m e nt e s pe c i a l l y w he n ut i l i z i ng a ni m a l m ode l s a s i nt r i ns i c a l l y va r i a bl e s uc h a s t he m i ddl e c e r e br a l a r t e r y oc c l us i on i n t he r a t M C A O r a t s t ha t di d not r e c e i ve H U C B C t r e a t m e nt s how e d e xt e ns i ve ne ur ona l l os s i n t he c or t e x a nd s t r i a t um of t he i s c he m i c he m i s phe r e w hi l e hi s t ol ogi c a l a na l ys i s of t he i s c he m i c br a i n he m i s phe r e f r om H U C B C t r e a t e d a ni m a l s r e ve a l e d t ha t hi s t ol ogi c a l be ne f i t w he n dos e s of 10 6 H U C B C or hi ghe r w e r e i nf us e d. H ow e ve r dos e s of 10 6 c e l l s w e r e not a bl e t o i nduc e a s t a t i s t i c a l l y s i gni f i c a nt r e duc t i on of i n f a r c t vo l um e A s i gni f i c a nt r e duc t i on of i nf a r c t s i z e w a s onl y obs e r va bl e w i t h dos e s of 10 7 a nd 3 5x10 7 I nt e r e s t i ngl y, t he s e r e s ul t s s how t ha t w hi l e be ha vi o r a l be ne f i t c a n be obs e r ve d a f t e r t r e a t m e nt w i t h 10 6 c e l l s onl y dos e s of 10 7 o r hi ghe r c a n i nduc e a s i gni f i c a nt r e duc t i on of a m ount of i s c he m i c da m a ge W hi l e t he r e a r e s t udi e s t ha t ha ve s how n hi s t ol ogi c a l a m e l i or a t i on i n t he M C A O br a i n pa r a l l e l i ng be ha v i or a l r e c ove r y ( O ba na P i t t s e t a l 1988) a num be r o f s t udi e s e m pl oyi ng t he r a t M C A O m ode l of s t r oke ha ve pr e vi ous l y de s c r i be d a l a c k i n c or r e l a t i on be t w e e n i m p r ove d b e ha vi or t e s t pe r f o r m a nc e a nd br a i n hi s t ol ogy ( G r ot t a P e t t i g r e w e t a l 1988; W a hl A l l i x e t a l 1992) I n pa r t i c ul a r G r ot t a e t a l ha ve s how n be ne f i c i a l e f f e c t of ne ur opr o t e c t i ve dr ugs i n i m pr ovi ng m e m or y i n t he a bs e nc e of hi s t ol ogi c a l be ne f i t ( G r ot t a P e t t i gr e w e t a l 1988) a nd W a hl e t a l r e por t e d no c or r e l a t i on be t w e e n pe r f o r m a nc e i n ne u r obe ha vi or a l t e s t s ( ne ur ol ogi c a l e xa m i na t i on, pa s s i ve a voi da nc e t a s k, a nd s pont a ne ous a c t i vi t y) a nd s i z e of i nf a r c t s i nduc e d by M C A O ( W a hl A l l i x e t a l 1992) T hi s l a c k i n c or r e l a t i on be t w e e n t he hi s t ol ogi c a l gr a de a nd t he

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60 be ha vi or a l out c om e s r a i s e s a num be r o f c ons i de r a t i ons e s pe c i a l l y s i nc e i t s e e m s a t odds w i t h t he e xpe c t a t i on of gr e a t e r c e r e br a l da m a ge m a ni f e s t i ng a s w or s e ni ng be ha vi or a l pe r f or m a nc e F i r s t l y, t he r e m a y be c om pe ns a t or y c ha nge s i n f unc t i on i n ot he r b r a i n r e gi ons A t c e l l ul a r l e ve l s ur vi vi ng c e l l s m a y c om pe ns a t e f or t he f unc t i on of l os t ne ur ons pr ovi di ng f unc t i on t o a n ot he r w i s e da m a ge d br a i n a r e a s S e c ond gi ve n t he he t e r oge ne ous a r c hi t e c t ur e of t he br a i n c o r r e l a t i on be t w e e n pa t hol ogy obs e r ve d a nd be ha vi or a l out c om e s houl d de pe nd m o r e on t he un i que ne s s of t he f unc t i on t o pa r t i c ul a r b r a i n a r e a s t ha n s t r i c t l y t o i n f a r c t vol u m e T hi r d, d i f f e r e nt m e a s ur e s of be ha vi or a l out c om e m a y ha ve di f f e r e nt s e ns i t i vi t y i n t he de t e c t i on of a c ha nge of i nf a r c t vol um e I nve s t i ga t i ons on t he l oc a l i z a t i on o f i nt r a ve nous l y de l i ve r e d c e l l s l e d t o t he c on c l us i on t ha t H U C B C r e a c he d t he i nj ur e d b r a i n a nd ve r y l i k e l y m i gr a t e d t o t he s pl e e n. T he ne s t e d P C R a na l ys i s pe r m i t t e d t he de t e c t i on of t he H U C B C D N A w i t h hi gh s e ns i t i vi t y, a l l ow i ng t he de t e c t i on of l e s s t ha n 10 H U C B C c e l l s w i t hi n t he D N A c ont a i ne d i n a l m o s t 40 000 c e l l s of t he s a m pl e d or ga n C onf i r m a t i on of t hi s f i ndi ng w i t h i m m unohi s t oc he m i s t r y us i ng a n a nt i body a ga i ns t hum a n nuc l e i a l l ow e d a m or phol ogi c a l a nd s pa t i a l a na l ys i s I t w a s ve r y s ur pr i s i ng t o f i nd t he i nj e c t e d c e l l s i n s pe c i f i c or ga ns a nd not s c a t t e r e d t hr oughout t he body. S pe c i f i c a l l y, br a i n a nd s pl e e n s e e m e d t o be t he t a r ge t o r ga ns O f e ve n gr e a t e r i nt e r e s t i s t he f a c t t ha t t he H U C B C c e l l s w e r e onl y f ound i n t he i nj u r e d br a i n he m i s phe r e A not he r i m po r t a nt f i ndi ng i s t he pr e s e nc e of c e l l s i n t he s pl e e n, w hi c h s ugge s t s a t r opi s m or m i g r a t i on of t he s e c e l l s t o t hi s or ga n. S i m i l a r f i nd i ng ha s be e n r e por t e d by o t he r i nve s t i ga t or s w ho ha ve e m pl oye d H U C B C i nt r a ve nous l y i n m ode l s of ne ur ode ge ne r a t i ve di s e a s e s F or e xa m pl e G a r buz ova D a vi s e t a l i n j e c t e d 10, 000

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61 H U C B C i n m ous e m ode l of a m yot r ophi c l a t e r a l s c l e r os i s a nd f ound 10 w e e ks pos t i nj e c t i on pl e nt y of t r a ns pl a nt e d c e l l s hom i ng t he s p l e e n ( G a r buz ova D a vi s W i l l i ng e t a l 2003) T he H U C B C pr e pa r a t i on c ons i s t s m a i nl y o f i m m a t ur e l ym phoc yt e s a nd m onoc yt e s w hi c h m a y pos s e s s i nna t e hom i ng a bi l i t i e s f or p r i m a r y or s e c onda r y i m m une or ga ns H ow e ve r w he n w e l ooke d a t t he pr i m a r y i m m une or ga ns s uc h a s t he t hym us a nd t he bone m a r r ow no H U C B C w e r e f ound T he s e f i ndi ngs s e e m t o s t r ongl y s uppor t a phe nom e non of t r opi s m of H U C B C e xc l us i ve l y f or t he s pl e e n. F i na l l y, hum a n D N A w a s a l s o l oc a l i z e d i n t he he a r t of t w o r a t s i n j e c t e d w i t h dos e s of 10 7 a nd 3 5x10 7 T he s i gni f i c a nc e of t hi s f i ndi n g f o r p hys i opa t hol ogi c a l a nd t he r a pe ut i c pr ope r t i e s of H U C B C r e m a i ns i n que s t i on. E i t he r s om e a ni m a l s ha d a he a r t i nj u r y a nd c he m oa t t r a c t a nt s dr e w H U C B C t o a n i nj ur y s i t e o r H U C B C w e r e t r a ppe d i n t he f i ne ve s s e l s of t he c or ona r y c i r c ul a t i on of t he c a r di o m u s c ul a r s t r uc t ur e I n c onc l us i on, w e ha ve s how n t ha t H U C B C c a n i n duc e f unc t i ona l a nd hi s t ol ogi c a l be ne f i t s i n M C A O r a t s i n a dos e de pe nde nt m a nne r M or e ove r a t one m ont h a f t e r t he i nt r a ve nous t r a ns pl a nt H U C B C w e r e m a i nl y l oc a l i z e d i n t he i nj u r e d br a i n a nd i n t he s pl e e n. T he s e f i ndi ngs s e t t he f ounda t i on f or our f ol l ow i ng i nve s t i ga t i ons di r e c t e d t o unde r s t a ndi ng how H U C B C e xe r t t he s e pr ot e c t i ve a c t i ons

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62 C H A P T E R F O U R E X A M I N A T I O N O F T H E E F F E C T O F H U C B C I N F U S I O N O N S T R O K E I N D U C E D A L T E R A T I O N S O F S P L E N O C Y T E T Y P E A N D F U N C T I O N 4. 1 I n t r od u c t i on C N S i s c he m i c i nj ur y i nduc e s a l oc a l i nf l a m m a t or y r e s pons e c ha r a c t e r i z e d by a c t i va t i on of r e s i de nt a nd r e c r ui t e d c e l l s ( s uc h a s m i c r ogl i a a nd i nf i l t r a t i ng l e ukoc yt e s ) t ha t pr oduc e va r i ous pr o i nf l a m m a t or y m o l e c ul e s i m pl i c a t e d i n t he m e di a t i on of ne ur ona l da m a ge ( D i r na gl I a de c ol a e t a l 1999 ) S t udi e s i n bot h t he c l i ni c a l s e t t i ng a nd i n a ni m a l m ode l s of s t r oke ha ve de m ons t r a t e d t ha t t he r e a r e a l t e r a t i o ns i n t he pe r i phe r a l i m m une s t a t us w hi c h a ppe a r t o f ol l ow t he C N S c a s c a de of pr o i n f l a m m a t or y e ve nt s ( I n t i s o, Z a r r e l l i e t a l 2004; S m i t h E m s l e y e t a l 2004) T he C N S a n d t he i m m une s ys t e m r e s pond t o t hi s pr o i nf l a m m a t o r y s t a t e w i t h a c om pl e x m ol e c ul a r a nd c e l l ul a r i nt e r pl a y, m e di a t e d i n pa r t by t he hypot ha l a m i c pi t ui t a r y a dr e na l a xi s a nd t he s ym pa t he t i c ne r vos s ys t e m ( W oi c i e c how s ky, S c honi ng e t a l 1999 ) T he r e l e a s e of gl uc oc or t i c oi ds a nd c a t e c hol a m i ne s t ha t f o l l ow s t he a c t i va t i on o f t he s e r e s pe c t i ve s ys t e m s m e di a t e t he pr oduc t i on of a nt i i n f l a m m a t or y i nt e r l e uki ns ( s uc h a s I L 10 ) f r om r e s i de nt m i c r ogl i a a nd i nf i l t r a t i ng m onoc yt e s w hi c h p r ovi de i nhi b i t or y f e e dba c k de c r e a s i ng t he s t r oke i nduc e d pr o i nf l a m m a t o r y C N S r e s pons e T hi s i nhi bi t or y s i gna l i s t hought t o a l s o i nduc e a n

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63 i m m unos uppr e s s i ve s t a t e w hi c h c l i ni c a l s t udi e s ha ve s ugge s t e d i s r e s pons i bl e f or t he i nf e c t i ous c om pl i c a t i ons s e e n i n t he pos t a c ut e pha s e of s t r oke ( P r a s s M e i s e l e t a l 2003) T he c om pl e x c ha nge s i n t he i m m une s ys t e m r e l a t e d t o br a i n i nj ur y ha ve a l s o be e n c ha r a c t e r i z e d a s a l t e r a t i ons i n t he c e l l ul a r c om pos i t i on a nd f unc t i on of pe r i phe r a l l ym phoi d or ga ns s uc h a s s pl e e n, t hym us a nd l ym p h node s ( P r a s s M e i s e l e t a l 2003) F or e xa m pl e M C A O i n t he r a t pr ovoke s a de c r e a s e i n t he a bs ol ut e num be r of s pl e noc yt e s w i t h a c onc om i t a nt i nc r e a s e i n t he r a t i o of C D 4 + / C D 8 + T l ym phoc yt e s ( G e ndr on, T e i t e l ba um e t a l 2002) I n t he s a m e s t udy, m i t oge n pr ol i f e r a t i on a s s a ys f or B a nd T c e l l s s how e d a hi ghe r pr ol i f e r a t i ve c a pa c i t y f or t he s e c e l l s w he n ha r ve s t e d f r om t he s pl e e n of s t r oke d r a t s M or e ove r t he c e l l ul a r a nd f unc t i ona l c ha nge s oc c ur r i ng i n t he s e pe r i phe r a l l ym phoi d or ga ns i s m i r r or e d i n t h e i s c he m i c C N S by a pe a k i n i nf i l t r a t i ng T l ym phoc yt e s a s w e l l a s a c t i va t e d m i c r ogl i a T he i m por t a nc e o f t he s e i nva di ng l e ukoc yt e s i n s t r oke i nduc e d C N S i nj ur y ha s be e n s how n by t he r e duc t i on of i s c he m i c vol um e i n m i c e de f i c i e nt i n t he l e ukoc yt e a dhe s i on m ol e c ul e C D 11a / C D 18 ( S or i a no, C oxon e t a l 1999 ) I n s e ve r a l r ode nt m o de l s of br a i n i nj ur y C N S s pe c i f i c a ut or e a c t i ve T c e l l s ha ve be e n s how n t o e i t he r r e s c ue ne ur ons f r om i nf l a m m a t i on r e l a t e d i nj ur y ( C he n, R ue t z l e r e t a l 2003 ) o r e nha nc e ne ur ot oxi c i t y de pe ndi ng on t he s ubt ype of T c e l l ( W ol f F i s he r e t a l 2002) I nt e r e s t i ngl y, s pl e noc yt e s de r i ve d f r om r a t s t ol e r i z e d w i t h C N S m ye l i n a nt i ge ns s uc h a s m ye l i n ba s i c pr ot e i n w e r e a bl e t o m e di a t e pr o t e c t i on a ga i ns t i s c he m i c br a i n i nj ur y w he n a dopt i ve l y t r a n s pl a nt e d i nt o na ve r a t s ( B e c ke r M c C a r r on e t a l 1997 ; B e c ke r K i nd r i c k e t a l 2003 ) N um e r ous r e por t s ha ve now de m ons t r a t e d t ha t s ys t e m i c i nf us i on of um bi l i c a l c or d bl ood c a n a t t e nua t e t he ne ur obe ha vi or a l de f i c i t s s e e n a f t e r M C A O s ur ge r y i n r ode nt s ( C he n,

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64 S a nbe r g e t a l 2001; W i l l i ng, L i xi a n e t a l 2003; W i l l i ng, V e nd r a m e e t a l 2003) H ow e ve r t he m e c ha ni s m ( s ) by w hi c h um bi l i c a l c o r d bl ood m e di a t e s t hi s be ne f i c i a l e f f e c t r e m a i ns unc l e a r O ne put a t i ve m e c ha ni s m i s t ha t t he C D 34+ s t e m c e l l popul a t i on ( c ons t i t ut i n g a bout 1 % of m ononuc l e a r f r a c t i on o f um bi l i c a l c or d bl ood ) t r a ns di f f e r e nt i a t e i nt o ne ur ons onc e w i t hi n t he i nj ur e d br a i n pa r e nc hym a ( S a nc he z R a m os S ong e t a l 2001; B i c kne s e G oodw i n e t a l 2002) H ow e ve r t he de t e c t i on of t he s e c e l l s i n br a i n i nj u r y m ode l s w he n de l i ve r e d e i t he r l oc a l l y o r i nt r a ve nous l y ha s not pr ovi de d c onvi nc i ng e vi de nc e t ha t t he c or d bl ood c e l l s s ur vi ve l ong t e r m l e t a l one r e pl a c e l os t c e l l s ( W i l l i ng, L i xi a n e t a l 2003 ) I t h a s be e n r e c e nt l y s how n t ha t i nt r a ve nous l y de l i ve r e d H U C B C not onl y m i gr a t e t o t he i nj u r e d C N S i n t he va r i ous m ode l s of ne ur ode ge ne r a t i on bu t t ha t t he y c a n a l s o be f ound i n pe r i phe r a l t i s s ue s i nc l udi ng t he bl ood a nd s pl e e n ( G a r buz ova D a vi s W i l l i ng e t a l 2003) I n t he p r e vi ous c ha pt e r w e s how e d t ha t t he i nf us e d c or d bl ood c e l l s w e r e a l s o de t e c t e d i n t he s pl e e n. M or e ove r t he m ononuc l e a r f r a c t i on of c or d bl ood i s m a i nl y c om pos e d of i m m a t ur e l ym phoc yt e s ( s e e C ha pt e r 1 ) w hi c h, a s d i s c us s e d a l s o i n t he pr e vi ous c ha pt e r m a y pos s e s s t r opi c pr ope r t i e s f or l ym phoi d o r ga ns G i ve n t he s e obs e r va t i ons w e ha ve hypot he s i z e d t h a t t he be ne f i c i a l i m p r ove m e nt s s e e n i n our a nd p r e vi ous w or k f ol l ow i ng H U C B C i nf us i on m a y be a r e s ul t of c or d bl ood m odul a t i on of pe r i phe r a l i m m une a l t e r a t i ons i nduc e d by t he s t r oke T o t e s t t hi s hypot he s i s w e e xa m i ne d phe not ypi c a nd f unc t i on a l c ha r a c t e r i s t i c s of s pl e e n c e l l s a f t e r s t r oke a nd t he s ubs e que nt i nf us i on of c or d bl ood c e l l s

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65 4. 2 M at e r i al s an d M e t h od s A dul t m a l e S p r a gue D a w l e y r a t s w e r e r a ndom l y a s s i gne d t o t he f ol l ow i ng e xpe r i m e nt a l gr oups : s ha m s ur ge r y ( n = 6 ) M C A O onl y ( n = 6) or M C A O pl us H U C B C ( n = 6) M C A O s ur ge r y w a s pe r f or m e d a s pr e vi ous l y de s c r i be d i n S e c t i on 2. 1 R a t s r e c e i ve d a t r a ns pl a nt of 10 000, 000 c or d b l ood c e l l s i nt o t he p e ni l e ve i n 24 hou r s a f t e r t he s t r oke s ur ge r y ( s e e G e ne r a l M a t e r i a l s a nd M e t hods s e c t i on 2. 2 3) O ne da y a f t e r H U C B C i nf us i on, a l l r a t s w e r e a ne s t he t i z e d w i t h i s of l ur a ne a nd ki l l e d by de c a pi t a t i on S p l e e ns w e r e ha r ve s t e d a nd di s s e c t e d f r e e f r o m t he s ur r oun di ng a di pos e t i s s ue T he ne t w e i ght of e a c h s pl e e n w a s m e a s ur e d w i t h t he us e of t he C ha us S c out I I s c a l e A f t e r w a r ds e a c h s pl e e n w a s s e pa r a t e d i nt o t w o pa r t s one of w hi c h w a s i m m e di a t e l y i m m e r s e d i n T r i z ol r e a ge nt f or R N A e xt r a c t i on, a nd t he o t he r one w a s us e d f or pr e pa r i ng s i ngl e c e l l s us pe n s i ons f or c ul t ur e e xpe r i m e nt s a nd f l ow c yt o m e t r y a na l ys i s F or e xa m i na t i on o f c or r e l a t i on o f s pl e e n w e i ght w i t h i nf a r c t vol um e a n i nde pe nde nt s e t of a ni m a l s w a s us e d. T w e l ve a dul t m a l e S p r a gue D a w l e y r a t s w e r e r a ndom l y a s s i gne d t o t he f ol l ow i ng e xpe r i m e nt a l gr oups : s ha m s ur ge r y ( n = 4) M C A O onl y ( n = 4 ) o r M C A O pl us H U C B C ( n = 4) M C A O s ur ge r y a nd H U C B C t r a ns pl a nt ( 10, 000 000 c e l l s ) w e r e pe r f or m e d a s f o r t he ot he r e xpe r i m e nt s i n t h i s c ha pt e r O ne da y a f t e r H U C B C i nf us i on, t he r a t s w e r e ki l l e d by de c a pi t a t i on a nd t he b r a i n w a s qui c kl y r e m ove d, s e c t i one d a nd s t a i ne d w i t h 2 3, 5 t r i phe nyl t e t r a z ol i um c hl or i de ( T T C )

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66 4. 2. 1 2, 3, 5 t r i p h e n yl t e t r az ol i u m c h l or i d e ( T T C ) s t ai n i n g an d i n f ar c t vol u m e q u an t i f i c at i on B r a i n 6x2 m m c o r ona l s l i c e s w e r e pr e pa r e d w i t h a br a i n m a t r i x ( A s t e r I ndus t r i e s I nc ) T he br a i n s e c t i ons w e r e s t a i ne d w i t h 2% T T C a t 3 7C f or 15 m i nut e s T he T T C s t a i ne d vi a bl e br a i n t i s s ue da r k r e d, w he r e a s i nf a r c t e d t i s s ue w a s uns t a i ne d. A f t e r s t a i ni ng t he s e c t i ons w e r e f i xe d i n phos pha t e buf f e r e d f o r m a l i n ( 10% ) a t 4C f ol l ow e d by s c a nni ng on a 1200 dpi U M A X s c a nne r T he bor de r be t w e e n i nf a r c t e d a nd non i nf a r c t e d t i s s ue w a s out l i ne d w i t h t he i m a ge a na l ys i s s ys t e m I m a ge J ( I m a ge J 1. 31v; ht t p: / / r s b. i nf o. ni h gov/ i j ; W a yne R a s ba nd, N a t i ona l I ns t i t ut e s of H e a l t h, B e t he s da M D ) B r i e f l y, on e a c h s l i c e t he uns t a i ne d a r e a ( i s c he m i c br a i n) w a s de l i ne a t e d a nd t he i nf a r c t vol um e w a s c a l c ul a t e d a c c or di ng t o t he t hi c kne s s of 2 m m pe r s l i c e A s s e s s m e nt of i nf a r c t i on s i z e w a s m a de i n dupl i c a t e a nd m e a n va l ue s w e r e c a l c ul a t e d. T he i nf a r c t a r e a s on e a c h s l i c e w e r e s um m e d a nd m ul t i pl i e d by s l i c e t hi c kne s s t o gi ve t he i nf a r c t vo l um e s T o a voi d ove r e s t i m a t i on of t he i nf a r c t vol um e t he i nf a r c t e d vol um e w a s r e l a t e d t o t he t ot a l vol um e o f t he b r a i n t o c a l c ul a t e % i nf a r c t e d v ol um e ( t ha t i s [ vo l um e of i nf a r c t e d he m i s phe r e / t ot a l vol um e of b r a i n] *100) 4. 2. 2 D e t e c t i on o f R N A e xp r e s s i on of c yt o k i n e ge n e s 4. 2. 2 1 R N A e xt r ac t i on R N A w a s i s ol a t e d e m pl oyi ng t he T r i z ol r e a ge nt ( I nvi t r oge n) T i s s ue s a m pl e s w e r e i nc uba t e d w i t h 1m l of T r i z ol f or 5 m i nut e s a t r oom t e m pe r a t ur e t o pe r m i t t he f ul l t i s s ue di s s oc i a t i on. C hl or of or m w a s t he n a dde d a t a c onc e nt r a t i on of 0. 2 m l pe r 1m l of T r i z ol R e a ge nt T ube s w e r e vor t e xe d f or 15 s e c onds a nd i nc uba t e d a t r oom t e m pe r a t ur e f or 3

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67 m i nut e s S a m pl e s w e r e t he n s pun a t 1, 000g f or 15 m i nut e s a t 4 C F ol l ow i ng t he c e nt r i f uga t i on s t e p, t he s ol u t i on s e pa r a t e d i nt o a l o w e r r e d ( phe nol c hl or of o r m pha s e ) a n i nt e r m e di a t e pha s e a nd a n uppe r a que ous pha s e ( c ol or l e s s ) w hi c h c ont a i ns t he R N A T hi s pha s e w a s t he n t r a ns f e r r e d t o a not he r t ube c o nt a i ni ng 500 l o f i s opr opyl a l c ohol S a m pl e s w e r e l e f t a t r oom t e m pe r a t ur e f or 10 m i n ut e s a nd t he n c e nt r i f uge d a t 1 000g f o r 10 m i nut e s a t 4 C S upe r na t a nt w a s r e m ove d a nd t he R N A pe l l e t w a s he d onc e w i t h 70% e t ha nol T he R N A pe l l e t w a s t he n d r i e d a nd r e di s s ol ve d i n R N a s e f r e e w a t e r ( G i bc o) 4. 2. 2 2 R N as e P r ot e c t i o n A s s ay A l l pr oc e dur e s f o r t he R N a s e pr ot e c t i on a s s a y w e r e c onduc t e d a t J a m e s A H a l e y V e t e r a ns r e s e a r c h c e nt e r unde r t he s upe r vi s i on o f D r C a r m e l i na G e m m a A l l pr oc e dur e s w e r e pe r f or m e d w i t h s t r i c t obs e r va t i on of t he r e gul a t i ons s e t by t he J a m e s A H a l e y V e t e r a ns r e s e a r c h c e nt e r c om m i t t e e f o r m a n i pul a t i on of r a di oa c t i ve m a t e r i a l T w e nt y g o f t o t a l R N A f r om e a c h s a m pl e w a s hy br i di z e d w i t h a nt i s e ns e r a di ol a be l e d pr obe s a f t e r w hi c h f r e e pr obe a nd r e m a i ni ng s i ngl e s t r a nde d R N A w e r e di ge s t e d w i t h R N a s e A / T 1. D oubl e s t r a nde d R N a s e pr ot e c t e d f r a gm e nt s w e r e r e s ol ve d on 5% de na t ur i ng pol ya c r yl a m i de ge l s T he pr obe t e m pl a t e us e d w a s pur c ha s e d f r om P ha r m i nge n ( S a n D i e go, C A ) a nd i nc l ude d r a t s pe c i f i c s e que nc e s f or I L 1a l pha I L 1be t a T N F be t a I L 4 I L 6, I L 10, T N F a l pha I L 2 a nd i nt e r f e r on ( I F N ) ga m m a A pos i t i ve c ont r ol t r a ns c r i pt w a s m a de us i ng a pr obe s pe c i f i c f or t he r i bos om a l p r ot e i n L 32, hous e ke e pi ng ge ne i n or de r t o c a l c ul a t e t he s pe c i f i c a c t i vi t y a nd a c hi e ve a s uf f i c i e nt e xc e s s of pr obe ove r t a r ge t f o r L 32. T he L 32 pr ob e w a s t he n a dde d t o t he pr obe t e m p l a t e be f or e t he hyb r i di z a t i on r e a c t i on s t a r t e d. Y e a s t t r a ns f e r R N A a nd r a t m R N A w e r e us e d a s ne ga t i ve a nd pos i t i ve c ont r ol s r e s pe c t i ve l y. D r i e d ge l s w e r e pl a c e d on a phos phor i m a ge r

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68 s c r e e n f or 1 6 20 hour s T he phos phor i m a gi ng s c r e e n w a s s ubs e que nt l y s c a nn e d w i t h a phos phor i m a ge r ( M ol e c ul a r I m a ge S ys t e m G S 36 3; B i o R a d, H e r c ul e s C A ) T he i m a ge s w e r e pr oc e s s e d us i ng M ol e c ul a r A na l ys t s of t w a r e ( B i o R a d) T he i nt e ns i t y of a ba nd i n t he c om put e r g e ne r a t e d i m a ge i s di r e c t l y p r opor t i ona l t o t he a m ount of r a di oa c t i vi t y w i t hi n t he ba nd. T he opt i c a l de ns i t y ( O D ) va l ue s obt a i ne d f r o m e a c h ba nd w e r e nor m a l i z e d a ga i ns t t he O D obt a i ne d f r om t he L 32 ba nd i n t ha t s a m pl e by t he f ol l ow i ng e xpr e s s i on: ( O D of t he s a m pl e ba nd / O D of t he L 32 ba nd X 100 ) 4. 2. 3 S p l e n oc yt e i s ol at i on an d c u l t u r e s F r e s h s pl e e n t i s s ue w a s hom oge ni z e d us i ng S t om a c he r pl a s t i c ba gs ( S e w a r d) A bout 5 m i nut e s of hom oge ni z a t i on w e r e ne c e s s a r y t o di s s oc i a t e t he s pl e e ns i nt o a hom oge ne ous c e l l s us pe ns i on. T he s us pe ns i on w a s c ol l e c t e d i nt o 15 m l t ube s a nd s e t t l e d on i c e f or 10 m i nut e s A f t e r w a r ds t he s upe r na t a nt w a s t r a ns f e r r e d i nt o a ne w t ube a nd c e nt r i f uge d a t 1, 500g f o r 5 m i nut e s T he s upe r na t a nt w a s t he n r e m ove d, t he c e l l pe l l e t w a s r e s u s pe nde d i nt o 10 m l of r e d b l ood c e l l l ys i s buf f e r ( G e nt r a s y s t e m s ) a nd m i xe d. T he s us pe ns i on w a s t he n c e nt r i f uge d f or 5 m i nut e s a nd w a s he d onc e w i t h H B S S / 10% he a t i na c t i va t e d f e t a l bovi ne s e r um ( F B S ) E a c h pe l l e t w a s r e s us pe nde d i n H B S S pa s s e d t hr ough a 80 um nyl on c e l l s t r a i ne r ( F i s he r S c i e nt i f i c ) a nd w a s he d a not he r t i m e w i t h H B S S V i a bl e c e l l s w e r e c ount e d us i ng t he t r ypa n bl ue dye e xc l us i on m e t hod, a n a l i quo t w a s s e t a s i de f or f l ow c yt om e t r y a na l ys i s a nd t he r e s t o f t he s a m pl e s w e r e r e s us pe nde d ( a bo ut 10 6 c e l l s / m l ) i n R P M I m e di um ( s uppl e m e nt e d w i t h 10 % f e t a l bovi ne s e r um 100 U / m l pe ni c i l l i n G 100 g/ m l s t r e pt om yc i n s ul f a t e 1 5 m M gl ut a m i ne ) S pl e noc yt e s w e r e

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69 c ul t ur e d i nt o 96 w e l l pl a t e s a t 1 x 10 5 c e l l s pe r w e l l a nd p l a t e s w e r e i nc uba t e d a t 37 C a nd 5% C O 2 4. 2. 4 P r ol i f e r at i on as s ay M i t oge ns i nc l ude d l i popol ys a c c a r i de ( L P S S i g m a ) a t a c onc e nt r a t i on of 1 g/ m l f o r s t i m ul a t i on of B l ym phoc yt e s a nd C onc a va l i n A ( C on A S i gm a ) a t a c onc e nt r a t i on o f 2. 0 g/ m l f o r s t i m ul a t i on of T l ym phoc yt e s C e l l s us pe ns i ons w e r e a dde d i n qua dr upl i c a t e t o f l a t bot t om 96 w e l l p l a t e s a t a vol um e of 100 l / w e l l a nd pl a t e s w e r e i nc uba t e d a t 37 C a nd 5% C O 2 A f t e r 72 h t he c e l l pr ol i f e r a t i on a s s a y w a s pe r f or m e d B r i e f l y, t he ye l l ow t e t r a z ol i um M T T ( 3 ( 4 5 d i m e t hyl t hi a z ol yl 2) 2 5 di phe nyl t e t r a z ol i um br om i de ) t e t r a z ol i um s ubs t r a t e s ol ut i on ( S i gm a 5 m g/ m l i n P B S ) w a s pr e pa r e d a nd 20 l w a s a dde d t o e a c h w e l l o f t he 9 6 w e l l pl a t e s L i vi ng c e l l s c onve r t t h i s s ubs t r a t e t o f or m a z a n, pr oduc i ng a br ow n pr e c i pi t a t e T he pl a t e s w e r e i nc uba t e d a t 37 C a nd 5% C O 2 f or 3 hou r s a f t e r w hi c h, t he s upe r na t a nt w a s ge nt l y r e m ove d us i ng a m ul t i c ha nne l pi pe t t e a nd a dhe r e nt c e l l s w e r e s ol ub i l i z e d i n 200 l o f 100 % D M S O T he r e s ul t i ng c ol or c ha nge s w e r e qua nt i f i e d by obt a i ni ng opt i c a l de ns i t y ( O D ) r e a di ngs a t 570 nm us i ng t he S pe c t r a m a x P l us s pe c t r ophot om e t e r 4. 2. 5 I m m u n op h e n ot y p i n g o f s p l e e n c e l l s S pe c i f i c l e ukoc yt e s ubt ype s w e r e m e a s ur e d by i m m unof l uor e s c e nt a nt i body s t a i ni ng a nd f l ow c yt om e t r y a na l ys i s w a s pe r f or m e d on a F A C S c a n ( B D B i os c i e nc e s ) us i ng C e l l Q ue s t s of t w a r e T c e l l s w e r e i de nt i f i e d us i ng m ous e a nt i r a t ( C D 4 a nd C D 8) f r om B D P ha r m i nge n ( S a n D i e go, C A ) B r i e f l y c e l l s w e r e r e s us pe nde d i n 200 l of f l ow

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70 c yt om e t r y buf f e r ( 0. 02% B S A 0. 05 m M E D T A i n P B S ) c ont a i ni ng F c bl oc k ( B D P ha r m i nge n) t o r e duc e non s pe c i f i c a nt i body bi ndi ng. C e l l s w e r e t he n i nc uba t e d i n t he da r k w i t h t he a ppr opr i a t e F I T C c onj uga t e d a nt i bo dy ( 50 g/ m L ) f o r 60 m i nut e s on i c e A f t e r w a r ds c e l l s w e r e w a s he d t hr e e t i m e s w i t h 50 0 l f l ow c yt om e t r y buf f e r a nd f l ow c yt om e t r y w a s pe r f or m e d on t he F A C S c a n s ys t e m D a t a w e r e a na l yz e d us i ng C E L L Q ue s t s of t w a r e ( B e c t on D i c ki ns on) 4. 3. 6 C yt ok i n e E n z ym e l i n k e d i m m u n oad s or b e n t as s ays C e l l f r e e s upe r na t a nt s f r om C on A t r e a t e d s pl e noc yt e s w e r e c ol l e c t e d a nd s t or e d a t 70 C unt i l a na l ys i s I F N ga m m a a nd I L 10 c yt oki n e l e ve l s i n t he s upe r na t a nt s w e r e e xa m i ne d us i ng s pe c i f i c e nz ym e l i nke d i m m unos o r be nt a s s a y ( E L I S A ) ki t s ( B i os our c e I nt e r na t i ona l ) i n s t r i c t a c c or da nc e t o t he f ol l ow i ng pr ot oc ol s 4. 3. 6 1 Q u a n t i f i c at i on of s am p l e p r ot e i n am ou n t s P r ot e i n c onc e nt r a t i on i n e a c h s a m pl e w a s m e a s ur e d w i t h t he B i or a d A s s a y. T he B i oR a d a s s a y dye w a s di l ut e d i n di s t i l l e d w a t e r a t a c onc e n t r a t i on 1: 5 S t a nda r ds w e r e p r e pa r e d f or c onc e nt r a t i on of 0 2 4 6 8, 10 g. S a m pl e s w e r e di l ut e d 1: 5000 i n t he B i or a d A s s a y a nd t r a ns f e r r e d t o a gl a s s c uve t t e S a m pl e s w e r e r e a d on t he s pe c t r ophot om e t e r S pe c t r a m a x P l us a t 595 nm w a ve l e ngt h 4. 3. 6 2 E n z ym e l i n k e d i m m u n oad s or b e n t as s ays I F N ga m m a a nd I L 10 c yt oki ne l e ve l s i n t he s u pe r na t a nt s w e r e e xa m i ne d us i ng s pe c i f i c E L I S A ki t s obt a i ne d f r om B i os our c e I nt e r na t i ona l T he a m ount of 50 L o f s t a nda r d a nd s a m pl e s w e r e l oa de d i nt o t he a ppr op r i a t e w e l l s t og e t he r w i t h 50 L of I nc uba t i on B uf f e r T he pl a t e w a s i nc uba t e d a t 4 C ove r ni gh t A f t e r w a r ds w e l l s w e r e w a s he d w i t h W o r ki ng

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71 W a s h B uf f e r f our t i m e s T he B i ot i n c onj uga t e d a nt i body w a s t he n a dde d t o e a c h w e l l ( 100 l pe r w e l l ) a nd t he pl a t e w a s i nc uba t e d f or 2 hour s a t r oom t e m pe r a t ur e A f t e r w a s hi ng t he w e l l s 100 L S t r e pt a vi di n H R P W or ki ng C onj uga t e w a s a dde d t o e a c h w e l l a nd t he pl a t e i nc uba t e d f or 30 m i nut e s a t r oom t e m pe r a t ur e A f t e r w a s hi ng, 100 L o f S t a bi l i z e d C hr om oge n w e r e l oa de d i n e a c h w e l l a nd t he pl a t e w a s i nc uba t e d a t r oom t e m pe r a t ur e i n t he da r k unt i l t he a ppe a r a nc e of t he bl ue c ol or F i na l l y, 100 L o f S t op S ol ut i on w e r e a dde d t o e a c h w e l l a nd t he pl a t e w a s r e a d w i t h t he S pe c t r a m a x P l us s pe c t r ophot om e t e r a t t he a bs or ba nc e a t 450 nm 4. 2 R e s u l t s 4. 3. 1 C or d b l ood r e s c u e s s t r ok e i n d u c e d s p l e e n w e i g h t r e d u c t i o n w h i c h c or r e l at e s w i t h b r ai n i n f ar c t vol u m e s I n F i gu r e 4. 1A i t i s c l e a r t ha t s pl e e ns f r om a ni m a l s s ubj e c t t o s t r oke s ur ge r y w e r e s m a l l e r t ha n t hos e f r o m s ha m a ni m a l s W he n t he s pl e e ns w e r e w e i ghe d t hi s de c r e a s e w a s s i gni f i c a nt ( p< 0. 05) ( F i gu r e 4 1B ) M or e i m p or t a n t l y, H U C B C i nf us i on r e ve r s e d t hi s w e i ght l os s a nd s i z e r e duc t i on t o va l ue s c om p a r a bl e t o t he non s t r oke d s ha m r a t s ( F i gur e 4 1A a nd 4. 1B ) W hi l e t he a ve r a ge s pl e e n w e i ght of a s t r oke r a t w a s 0. 53 0. 049 gr a m s s pl e e ns f r om s t r oke d r a t s t r e a t e d w i t h H U C B C h a d a n a ve r a ge w e i ght of 0. 67 0. 06 gr a m s w hi c h w a s s i gni f i c a nt l y gr e a t e r t ha n t he w e i ght f or M C A O onl y a ni m a l s ( p < 0. 05 ) T T C s t a i ni ng o f t he br a i n r e ve a l e d t ha t H U C B C i nf us i on a l s o s i gni f i c a nt l y m i t i ga t e d t he e xt e nt of i s c he m i c da m a ge s e e n a f t e r M C A O ( F i g u r e 4. 2A ) A s s how n i n F i gur e 4 2B

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80 ha r ve s t e d f r om s ha m M C A O a nd H U C B C t r e a t e d M C A O r a t s de m ons t r a t e d t ha t s t r oke i nc r e a s e d t he s e ns i t i vi t y of s pl e noc yt e s t o t he T c e l l m i t oge n, C on A ( p< 0 05) ( s e e F i gur e 4. 7) I n c ont r a s t i nf us i on of c o r d bl ood c e l l s 24 h our s pr i or t o ha r ve s t i ng t he s pl e e ns of M C A O r a t s l e d t o a s i gni f i c a nt r e duc t i on i n T c e l l pr ol i f e r a t i on i n r e s pons e t o C on A ( p< 0. 05) ( s e e F i gur e 4. 7) B c e l l p r ol i f e r a t i on a s t e s t e d w i t h t he s t i m ul a t i on o f c ul t u r e d s pl e noc yt e s w i t h L P S w a s not s i gni f i c a nt l y a l t e r e d by s t r oke s ur ge r y or w i t h s ubs e que nt i nj e c t i on of c o r d bl ood ( s e e F i gur e 4. 7 ) G i ve n t he dr a m a t i c e f f e c t of c or d bl ood i n f us i on o n t he s pl e noc yt e r e s pons e t o a T c e l l m i t oge n ( C on A ) s upe r na t a nt f r om C on A t r e a t e d s pl e noc yt e c ul t ur e s of a l l e xpe r i m e nt a l c ondi t i ons w a s c ol l e c t e d a nd E L I S A pe r f or m e d S t r oke di d no t s i gni f i c a nt l y i nc r e a s e t he pr oduc t i on of I F N ga m m a f r om C on A s t i m ul a t e d s pl e noc yt e s a s c om pa r e d t o c ul t ur e s of s ha m r a t s w he r e a s I L 10 r e l e a s e w a s dr a m a t i c a l l y de c r e a s e d ( p< 0. 01) ( s e e F i gu r e 4 8A a nd 4. 8B ) I n c ont r a s t H U C B C t r e a t m e nt dr a m a t i c a l l y s uppr e s s e d t he e xpr e s s i on of I F N ga m m a a nd c ons i de r a bl y e nha nc e d t he p r oduc t i on of I L 10 ( p< 0 01) c om pa r e d t o M C A O de r i ve d s pl e noc yt e s ( s e e F i gur e 4. 8A a nd 4. 8B ) 4. 4 D i s c u s s i on I n our pr e vi ous s t udi e s ( s e e C ha pt e r 3) ( W i l l i ng L i xi a n e t a l 2003; W i l l i ng, V e ndr a m e e t a l 2003) w e de s c r i be d a ne ur o p r ot e c t i ve e f f e c t of H U C B C i n t he r a t M C A O m ode l of s t r oke I n t hi s s t udy w e ha ve e xt e nde d t hos e f i ndi ng by s how i ng a n e a r l i e r r e s c ue f r om M C A O i nduc e d br a i n da m a ge t ha n pr e vi ous l y r e p or t e d. M or e ove r w e de s c r i be a di r e c t e f f e c t of H U C B C on t he s pl e e n a s de m ons t r a t e d b y t he r e ve r s a l o f M C A O a s s oc i a t e d

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83 s pl e e n s i z e r e duc t i on; i nt e r e s t i ngl y, a m ong a l l t he M C A O a ni m a l s w e f ound a n i nve r s e c or r e l a t i on be t w e e n s pl e e n w e i ght a nd t he e xt e nt o f br a i n i nf a r c t i on T he s pl e e n s hr i nka ge f ol l ow i ng s t r oke ha s be e n pr e vi ous l y pr opos e d t o m i r r or a m obi l i z a t i on of l ym phoc yt e s f r om t he s pl e e n ( G e ndr on, T e i t e l ba um e t a l 2002) A ddi t i ona l l y, t he C D 8+ l ym phoc yt e s w hi c h a r e T l ym phoc yt e s w i t h c yt ot oxi c pr ope r t i e s ha ve be e n s how n t o be s i gni f i c a nt l y e l e va t e d i n t he i s c he m i c he m i s phe r e 48 hour s f ol l ow i ng M C A O i n t he r a t ( S c h r oe t e r J a nde r e t a l 1994) I n s uppor t o f t he s e obs e r va t i ons w e a l s o f ound de c r e a s e d num be r s of C D 8+ l ym phoc yt e s i n t he s pl e e ns f r om M C A O r a t s ; how e ve r H U C B C t r e a t m e nt r e t ur ne d s pl e e n C D 8+ pe r c e nt a ge s t o pr e s t r oke l e ve l s T he s e r e s ul t s s ugge s t t ha t t he s pl e e n c oul d be a r e s e r voi r f or t he C D 8+ T l ym phoc yt e s t ha t a r e m obi l i z e d f ol l ow i ng C N S i nj ur y a nd i m po r t a nt l y t ha t H U C B C pr ot e c t i on i n M C A O r a t c oul d be pa r t l y de r i ve d f r om t he m odul a t i on of s pl e e n C D 8+ T l ym phoc yt e f unc t i on. F unc t i ona l c ha r a c t e r i z a t i on of t he s pl e noc yt e s us i ng t he l ym phoc yt e m i t oge n C on A s ugge s t e d t ha t f ol l ow i ng M C A O t he s pl e e n w a s s e ns i t i z e d ( i n t e r m s o f pr o l i f e r a t i ve r e s pons e ) t o s uc h s t i m ul i A ddi t i ona l l y t he p r of i l e of c yt oki ne s e l i c i t e d by C on A t r e a t m e nt of s pl e noc yt e s de r i ve d f r om t he M C A O r a t s s how e l e va t e d l e ve l s of I F N a nd de c r e a s e d I L 10 pr o duc t i on. I n c ont r a s t w he n c ha l l e nge d w i t h C on A s pl e noc yt e s f r om M C A O r a t s t ha t r e c e i ve d H U C B C i nf us i on i nduc e d i nc r e a s e d I L 10 a nd de c r e a s e d I F N r e l e a s e c om pa r e d t o M C A O r a t s T hi s f unc t i ona l c ha r a c t e r i s t i c of t he s pl e e n c e l l s i ndi c a t e s t ha t f ol l ow i ng s t r oke i nj ur y t he pe r i phe r a l i m m une r e s pons e ( a s gove r ne d by t he s pl e e n) m a y be s ke w e d t ow a r ds a T h1 t ype r e s pons e i e t he p r o i nf l a m m a t or y r e a c t i on t o t he i nj u r y. H U C B C i nf us i on c ha nge d t hi s r e s pons e t o a T h2 t ype r e s pons e ( gr e a t e r

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84 r a t i o of C D 8+ / C D 4+ s pl e noc yt e s t ha t r e l e a s e I L 1 0 w hi l e de m ons t r a t i ng on l y m i ni m a l pr ol i f e r a t i on) S uc h phe nom e non m a y r e pr e s e nt a m e c ha ni s t i c e xpl a na t i on f or t he be ne f i c i a l e f f e c t i nduc e d by c or d bl ood i n f us i on a s s how n i n our s t udi e s r e l a t i ng t o s t r oke a nd by ot he r s i n va r i ous m ode l s o f br i a n i nj ur y I n t e r e s t i ngl y, I L 10 ha s be e n r e por t e d t o di r e c t l y m e di a t e i n f a r c t s i z e r e duc t i on w he n s ys t e m i c a l l y de l i ve r e d i nt o M C A O r a t s ( S pe r a E l l i s on e t a l 1998) or w he n s e c r e t e d e ndo ge nous l y by m obi l i z e d C D 4+ T l ym phoc yt e s ( F r e nke l H ua ng e t a l 2003 ) I n a ddi t i on t o t he i nc r e a s e d pr ot e i n l e ve l s o f I L 10 i n t he c ul t ur e s upe r na t a nt o f C on A t r e a t e d s pl e noc yt e s de r i ve d H U C B C r e c i pi e nt M C A O r a t s R N A s e pr ot e c t i on a s s a y de m ons t r a t e d t ha t m R N A l e ve l s of I L 10 f r om s pl e e ns of t he s a m e gr oup w e r e a l s o s i gni f i c a nt l y i nc r e a s e d c om pa r e d t o c ont r ol or M C A O r a t s H U C B C i s w e l l know n f or i t s l ow i m m unoge ni c i t y ; none t he l e s s gi ve n t he l a c k o f i m m unos uppr e s s i ve t he r a py i n t he e xpe r i m e nt s ( C h a pt e r 4 a nd 5 ) e xa m i ni ng H U C B C e f f e c t s on M C A O a s s oc i a t e d i m m une a l t e r a t i ons i t i s pos s i bl e t ha t s om e of t he out c om e w e de s c r i be d m i ght be r e l a t e d t o a gr a f t ve r s us hos t phe nom e na H ow e ve r t he i m m unom odul a t or y e f f e c t s w e de s c r i be a r e c ons i s t e nt w i t h r e po r t s s how i ng t ha t H U C B C c a n f unc t i ona l l y r e popul a t e t he l ym phoi d or ga ns ( i nc l udi ng s pl e e n) i n m i c e w hi c h a r e ge ne t i c a l l y i m m unode f i c i e nt ( T r a ggi a i C hi c ha e t a l 2004 ) W e s ugge s t t ha t t hi s i m m unom odul a t or y f unc t i on o f H U C B C m a y be c om pa r a bl e t o t ha t of a dopt i ve t r a ns f e r of s pl e noc yt e s a s t he r a py f or M C A O a nd ot he r di s e a s e s of br a i n i nj ur y T hi s phe nom e non m i ght r e p r e s e nt a pr ot ot ype m e c ha ni s m by w hi c h H U C B C m e di a t e t he ne ur obe ha vi or a l r e c ove r y a s de s c r i be d not onl y i n s t r oke but a l s o i n ot he r ne ur o i nf l a m m a t or y/ de ge ne r a t i ve di s e a s e m ode l s

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85 C H A P T E R F I V E M O D U L A T I O N O F S T R O K E I N D U C E D B R A I N I N F L A M M A T O R Y E V E N T S B Y C O R D B L O O D I N F U S I O N 5. 1 I n t r od u c t i on B r a i n i s c he m i a i nduc e s a c a s c a de of i nf l a m m a t or y e ve nt s w hi c h c ont r i but e s t o t he e xt e nt of C N S da m a ge R e s i de nt m a c r opha ge s s uc h a s m i c r ogl i a a r e a c t i va t e d a nd pa r t i c i pa t e i n t he pr oduc t i on o f a nu m be r o f s ubs t a nc e s i nc l udi ng pr o i n f l a m m a t or y c yt oki ne s s uc h a s T N F a l pha a nd ot he r m ol e c ul e s w i t h ne ur ot oxi c pr ope r t i e s l i ke t ox i c f r e e r a di c a l s ( L i u, C l a r k e t a l 1994; B ut t i n i A ppe l e t a l 1996; L i u G r os s m a n e t a l 2001) C onc om i t a nt l y, a num be r of di f f e r e nt t ype s of c i r c ul a t i ng i nf l a m m a t o r y c e l l s i nf i l t r a t e t he i nf a r c t e d br a i n t i s s ue E xa m pl e s of t hos e a r e l ym p hoc yt e s ne ut r ophi l s a nd m a c r opha ge s ( S c hr oe t e r J a nde r e t a l 1994; J a nde r K r a e m e r e t a l 1995; S t e ve ns B a o e t a l 2002 ) T he l i t e r a t u r e r e ga r di ng t he t i m e l i ne of e xt r a va s a t i on f r om bl ood i nt o b r a i n of t he s e di f f e r e nt c e l l popul a t i ons i s qui t e he t e r oge ne ous F or e xa m pl e u s i ng f l ow c yt om e t r y t o i de nt i f y m a c r opha ge s i n t he m ous e br a i n a f t e r i s c he m i a S t e ve ns e t a l r e por t e d a n i nc r e a s e i n num be r of m a c r opha ge s ( e i t he r r e s i de nt m i c r ogl i a or b l ood bor ne m a c r opha ge s ) s t a r t i ng a t 18 hour s a f t e r M C A O w hi c h pe a ke d a t f our f ol d a bov e t ha t of c ont r ol non s t r oke a ni m a l s a t 48 hou r s a f t e r M C A O ( S t e ve ns B a o e t a l 2002) I n a not he r s t udy, i m m unohi s t oc he m i s t r y t e c hni que s ha ve s ho w n t ha t t he r e i s a n i nc r e a s e i n i nf i l t r a t i ng m a c r opha ge s a t 1 t o 2 da ys a f t e r s t r oke w hi c h i nc r e a s e s pr ogr e s s i ve l y ove r t he f ol l ow i ng 5 da ys ( J a nde r K r a e m e r e t a l 1995 ) O t he r s t udi e s ha ve s how n t ha t t hi s

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86 i nc r e a s e c a n be pr ol onge d up t o 14 da ys a f t e r s t r ok e a nd m a c r opha ge s c a n be i de nt i f i e d w i t hi n t he i s c he m i c t i s s ue up t o 30 da ys a f t e r t he s t r oke i nduc t i on ( S c hr oe t e r J a nde r e t a l 1994) I n t he s t udy by S t e ve ns e t a l T c e l l s w e r e s how n t o be s i gni f i c a nt l y i nc r e a s e d 48 hour s a f t e r M C A O t o r e a c h t he i r pe a k o f e xpr e s s i on a t t he 72 hour t i m e poi nt a nd r e m a i n e l e va t e d up t o 4 da ys pos t i s c he m i a ( S t e ve ns B a o e t a l 2002) S i m i l a r l y, i m m unohi s t oc he m i c a l s t udi e s of S c hoe t l e r e t a l ( S c hr oe t e r J a nde r e t a l 1994) a nd J a nde r e t a l ( J a nde r K r a e m e r e t a l 1995 ) f ound T c e l l i nf i l t r a t i on a t 48 t o 72 hour s o f s t r oke i nduc t i on t ha t c ont i nue d t o i nc r e a s e ove r t he f ol l ow i ng 7 da ys C ont r a r y t o T c e l l s t he pr e s e nc e of B c e l l s i n t he br a i n i s not doc um e nt e d a s s i gni f i c a nt l y a ugm e nt e d pos t i s c he m i a ( S t e ve ns B a o e t a l 2002) F l ow c yt om e t r y s t udi e s ha ve s how n t ha t ne ut r oph i l s i nf i l t r a t e i n t o s t r oke d b r a i n pe a ki ng a t 48 hou r s po s t i s c he m i a ( S t e ve ns B a o e t a l 2002) I nf l a m m a t or y c e l l i nf i l t r a t i on i nt o t he br a i n a f t e r i s c he m i c i ns ul t ha s be e n s t udi e d qui t e e xt e ns i ve l y be c a us e c ont r ol l i ng t he i nf i l t r a t i on o f t he s e c e l l s c oul d c ons t i t ut e a pr ot e c t i ve i nt e r ve nt i o n i n t he i s c he m i c br a i n I nde e d t he pr o c e s s of t i s s ue l os s a f t e r a n i s c he m i c i ns ul t i s t hought not t o be c om pl e t e d f o r hou r s or d a ys A s e xpl a i ne d i n t he I nt r oduc t i on c ha pt e r t he s o c a l l e d pe num br a i s t he a r e a of b r a i n i nf a r c t i n w hi c h h i ghl y c om pl e x pa t hol ogi c a l pr oc e s s e s i nduc e d by t he i s c he m i a a r e s l ow l y pr ogr e s s i ng a nd a s s uc h a r e s t i l l r e ve r s i bl e W i t h t he i de a t ha t t he pr og r e s s i on of t he i nj u r y i n t he pe num b r a c a n be a f f e c t e d by m a ni pul a t i on of t he i nf l a m m a t or y r e s pons e a ge nt s t ha t bl oc k t he m i gr a t i on of c e l l s i nt o t he br a i n ha ve be e n e m pl oye d i n s e ve r a l s t udi e s A nt i bodi e s t ha t p r e ve nt l e ukoc yt e a dhe s i on t o e ndot he l i a s uc h a s a nt i bodi e s t o t he i nt e r c e l l ul a r a dhe s i on m ol e c ul e 1 ( I C A M 1) ha ve be e n r e por t e d t o de c r e a s e i s c he m i c da m a ge i n a ni m a l m ode l s of s t r oke ( Z ha ng, C hopp e t a l 1994 ) S i m i l a r l y, t h e r a pe ut i c i nt e r ve nt i ons bl oc k i ng t he

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87 C D 11b m ol e c ul e t he l i ga nd of I C A M 1, ha ve be e n de m ons t r a t e d t o r e duc e i nf a r c t vol um e a nd a m e l i or a t e f unc t i ona l de f i c i t s i n t he M C A O m ode l of s t r oke ( C he n, C hopp e t a l 1994) H ow e ve r a s I a l s o poi nt e d out i n t he C h a pt e r 1, t he us e of s uc h a ge nt s i n c l i ni c a l t r i a l s ha s pr oduc e d e qui voc a l r e s ul t s D e s pi t e t hi s i n t he r e s e a r c h s e t t i ng t he r e a r e s t i l l hope s f o r t he d i s c ove r y of a n e f f e c t i ve bl o c ka de of t he p r o i nf l a m m a t o r y c e l l i nf i l t r a t i on T a ki ng i nt o a c c ount our r e s ul t s i l l us t r a t e d i n t he pr e vi ous c ha pt e r s ( C ha pt e r 3 a nd C ha pt e r 4) w e s ought t o i nve s t i ga t e w he t he r H U C B C i nf us i on c oul d s om e how ha ve a n e f f e c t on t he c e l l ul a r e ve nt s t ha t oc c ur i n t he br a i n a f t e r i s c he m i c i nj ur y I n pa r t i c ul a r t he f a c t t ha t i nt r a ve nous l y de l i ve r e d H U C B C ha d s ys t e m i c i m m une r e gul a t or y e f f e c t s by m odul a t i ng t he f unc t i on of s pl e ni c c e l l s of s t r oke d r a t s l e d us t o i nve s t i ga t e w he t he r t he s e c e l l s c oul d m od ul a t e t he pr e s e nc e of i m m une / i nf l a m m a t or y c e l l s w i t hi n t he i s c he m i c br a i n. T o a ddr e s s t hi s que s t i on, w e us e d f l ow c yt om e t r y t o c ha r a c t e r i z e i m m une / i n f l a m m a t or y c e l l s i n t he s t r oke b r a i n be c a us e i t of f e r s a num be r of a dva nt a ge s ove r i m m unohi s t oc he m i s t r y. F i r s t of a l l i t a l l ow s a qua nt i t a t i ve a na l ys i s on t he e nt i r e he m i s phe r e w hi c h i s s e ns i t i ve a nd f a s t S e c ondl y t he pos s i bi l i t y of pe r f o r m i ng i t e xc l us i ve l y on l i ve c e l l s a l l ow s us t o e xc l ude pos s i bl e a r t i f a c t s due t o non s pe c i f i c a nt i body bi ndi ng o r ba c k gr ound s i gna l s of i nt e r f e r e nc e H ow e ve r t he di s a dva nt a ge t ha t f or ye a r s ha s he l d i nve s t i ga t or s ba c k f r om us i ng i t on t i s s ue i s t ha t t he s i ngl e c e l l s us pe n s i on i s di f f i c ul t t o obt a i n a nd m a y ha r m t he c e l l s a nd c e l l s ur f a c e m a r ke r s R e c e nt l y s e ve r a l i nve s t i ga t or s ha ve a t t e m pt e d t o i s ol a t e di f f e r e nt br a i n c e l l s ubpopul a t i ons f or t he f l ow c yt om e t r y a na l ys i s ( F o r d, G oods a l l e t a l 1995; C a m pa ne l l a

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88 S c i or a t i e t a l 2002; S t e ve ns B a o e t a l 2002) T he s e t e c hni que s w e r e m odi f i e d i n ou r l a bor a t or y t o pr ovi de r e l i a bl y r e pr oduc i bl e c e l l s us pe ns i ons f r om t he i s c he m i c br a i n A not he r a ppr oa c h t ha t w e a dopt e d t o e xa m i ne t he e f f e c t of c or d bl ood i nf us i on i n t he m odul a t i on of br a i n i nf l a m m a t or y e ve nt s f ol l ow i n g s t r oke w a s ba s e d on t he i nv e s t i ga t i on of br a i n c yt oki ne pr o f i l e s B r a i n c e l l s i nc l udi ng m i c r ogl i a a s t r oc yt e s e ndot he l i a l c e l l s a nd ne ur ons c a n pr oduc e c yt oki ne s T he m os t pot e nt pr o i n f l a m m a t or y c yt oki ne t u m or ne c r os i s f a c t or a l pha ( T N F a l pha ) i s pr oduc e d by m i c r ogl i a l c e l l s w i t hi n t he f i r s t 3 hour s pos t i s c he m i c i ns ul t ( B ut t i ni A ppe l e t a l 1996 ) A l t hough s om e s t udi e s ha ve s how n t ha t t hi s c yt oki ne c a n pos s e s s ne ur opr ot e c t i ve pr op e r t i e s t he s e a r e t hought t o be c onf i ne d t o r e gul a t i on o f c a l c i um hom e os t a s i s i n t he ne ur on ( C he ng, C hr i s t a kos e t a l 1994 ) G e ne r a l l y, T N F a l pha i s know n t o be ne u r ot ox i c a n d i t s upr e gul a t i on i s a s s oc i a t e d w i t h ne ur ode ge ne r a t i on a nd m i c r ogl i os i s ( S r e dni K e ni g s buc h 2002) T oge t he r w i t h T N F a l pha t he i nt e r l e uki n 1 be t a ( I L 1be t a ) i s c ons i de r e d a not he r i m po r t a nt pl a ye r i n t he i s c he m i c br a i n. I nc r e a s e d l e ve l s of t hi s c yt oki ne ha ve be e n doc um e nt e d a s s oon a s 2 hour s pos t M C A O ( Z ha i F ut r e l l e t a l 1997) a nd s t udi e s on M C A O m i c e l a c ki ng i t s r e c e pt or ( I L 1be t a r e c e pt or knoc k out m i c e ) ha ve s how n a s i gni f i c a nt de c r e a s e i n oxi da t i ve ne ur ona l da m a ge c om pa r e d t o w i l d t ype m i c e ( O ht a ki T a ka ki e t a l 2003) B l oc ka ge of pr o i n f l a m m a t or y c yt oki ne s e i t he r by bl oc ki ng t he i r e xpr e s s i on or a br oga t i ng t he i r f unc t i on, ha s be e n t he f oc us of s e v e r a l t he r a pe ut i c s t r a t e gi e s i n t he pa s t F or i ns t a nc e ove r e xp r e s s i on of t he I L 1 r e c e pt or a nt a goni s t i nduc e d w i t h a v i r a l ve c t or ha s be e n s how n t o a t t e nua t e t he i n f l a m m a t or y r e s pons e t ha t f ol l ow s M C A O ( Y a ng, L i u e t

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89 a l 1998) C ur i ous l y, t he bl oc ka ge of I L 1be t a p r o duc t i on i s t hought t o be t he m e c ha ni s m by w hi c h e l e c t r oa c upunt ur e i s a n e f f e c t i ve t r e a t m e nt a f t e r i s c he m i c br a i n i nj u r y i n C hi na ( X u, W u e t a l 2002 ) O t he r t he r a pe ut i c a nd e xpe r i m e nt a l a ppr oa c he s ha ve be e n ba s e d on t he bl oc ka ge of pr o i nf l a m m a t or y c yt oki ne s e i t he r by t he us e of a nt i bodi e s ne ut r a l i z i ng t he i r a c t i vi t y, or by t he i nhi bi t i on of e nz ym e s i nvol ve d i n t he i r pr oduc t i on F or e xa m pl e i nhi bi t i on of T N F a l pha w i t h a nt i bodi e s a dm i ni s t e r e d i nt r a ve nt r i c ul a r l y t o M C A O m i c e i s e f f e c t i ve i n r e duc i ng t he l e s i on vol um e ( Y a ng, G ong e t a l 1998 ) S i m i l a r l y, pha r m a c ol ogi c a l i nh i bi t i on o f t he T N F a l pha c onve r t i ng e nz ym e 30 m i nut e s be f or e t he i nduc t i on of t he s t r oke c a n m i ni m i z e t h e i nf a r c t vol u m e a nd p r om ot e f unc t i ona l be ne f i t s i n t he M C A O r a t s ( W a ng, F e ue r s t e i n e t a l 2004) T o e xa m i ne w he t he r c or d bl ood c e l l s c oul d ha ve a r ol e i n t he r e gul a t i on of t he e xpr e s s i on of pr o a nd/ or a nt i i n f l a m m a t or y c yt oki ne s w e de t e r m i ne d t he m e s s e nge r R N A a nd pr ot e i n l e ve l s of a n a r r a y of c yt oki ne s w i t hi n t he e nt i r e i s c he m i c br a i n. 5. 2 M at e r i al s an d M e t h od s A dul t m a l e S p r a gue D a w l e y r a t s w e r e r a ndom l y a s s i gne d t o t he f ol l ow i ng e xpe r i m e nt a l gr oups : s ha m s ur ge r y ( n = 16 ) M C A O onl y ( n = 1 8) or M C A O pl us H U C B C ( n = 18) M C A O s ur ge r y w a s pe r f or m e d a s pr e vi ous l y de s c r i be d i n S e c t i on 2. 1 R a t s r e c e i ve d c or d bl ood c e l l s t r a ns pl a nt 24 hour s a f t e r t he s t r ok e s ur ge r y. A n a m ount o f 10 000, 000 c e l l s w a s i nj e c t e d a s e xpl a i ne d i n de t a i l ( C ha pt e r 2 pa r a gr a ph 2. 2 3) T he f ol l ow i ng da y a l l r a t s w e r e ki l l e d by de c a pi t a t i on a f t e r i nduc t i on of a ne s t he s i a w i t h i s of l uor a ne H a l f o f t he a ni m a l s unde r w e nt R N A a na l ys i s a nd ha l f p r ot e i n a na l ys i s w i t h e i t he r c yt oki ne s a r r a y

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90 or E L I S A s T he r a t b r a i n w a s r e m ove d a nd t he e nt i r e r i ght he m i s phe r e w a s r a pi dl y di s s e c t e d. W e de c i de d not t o di s s e c t s pe c i f i c br a i n a r e a s but t o a na l yz e t he r i ght he m i s phe r e s i nc e pe r m a ne nt m i ddl e c e r e br a l a r t e r y oc c l us i on ge ne r a l l y pr o voke s a l e s i on t ha t e xt e nds a l ong t he e nt i r e he m i s phe r e a nd c yt oki ne s r a pi dl y di f f us e w i t hi n t he m i c r oe nvi r om e nt ( B ut t i ni A ppe l e t a l 1996 ) T he br a i n he m i s phe r e s of a ni m a l s f or R N A a na l ys i s w e r e i m m e di a t e l y i m m e r s e d i n T r i z ol r e a ge nt i n 1 5 m l R N A s e f r e e E ppe ndor f t ube s R N A w a s i s ol a t e d a nd R N A s e pr ot e c t i on a s s a y w a s c onduc t e d a s pr e vi ous l y de s c r i be d f or t he s pl e e n s a m pl e s ( f or de t a i l s s e e C ha pt e r 4, pa r a gr a ph 4 3) T he b r a i ns f or p r ot e i n a na l ys i s w e r e s t or e d i n 1. 5m l of l ys i s b uf f e r a t 80 C unt i l us a ge B r a i ns w e r e hom oge ni z e d a nd c yt oki ne s l e ve l s w e r e m e a s ur e d a s e xpl a i ne d i n de t a i l i n C ha pt e r 4 pa r a gr a p h 4 3. F or t he f l ow c yt om e t r y s t udi e s a not he r s e t of 12 a ni m a l s w a s e m pl oye d. T he s e w e r e a s s i gne d t o t he f ol l ow i ng e xpe r i m e nt a l gr oups : s ha m s ur ge r y ( n = 4 ) M C A O onl y ( n = 4) or M C A O pl us H U C B C ( n = 4) R a t s unde r w e nt s t r oke s ur ge r y a nd H U C B C t r a ns pl a nt 24 hour s a f t e r a c c or di ng t o t he m e t hods de s c r i be d pr e vi ous l y ( C ha pt e r 2 pa r a gr a ph 2 a nd 3) T he da y a f t e r t he t r a ns pl a nt r a t s w e r e ki l l e d by de c a pi t a t i on, t he br a i ns w e r e r a pi dl y r e m ove d a nd f r e e d f r o m t he m e ni nge s S i ngl e c e l l s us pe n s i ons w e r e obt a i ne d w i t h P e r c ol l gr a di e nt s e pa r a t i on a s de s c r i be d be l ow 5. 2. 1 S e p ar at i on of c e l l s b y P e r c ol l gr ad i e n t

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91 T he br a i n he m i s phe r e s w e r e s e pa r a t e d f r om t he m e ni nge s a nd pl a c e d i nt o 15 m L of i c e c ol d H B S S c ont a i ni ng 0 2% bovi ne s e r um a l bum i n ( B S A ) ( S i gm a ) 0 01 m o l / L E D T A a nd 10 m g/ m L de oxy r i bonuc l e a s e I ( S i gm a ) F i gu r e 5. 1 r e pr e s e nt s a s c he m a of t he pr oc e dur e B r a i n t i s s ue w a s hom oge ni z e d us i ng a D ounc e hom oge ni z e r a nd pa s s e d t hr ough a 40 m ny l on c e l l s t r a i ne r ( B e c t on D i c ki ns on) T he s us pe ns i on w a s c e nt r i f uge d a t 400g f o r 10 m i nut e s a t r oom t e m pe r a t u r e ; t he pe l l e t w a s r e s us pe nde d i n 10 m L of H B S S c ont a i ni ng 20U / m L c ol l a ge ns e I I ( S i gm a ) a nd c e nt r i f uge d a t 400g f or 10 m i nut e s a t r oom t e m pe r a t ur e F i na l l y t he pe l l e t w a s r e s us pe nde d i n 2 m L of 70 % P e r c ol l ( G i bc o ) i n a 15 m L t ube w hi c h w a s ove r l a i d w i t h a g r a di e nt c ont a i ni ng 2 m L of 30% o f P e r c ol l s ol ut i on a nd 2 m L o f P B S P e r c ol l w a s pr e pa r e d b y di l ut i on i n P B S T he g r a di e nt w a s c e nt r i f uge d a t 600g f or 45 m i nut e s a t r oo m t e m pe r a t ur e ; c e l l s w e r e c ol l e c t e d f r om t he 30% l a ye r t o t he P B S i nt e r f a c e ( a bout 5 m L ) a nd w a s he d w i t h H B S S c ont a i ni ng 10% f e t a l bovi ne s e r um ( F B S ) ( G i bc o) 5. 2. 2 I m m u n ol ab e l i n g p r o t oc ol an d p r e p ar at i on o f s am p l e s f or f l ow c yt om e t r y an al ys i s T he f ol l ow i ng a nt i bodi e s w e r e us e d i n t he f l ow c yt om e t r y a s s a y: m onoc l ona l a nt i r a t C D 11b f l uor e s c e i n i s ot hi oc ya na t e ( F I T C ) c onj uga t e d a nt i body, m onoc l ona l a nt i r a t C D 45 phyc oe r yt hr i n c onj uga t e d a nt i body m onoc l ona l a nt i r a t C D 3 a nt i body c onj uga t e d w i t h phyc oe r yt hr i n ( P ha r M i nge n) m onoc l ona l a nt i r a t B 220 p hyc oe r yt h r i n c onj uga t e d a nt i body, m onoc l ona l a nt i r a t ne ut r ophi l s a nt i body c onj uga t e d w i t h phyc oe r yt h r i n ( P ha r M i nge n) I s ot ype c ont r ol s ( P ha r m i nge n) w e r e us e d i n pa r a l l e l

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92 C e l l s w e r e t he n i nc uba t e d i n t he da r k w i t h t he a pp r opr i a t e f l uo r oc hr om e c onj uga t e d a nt i body f or 60 m i nu t e s on i c e A f t e r w a r ds c e l l s w e r e w a s he d t hr e e t i m e s w i t h 500 l of f l ow c yt om e t r y buf f e r ( 0 02% B S A 0 05 m M E D T A i n P B S ) 5. 2. 3 A c q u i s i t i on an d an al ys i s of d at a T he c e l l s w e r e r e s us pe nde d i n 200 l o f f l ow c yt o m e t r y buf f e r c ont a i ni ng F c bl oc k ( B D P ha r m i nge n) t o r e duc e non s pe c i f i c a nt i body bi ndi ng. A c qui s i t i on w a s pe r f or m e d w i t h t he F A C S C a l i bur ( B e c t on D i c ks on) a nd C E L L Q ue s t s of t w a r e ( B e c t on D i c ki ns on) D e a d c e l l s w e r e e xc l ude d f r om t he a na l ys i s by T o P r o 3 s t a i ni ng D a t a w e r e a na l y z e d us i ng C E L L Q ue s t s of t w a r e ( B e c t on D i c ki ns on) 5. 3 R e s u l t s 5. 3. 1 H U C B C i n f u s i on r e d u c e s t h e s t r ok e i n d u c e d u p r e gu l at i on of C D 45/ C D 11b p os i t i ve an d B 220 p os i t i ve c e l l s w i t h i n t h e b r ai n F A C S r e ve a l e d t ha t C D 45/ C D 11b pos i t i ve c e l l s w e r e i nc r e a s e d i n s t r oke b r a i ns r e l a t i ve t o br a i ns f r om s ha m c ont r ol s w i t h a n a ve r a ge of 5 f ol d i nc r e m e nt ( F i gu r e 5. 2 a nd 5 6) W he n H U C B C w e r e de l i ve r e d 24 hour s a f t e r t he s t r oke s ur ge r y, t hi s popul a t i on of C D 11b/ C D 45 pos i t i ve c e l l s w a s dr a m a t i c a l l y di m i ni s he d ( F i gur e 5 2 a nd F i gur e 5. 6) S t a t i s t i c a l a na l ys i s c onduc t e d on t he r e s ul t s of f our di s t i nc t obs e r va t i ons r e ve a l e d t ha t t h i s r e duc t i on w a s s i gni f i c a nt ( p< 0 01) F or t he a na l ys i s of B c e l l s t he B 220/ C D 45R m a r ke r w a s us e d. A na l ys i s s how e d a 2 f ol d i nc r e m e nt i n B 220 pos i t i ve c e l l s i n br a i ns t ha t ha d unde r gone M C A O ( F i gur e 5 3 a nd

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99 5. 6) I n br a i ns of M C A O r a t s t ha t r e c e i ve d H U C B C i nf us i on t he r e w e r e s i gni f i c a nt l y f e w e r B 220 c e l l s c om pa r e d t o br a i ns of M C A O onl y r a t s ( p< 0 05) ( F i gu r e 5. 3 a nd 5 6) 5. 3. 2 H U C B C i n f u s i on d oe s n o t al t e r t h e p r e s e n c e of C D 45/ C D 3 p os i t i ve c e l l s w i t h i n t h e i s c h e m i c b r ai n L ym phoc yt e popul a t i ons w e r e s t udi e d w i t h t he us e of a n a nt i body s pe c i f i c f or t he C D 3 m a r ke r A f t e r s t r oke s ur ge r y t he pe r c e nt a ge of c e l l s t ha t w e r e C D 45 a nd C D 3 pos i t i ve w a s a ugm e nt e d by 2 f ol d ( F i gu r e 5 4 a nd 5 6) W he n H U C B C w e r e i nj e c t e d, t he r e w a s no s i gni f i c a nt a l t e r a t i on i n t he num be r of C D 45/ C D 3 pos i t i ve c e l l s ( F i gu r e 5. 4 a nd 5 6) 5. 3. 3 T h e p e r c e n t age of N e u t r op h i l s i s n o t al t e r e d 48 h ou r s af t e r s t r ok e s u r ge r y. A l t hough t he a m ount of ne ut r ophi l s w a s i nc r e a s e d i n s om e br a i ns f r om s t r oke r a t s t h i s w a s not a c ons i s t e nt obs e r va t i on. W he n t he r e s ul t s w e r e a ve r a ge d a c r os s a ni m a l s t he r e w a s no s i gni f i c a nt i nc r e m e nt i n t he pe r c e nt a ge of n e ut r ophi l s pr e s e nt a f t e r M C A O ( F i gur e 5 5 a nd 5. 6 ) A ddi t i ona l l y a f t e r t r e a t m e nt w i t h H U C B C t hi s pe r c e nt a ge w a s not f ur t he r a l t e r e d ( F i gur e 5 5 a nd 5. 6 ) 5. 3. 4 M e s s e n ge r R N A e xp r e s s i on of p r o i n f l am m a t or y c yt ok i n e s i s r e gu l at e d b y t h e i n j e c t i on o f c or d b l ood c e l l s U s i ng t he R N a s e pr ot e c t i o n a s s a y w e w e r e a bl e t o a na l yz e of a w i de a r r a y o f c yt oki ne s i nc l udi ng: T N F a l pha I L 1be t a T N F be t a I L 2 I L 6, I L 10 I L 4 I N F ga m m a a nd T G F be t a I N F ga m m a a nd T F G be t a w e r e unde t e c t a bl e i n a ny e xpe r i m e nt a l c ondi t i ons

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103 5. 6) I n br a i ns of M C A O r a t s t ha t r e c e i ve d H U C B C i nf us i on t he r e w e r e s i gni f i c a nt l y f e w e r B 220 c e l l s c om pa r e d t o br a i ns of M C A O onl y r a t s ( p< 0 05) ( F i gu r e 5. 3 a nd 5 6) 5. 3. 2 H U C B C i n f u s i on d oe s n o t al t e r t h e p r e s e n c e of C D 45/ C D 3 p os i t i ve c e l l s w i t h i n t h e i s c h e m i c b r ai n L ym phoc yt e popul a t i ons w e r e s t udi e d w i t h t he us e of a n a nt i body s pe c i f i c f or t he C D 3 m a r ke r A f t e r s t r oke s ur ge r y t he pe r c e nt a ge of c e l l s t ha t w e r e C D 45 a nd C D 3 pos i t i ve w a s a ugm e nt e d by 2 f ol d ( F i gu r e 5 4 a nd 5 6) W he n H U C B C w e r e i nj e c t e d, t he r e w a s no s i gni f i c a nt a l t e r a t i on i n t he num be r of C D 45/ C D 3 pos i t i ve c e l l s ( F i gu r e 5. 4 a nd 5 6) 5. 3. 3 T h e p e r c e n t age of N e u t r op h i l s i s n o t al t e r e d 48 h ou r s af t e r s t r ok e s u r ge r y. A l t hough t he a m ount of ne ut r ophi l s w a s i nc r e a s e d i n s om e br a i ns f r om s t r oke r a t s t h i s w a s not a c ons i s t e nt obs e r va t i on. W he n t he r e s ul t s w e r e a ve r a ge d a c r os s a ni m a l s t he r e w a s no s i gni f i c a nt i nc r e m e nt i n t he pe r c e nt a ge of n e ut r ophi l s pr e s e nt a f t e r M C A O ( F i gur e 5 5 a nd 5. 6 ) A ddi t i ona l l y a f t e r t r e a t m e nt w i t h H U C B C t hi s pe r c e nt a ge w a s not f ur t he r a l t e r e d ( F i gur e 5 5 a nd 5. 6 ) 5. 3. 4 M e s s e n ge r R N A e xp r e s s i on of p r o i n f l am m a t or y c yt ok i n e s i s r e gu l at e d b y t h e i n j e c t i on o f c or d b l ood c e l l s U s i ng t he R N a s e pr ot e c t i o n a s s a y w e w e r e a bl e t o a na l yz e of a w i de a r r a y o f c yt oki ne s i nc l udi ng: T N F a l pha I L 1be t a T N F be t a I L 2 I L 6, I L 10 I L 4 I N F ga m m a a nd T G F be t a I N F ga m m a a nd T F G be t a w e r e unde t e c t a bl e i n a ny e xpe r i m e nt a l c ondi t i ons

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106 S i m i l a r l y, m R N A l e ve l s of I L 4 w e r e de c r e a s e d i n br a i ns of r a t s s ubj e c t e d t o s t r oke ( p< 0. 05) ( F i gu r e 5. 9 ) but t he i nj e c t i on o f H U C B C di d not m odi f y t hi s e f f e c t l e a vi ng I L 4 m R N A a t l e ve l s c om pa r a bl e t o M C A O onl y a ni m a l s ( F i gur e 5 9) 5. 3. 5 H U C B C i n f u s i on d e p r e s s e s t h e e xp r e s s i on of p r o i n f l am m at or y c yt ok i n e s t h at ar e u p r e gu l at e d w i t h i n t h e i s c h e m i c b r ai n C yt oki ne s E L I S A s w e r e pe r f or m e d f or T N F a l pha I L 1be t a I L 2, I L 6, I L 10 a nd i nt e r f e r on ga m m a ( I F N ga m m a ) T he I F N ga m m a w a s t he onl y c yt oki ne w hi c h w a s not de t e c t a bl e i n a ny e xpe r i m e nt a l c ondi t i ons T he e xpr e s s i on of T N F a l pha w a s s i gni f i c a nt l y h i g he r i n t he s t r oke a ni m a l s ( M C A O gr oup) c om pa r e d t o t he s ha m a ni m a l s ( p< 0. 05 ) ( F i gur e 5. 10A ) W he n c or d bl ood w a s i nj e c t e d, t he l e ve l s of T N F a l pha dr oppe d t o a l e ve l c om pa r a bl e t o t ha t of t he no r m a l s ha m r a t s I nt e r e s t i ngl y t hi s c o r d bl ood de pe nde nt dr op w a s s t a t i s t i c a l s i gni f i c a nt ( p< 0 05) ( F i gur e 5 10A ) W he n I L 1be t a w a s m e a s ur e d, i t w a s hi ghe r i n t he br a i ns of t he M C A O r a t s r e l a t i ve t o br a i ns f r om s ha m r a t s ( p< 0 05) ( F i gur e 5. 10B ) A f t e r i nj e c t i on o f c or d bl ood, t he r e w a s a s i gni f i c a nt de c r e a s e of I L 1be t a c om pa r e d t o t he n on t r e a t e d M C A O onl y r a t s ( p< 0. 05) ( F i gur e 5 10B ) I nt e r l e uki n 2 w a s de t e c t e d i n hi ghe r a m ount s f r o m i s c he m i c br a i ns i n c om pa r i s on t o non i s c he m i c br a i ns ( p< 0. 05) ( F i gu r e 5 11A ) W hi l e i n j e c t i on of c or d bl ood a ppe a r e d t o r e duc e t he s e a m ount s t hi s de c r e a s e w a s n ot s t a t i s t i c a l l y s i gni f i c a nt ( p> 0. 05 ) ( F i gur e 5. 11A )

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107 S i m i l a r t o t he r e s ul t s on t he I L 6 m R N A t he de t e c t i on of I L 6 r e ve a l e d a c ons i de r a bl e va r i a bi l i t y a c r os s t he a ni m a l s s ubj e c t t o s t r oke T h e l e ve l s of I L 6 s e e m e d hi ghe r i n gr oup of a ni m a l s r e l a t i ve t o t he non s t r oke a ni m a l s but t hi s di f f e r e nc e w a s not s t a t i s t i c a l l y s i gni f i c a nt ( p> 0. 05) ( F i gur e 5 11B ) T he r e w a s a r e duc t i on of I L 6 l e ve l s i nduc e d by t he i nj e c t i on of c o r d bl ood but a l s o t hi s r e duc t i on di d not r e a c h s i gni f i c a nc e ( F i gur e 5 11B ) W he n I L 10 w a s m e a s ur e d, i t s l e ve l s w e r e hi ghe r i n t he M C A O br a i n r e l a t i ve t o s ha m br a i ns t hi s di f f e r e nc e w a s not s i gni f i c a nt ( p> 0. 05 ) ( F i gu r e 5. 11C ) L e ve l s of I L 10 i n t he br a i ns of r a t s t r e a t e d w i t h c or d bl ood c e l l s w e r e c o m pa r a bl e t o t hos e of M C A O r a t s ( p> 0. 05 ) ( F i gu r e 5. 11C ) 5. 4 D i s c u s s i on T he s e da t a s ugge s t t ha t i n i s c he m i c r a t br a i n, C D 11b/ C D 45 pos i t i ve c e l l s a r e i nc r e a s e d i n num be r a nd i nf us i on o f H U C B C de pr e s s e s t hi s phe nom e non. I n pa r t i c ul a r t he r e duc t i on i n pe r c e nt a ge of C D 11b/ C D 45 pos i t i ve c e l l s a ppe a r e d t o be qui t e d r a m a t i c ( f r om a 5 f ol d i nc r e a s e t o ba s e l i ne l e ve l s ) P r e vi ous s t udi e s ha ve s ugge s t e d t ha t t hi s popul a t i on of C D 11b/ C D 45 pos i t i ve c e l l s t ha t i s m a i nl y c ons t i t u t e d by t he s o c a l l e d C D 11b/ C D 45 hi ghl y br i ght c e l l s ( i e di s pl a yi ng b r i ght e r f l uo r e s c e nc e f or t he C D 45m a r ke r ) a r e a c t ua l l y m i c r ogl i a l c e l l s ( F or d G oods a l l e t a l 1 99 5) A c c e pt i ng t hi s i de a t he f i ndi ng t ha t H U C B C c a n r e duc e t he num be r o f m i c r ogl i a l c e l l s t ha t a r e a ugm e nt e d by t he s t r oke i s a r e s ul t i s of pa r t i c ul a r i nt e r e s t gi ve n t ha t m i c r o gl i os i s i s t hought t o t a ke pa r t i n ne ur ona l da m a ge not onl y i n c ons e que nc e of i s c he m i c i nj ur y but a l s o i n ot he r ne ur ode ge ne r a t i ve di s e a s e s ( S t r e i t W a l t e r e t a l 19 99) C or d bl ood m a y b l oc k t hi s

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108 phe nom e non, a nd t hi s e f f e c t m a y be r e l a t e d t o t he be ne f i c i a l ne ur obe ha vi or a l e f f e c t s t ha t c or d bl ood i s s how n t o pr o m ot e F ut u r e s t udi e s w i l l be di r e c t e d i n i nve s t i ga t i ng t hi s phe nom e non m or e c l os e l y. B c e l l s w e r e de t e c t e d i n hi ghe r a m ount i n t he M C A O br a i ns r e l a t i ve l y t o s ha m ope r a t e d r a t br a i ns a f i ndi ng t ha t i s un c om m on i n t he l i t e r a t ur e ( S t e ve ns B a o e t a l 2002) M or e ove r t he r ol e t ha t B c e l l s c oul d ha ve i n t he p a t hoge ne s i s of t he i s c he m i c br a i n i nj ur y i s c om pl e t e l y unknow n. A l t hough hum o r a l r e s pons e s ( a s B c e l l i m m une r e s pons e s r e pr e s e nt ) a r e ge ne r a l l y s e e n m uc h l a t e r a f t e r a n i n j ur y, a n e a r l y hu m or a l r e s pons e c oul d be e voke d a f t e r a br a i n i nj ur y a nd t he pr e s e nc e of B c e l l s w i t hi n t he b r a i n pa r e nc hym a c oul d r e pr e s e nt t he m i g r a t i on o f t he s e c e l l s t hr oug h t he di s r upt e d bl o od br a i n ba r r i e r ( B B B ) T he s ubs e que nt i nf us i on o f H U C B C de c r e a s e d t he num be r of B c e l l s t o p r e s t r oke l e ve l s but gi ve n t he a bove obs e r va t i ons t he s i gni f i c a nc e of t hi s f i ndi ng r e m a i ns unc l e a r H ow e ve r H U C B C m i ght pl a y a r o l e i n d e c r e a s i ng t he hum or a l r e s pons e s e e n a f t e r a b r a i n i ns ul t a nd t hi s p h e nom e non c oul d be r e f l e c t e d i n a de c r e a s e of B c e l l s i n t he br a i n. W he n T c e l l s w e r e a na l yz e d, a l t hough t he r e w a s a 2 f ol d i nc r e m e nt i nduc e d w i t h s t r oke s ur ge r y, no a ddi t i ona l va r i a t i ons w e r e obs e r ve d w h e n c or d bl ood w a s i nf us e d. I n s om e a ni m a l s t he pe r c e nt a ge of T c e l l s w a s i nc r e a s e d w i t h M C A O s ur ge r y, but t hi s phe nom e non w a s not obs e r ve d f or a l l a ni m a l s T c e l l s ha ve be e n s how n t o pos s e s s ne ur opr ot e c t i ve pr ope r t i e s i n t he i s c he m i c br a i n ( W ol f F i s he r e t a l 2002) bu t w he t he r c or d bl ood c e l l i nf us i on m a y m odul a t e t he i r e xpr e s s i on w i t hi n t he s t r oke br a i n i s not c l e a r F i na l l y, t he ne ut r oph i l i c popul a t i ons w e r e n ot f ound s i gni f i c a nt l y a l t e r e d by e i t he r

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109 s t r ok e s ur ge r y or t he s ubs e que nt i nj e c t i on of c e l l s F ur t he r r e s e a r c h m a y be di r e c t e d t o t he a na l ys i s of t he s a m e e ve nt s a t f u r t he r t i m e poi n t s T o s um m a r i z e of t he f l ow c yt om e t r y s t udi e s t he i n hi bi t i on of s t r oke i nduc e d up r e gul a t i on of C D 45/ C D 11b pos i t i ve c e l l s by i n f us i on of c o r d bl ood m a y be o f i nt e r e s t a s a put a t i ve m e c ha ni s m of a c t i on of t he H U C B C T hi s r e s ul t hol ds pa r t i c ul a r s i gni f i c a nc e gi ve n t ha t s uc h a i ndi r e c t m e c ha ni s m of a c t i on o f t he H U C B C i s s uppor t e d by t he pr e vi ous l y pr e s e nt e d i m m unohi s t o c he m i c a l f i ndi n gs of s c a r c e c e l l s w i t hi n t he br a i n pa r e nc hym a or w i t hi n t he c e r e br ova s c ul a t ur e T he i nve s t i ga t i ons of c yt oki ne pr oduc t i on i n t he br a i ns of s t r oke r a t s a nd of s t r oke r a t s t ha t r e c e i ve d t he H U C B C t r e a t m e nt a l s o p r ovi de d i nt e r e s t i ng r e s ul t s O ve r e xpr e s s i on of T N F a l pha bot h a t t he l e ve l of R N A s ynt he s i s a nd pr ot e i n pr oduc t i on w a s i nduc e d by t he i s c he m i c i ns ul t I nj e c t i on of c or d bl ood s i gni f i c a nt l y de pr e s s e d t he s e phe nom e na i nduc i ng a de c r e a s e i n R N A pr oduc t i on a nd a c onc om i t a nt de c r e a s e a l s o i n pr ot e i n pr oduc t i on. T he s e f i nd i ngs a r e of c ons i de r a bl e i nt e r e s t gi ve n t he r ol e t ha t i s a t t r i bu t a bl e t o T N F a l pha i n ne ur ona l de ge ne r a t i on ( S r e dni K e n i gs buc h 2002) C or d bl oo d m a y e xe r t i t s ne ur opr ot e c t i ve pr ope r t i e s i n t he i s c he m i c br a i n by m odul a t i ng t he e xpr e s s i on of T N F a l pha I n pa r t i c ul a r t hi s f i ndi ng i s c ons i s t e nt w i t h t he pr e vi ous f i ndi ng f r om t he f l ow c yt om e t r y a na l ys i s s ugge s t i ng t ha t t ha t H U C B C m i ght ha ve a n i nhi bi t or y a c t i on on t he s t r o ke i nduc e d m i c r ogl i os i s

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110 T he m R N A l e ve l o f T N F be t a w a s a l s o f ound t o be s i gni f i c a nt l y de c r e a s e d a f t e r H U C B C t r e a t m e nt T N F be t a ( a l s o t e r m e d l ym phot ox i n) i s a c yt oki ne t ha t ha s a l s o be e n de s c r i be d a s a n i m por t a nt i n f l a m m a t or y m e di a t or s i m i l a r t o T N F a l ph a ( E r m e r t P a nt a z i s e t a l 2003) A l t hough t he r e i s l i t t l e l i t e r a t ur e on t he r ol e of T N F be t a i n t he pa t hoge ne s i s of s t r oke i t s de pr e s s i on by H U C B C t r e a t e d M C A O r a t s i s i n a c c or da nc e w i t h t he e f f e c t s on T N F a l pha e xpr e s s i on. T he I L 2 m R N A a nd pr ot e i n l e ve l s w e r e s i gni f i c a n t l y upr e gul a t e d i n t he C N S a f t e r t he i s c he m i c i ns ul t H ow e ve r t he de c r e a s e d I L 2 m R N A e xpr e s s i on a f t e r t he c or d bl ood i nf us i on ( a s s e e n by R N A s e pr ot e c t i on a s s a y) w a s not m i r r or e d by a s i m i l a r de c r e a s e i n i t s pr ot e i n l e ve l s T hi s m i s m a t c h c a n be r e l a t e d t o di f f e r e nc e s i n t i m i ng be t w e e n m R N A t r a ns c r i pt i on a nd p r ot e i n t r a ns l a t i on, or m a y s i m pl y r e f l e c t c i r c ul a t i ng l e ve l s of I L 2 pr ot e i n f r om t he pe r i phe r y I t i s i m po r t a nt t o r e m e m be r t ha t a f t e r a n i s c he m i c i ns ul t b r a i n a nd pe r i phe r a l s ys t e m s e nga ge i n a c om pl e x i nt e r pl a y m e di a t e d by c yt oki ne s c he m oki ne s a nd ne ur ot r a ns m i t t e r s ( W oi c i e c how s ky, S c honi ng e t a l 1 999) I L 2 m a y be i nvol ve d i n s uc h a phe nom e non e i t he r m a ni f e s t i ng i t s pr o i nf l a m m a t or y pr ope r t i e s or s i m pl y or c he s t r a t i ng t he di a l ogue be t w e e n br a i n c e l l s a nd i m m une pe r i phe r a l a c t or s dur i ng t he pos t i s c he m i c s ys t e m i c i nf l a m m a t or y r e a c t i on. W hi l e t he r e w a s a t e nde nc y i n bot h pr ot e i n l e ve l s a nd m R N A l e ve l s of I L 6 t o be upr e gul a t e d i n t he s t r oke br a i ns ; a na l ys i s r e ve a l e d t ha t t hi s e f f e c t di d not r e a c h s i gni f i c a nc e A nt i i nf l a m m a t or y c yt oki ne s w e r e a l s o e xa m i ne d. M e s s e nge r l e ve l s of I L 10 a nd I L 4 w e r e s i gni f i c a nt l y r e duc e d a f t e r s t r oke s ur ge r y. V e r y l i ke l y t hi s dr a m a t i c de c r e a s e r e pr e s e nt s t he de pr e s s i on of t he a nt i i nf l a m m a t or y r e a c t i on t ha t t a ke s pl a c e c onc om i t a nt l y

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111 t o t he a c t i va t i on o f t he pr o i nf l a m m a t or y r e s pons e I nt e r e s t i ngl y, p r ot e i n l e ve l s o f I L 10 w e r e hi ghe r i n s t r oke a ni m a l s c om pa r e d t o s ha m a ni m a l s W hi l e t hi s e f f e c t di d not r e a c h s t a t i s t i c a l s i gni f i c a nc e i t m a y r e f l e c t a s i m i l a r phe nom e non t o t ha t obs e r ve d w i t h t he m i s m a t c h i n I L 2 m R N A a nd pr ot e i n l e ve l s I t i s w e l l doc um e nt e d t ha t a f t e r a b r a i n i s c he m i c e ve nt I L 10 i s r a pi dl y pr oduc e d by pe r i p he r a l c e l l s ( W oi c i e c how s ky, S c honi ng e t a l 1999 ) a nd t hi s c i r c ul a t i ng I L 10 r e a c he s t he br a i n. G i ve n t hi s t he hi gh l e ve l s of I L 10 w e de t e c t e d w i t hi n t he br a i n m a y be a c t ua l l y I L 10 pr oduc e d i n t he pe r i phe r y; s uppor t i ng t hi s i de a w e f ound t ha t I L 10 m R N A l e ve l s i n t he br a i n di d no t pa r a l l e l t hos e I L 10 pr ot e i n e xpr e s s i on. A f t e r t he i nj e c t i on o f H U C B C m R N A l e ve l s of bot h I L 10 a nd I L 4 w e r e i nc r e a s e d c om pa r e d t o M C A O r a t b r a i ns a l t hough not s i gni f i c a nt l y H U C B C t r a ns pl a nt a t i on m a y be pr om ot i ng t he a nt i i nf l a m m a t or y r e s pons e i n t he b r a i n, w hi l e i n pa r a l l e l i nduc i ng a de pr e s s i on of t he p r o i nf l a m m a t or y phe nom e na T a ke n t oge t he r t he s e obs e r va t i ons s ugge s t t ha t f ol l ow i ng t he H U C B C i nt r a ve nous t r a ns pl a nt t he r e i s a bl oc ka ge of t he b r a i n pr o i nf l a m m a t or y e ve nt s i nduc e d by t he i s c he m i c i ns ul t T hi s bl oc ka ge m a y r e s ul t i n hi s t ol ogi c a l a nd f ur t he r be ha vi or a l be ne f i t s t ha t w e a nd ot he r s ha ve obs e r ve d.

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112 C H A P T E R S I X C O N C L U D I N G R E M A R K S T he f i r s t t i m e c o r d bl ood c e l l s w e r e us e d i n a ni m a l m ode l s of ne u r ode ge ne r a t i ve di s e a s e s t ook pl a c e f our ye a r s a go, w he n i nt r a ve nous l y i nj e c t e d H U C B C w e r e s how n t o pr ol ong l i f e s pa n i n m ode l s o f P a r ki ns on s A l z he i m e r s a nd H ungt i ngt on s di s e a s e ( E nde a nd C he n 2001; E nde C he n e t a l 2001; E nde a nd C he n 2002) W hi l e t he s e pr e l i m i na r y s t udi e s s how e d a n ove r a l l non s pe c i f i c e f f e c t o f H U C B C a c r os s t he s e m ode l s f ur t he r r e s e a r c h de m ons t r a t e d t ha t i nt r a ve nous l y de l i ve r e d c or d bl oo d c e l l s w e r e a bl e t o s pe c i f i c a l l y a m e l i or a t e ne ur obe ha vi or a l de f i c i t s a s s oc i a t e d w i t h br a i n i s c he m i a ( C he n, S a nbe r g e t a l 2001; W i l l i ng, L i x i a n e t a l 2003 ) T he f i r s t r e por t i n a r ode nt s t r oke m ode l s how e d t ha t 1, 000, 000 c e l l s t r a ns pl a nt e d i nt r a ve no us l y 24 hour s a f t e r t he i nduc t i on of t he s t r oke i nduc e d a c ons i de r a bl e r e duc t i on of t he s t r o ke i nduc e d i m pa i r m e nt s i n m ot or c oor di na t i on t e s t s ( s uc h a s t he r ot a r od t e s t ) a nd a m e l i or a t e d t he m ot o r s e ns or y pa r a m e t e r s e va l ua t e d w i t h t he m N S S s c a l e ( C he n, S a nbe r g e t a l 2001) T he s a m e i nve s t i ga t or s s how e d t ha t i nj e c t e d c e l l s w e r e de t e c t e d w i t hi n t he br a i n pa r e nc hym a a s f a r a s 14 da ys a f t e r t he t r a ns pl a nt F ur t he r m or e m i gr a t i on s t udi e s s uppor t e d t he hypot he s i s t ha t t he s e c e l l s pos s e s s t r opi c pr ope r t i e s a nd m i g r a t e d t o t he i nj ur e d br a i n ( C he n, S a nbe r g e t a l 2001) T he i nt r i gui ng que s t i on t ha t r e m a i ne d a f t e r t hi s s t udy w a s w he t he r t he s e c e l l s w e r e c a pa bl e of i nduc i ng t he s e e f f e c t s by di f f e r e nt i a t i ng i nt o ne u r a l or gl i a l c e l l t ype s o r

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113 w he t he r t he i r e f f e c t w a s r e l a t e d t o ot he r m e c ha ni s m s out s i de ne ur a l di f f e r e nt i a t i on. S ubs e que nt s t udi e s be ga n t o poi nt t o ot he r m e c ha ni s m s I t w a s s ur pr i s i ng t o di s c ove r t ha t w he t he r H U C B C w e r e de l i ve r e d di r e c t l y i nt o t he c or pus s t r i a t um or i nf us e d vi a t he f e m or a l ve i n t he ne ur obe ha vi or a l be ne f i t a s s e s s e d w i t h t e s t s of s pont a ne ous a c t i vi t y or m ot or a s ym m e t r y w a s e qua l ( W i l l i ng L i xi a n e t a l 2003) T he i nve s t i ga t or s poi nt e d out t ha t i n s om e t e s t s t he i m p r ove d m ot o r f unc t i on i nd uc e d by i nt r a ve nous de l i ve r y w a s e ve n be t t e r t ha n t ha t i nduc e d w i t h i nt r a c e r e br a l t r a ns pl a nt ( W i l l i ng L i xi a n e t a l 2003) H ow e ve r t he c e l l s w e r e no t de t e c t e d w i t hi n t he b r a i n pa r e nc hym a f ol l o w i ng e i t he r o f t he t w o t r a ns pl a nt m oda l i t i e s l e a di ng t he i nve s t i ga t or s t o hypot he s i z e t ha t H U C B C c oul d e xe r t t he i r be ne f i c i a l a c t i on t hough i ndi r e c t m e c ha ni s m s on t he br a i n 6. 1 N e u r ob e h avi or al an d N e u r op a t h ol ogi c al b e n e f i t s i n d u c e d b y H U C B C i n f u s i on i n t h e M C A O an i m al m od e l o f s t r ok e T he f i r s t i m por t a nt f i ndi ng of t he s t udi e s r e por t e d he r e i n i s t ha t H U C B C c a n c ont r ol t he ne ur obe ha vi or a l de f i c i t s i nduc e d by br a i n i s c he m i a i n a m a nne r t ha t i s de pe nde nt on t he num be r of c e l l s de l i ve r e d. A t l e a s t 1 m i l l i on c e l l s a r e ne c e s s a r y t o a c hi e ve a s t a bl e r e s c ue of de f i c i t s W he n 10 m i l l i on c e l l s a r e e m pl oye d, a n e ve n be t t e r e f f e c t c a n be a c hi e ve d. H ow e ve r pa r t i c ul a r l y t he s pont a ne ous a c t i vi t y m e a s ur e m e nt r e ve a l e d t ha t hi ghe r c e l l s dos e s s uc h a s c e l l a m ount s i n t h e r a nge of 30 t o 5 0 m i l l i on do not i nduc e a f ur t he r r e c ove r y. N ot onl y w e r e t he r e s i gni f i c a nt i m p r ove m e nt s i n be ha vi or but c o r d bl ood i nj e c t i on a l s o i nduc e d hi s t opa t hol ogi c a l r e c ove r y i n t he i s c he m i c br a i n; t hi s ha d not be e n r e por t e d be f or e M or e ove r w he n 10 m i l l i on H U C B C w e r e i nf us e d 24 hour s a f t e r t he

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114 s t r oke s ur ge r y, t he l e s i on vol um e w a s s i gni f i c a nt l y r e duc e d a s c om pa r e d t o t he non t r e a t e d s t r oke d a ni m a l s ; w he r e a s a ni m a l s t r e a t e d w i t h l e s s t ha n 1 m i l l i on c e l l s w hi l e s how i ng s om e r e duc t i on i n da m a ge ( a s e vi de nc e d by c yt oa r c hi t e c t ur e s pa r i ng a nd l e s s pr e s e nc e of l a c una r s pa c e s ) T he s e a m e l i or a t i ons how e ve r di d not t r a ns l a t e i nt o a s i gni f i c a nt r e duc t i on i n vol um e o f da m a ge G i ve n a l l t he H U C B C dos e s e m pl oye d, onl y t he t w o hi ghe s t dos e s of H U C B C w e r e f ound t o i nduc e bot h be ha vi or a l be ne f i t a nd i nf a r c t vol um e r e duc t i on O ne i m po r t a nt obs e r va t i on r e ga r ds t he m i s m a t c h be t w e e n t he be ha vi or a l be ne f i t a nd i nf a r c t vol um e r e duc t i on w he n dos e s of 1 m i l l i on c e l l s a r e us e d. W hi l e t hi s dos e c a n i nduc e a s i gni f i c a nt be ne f i t i n t e r m s of s pont a ne ous a c t i vi t y m e a s ur e m e nt s a nd m ot or a bi l i t i e s t he s a m e dos e c a nnot i nduc e a r e duc t i on of l e s i on vo l um e O ne e xpl a na t i on i s t ha t a t t hi s dos e H U C B C t he r e m a y not be s uf f i c i e nt e f f e c t or c e l l s t o pr ovi de pr ot e c t i on t o s i g ni f i c a nt l y a m e l i or a t e i s c he m i c da m a ge A s a l s o poi n t e d i n C ha pt e r 1, t he s e r e s ul t s de m ons t r a t e s t he i m por t a nc e of us i ng a ba t t e r y of t e s t s t o e xa m i ne f unc t i ona l out c om e s i n t he s e a ni m a l m ode l s of i s c he m i a I nde e d, t he M C A O m ode l i s c ha r a c t e r i z e d by a n i n t r i ns i c va r i a bi l i t y ( i e l e s i ons c a n ha ve di f f e r e nt di s t r i but i on due t o v a r i a bi l i t y of t he m i c r ova s c ul a r s t r uc t ur e s a c r os s t he a ni m a l s ) a nd by t he us e of m u l t i pl e m e a s ur e s of f unc t i ona l out c om e s de f i c i t s r e l a t e d t o l e s i ons i n di f f e r e nt l o c a t i ons a r e pi c k e d up by di f f e r e nt be ha vi or a l t e s t s

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115 6. 2 Wh at i s t h e m e c h an i s m b y w h i c h H U C B C e xe r t t h e i r b e n e f i c i al e f f e c t s ? T he s e f i ndi ngs l e d us t o i nve s t i ga t e t he m e c ha ni s m s t hr ough w hi c h H U C B C m a y i nduc e f unc t i ona l a nd s t r uc t ur a l a m e l i or a t i ons i n t he M C A O r a t m o de l of i s c he m i c br a i n i nj u r y. T he f i r s t a ppr oa c h w a s t o e xa m i ne w he r e i nt r a ve nous l y i nj e c t e d c e l l s w e r e l oc a t e d w i t hi n t he body a t one m ont h a f t e r t he t r a ns pl a nt T he m a i n que s t i on w a s w he t he r H U C B C c oul d t a r ge t a s pe c i f i c o r ga n a nd m i gr a t e t o i t or w he t he r t he y a r e di s pe r s e d ( vi a t he va s c ul a t ur e ) non s pe c i f i c a l l y t hr oughout t he body O ne m ont h a f t e r t he t r a ns pl a nt c e l l s w e r e m a i nl y l oc a t e d w i t hi n t he i n j ur e d s i de o f t he br a i n, a nd i n t he s pl e e n. B ot h t he s e f i ndi ngs a r e of r e l e va nc e F i r s t t he f a c t t ha t H U C B C m a y r e a c h t he i nj u r e d br a i n s uppor t s pr e vi ous t he or i e s of t r opi c p r ope r t i e s i f H U C B C f or t he i nj u r e d t i s s ue ( C he n, S a nbe r g e t a l 2001) H U C B C m a y be a t t r a c t e d t o t he s i t e of t he l e s i on by c he m oki ne s a nd s pe c i f i c a l l y e xe r t t he i r a c t i on i n s i t u W i t hi n t h e br a i n, t he H U C B C s e e m e d t o be m a i nl y l oc a l i z e d a t t he l e ve l of t he c e r e br ova s c ul a t ur e T hi s f i ndi ng m a y i ndi c a t e t ha t H U C B C c a n a c t w i t hout be c om i ng i nt e gr a t e d i n t h e pa r e nc hym a a n i de a i n di r e c t c ont r a s t t o t he o r i gi na l hypot he s i s H U C B C pe r f or m e d c e l l r e pl a c e m e nt R e s ul t s f r om t he f i r s t w or k e m pl oyi ng H U C B C i n a s t r oke m ode l ( C he n S a nbe r g e t a l 2001) s uppor t e d t he i de a t ha t H U C B C m a y i nt e gr a t e a nd s ubs t i t ut e f or l os t o r dyi ng ne ur ons w i t hi n t he br a i n pa r e nc hym a I nt r a ve nous l y t r a ns pl a nt e d H U C B C w e r e f ound i n t he C N S a nd i m m unohi s t oc he m i c a l a na l ys i s s how s t he e xpr e s s i on of ne u r a l m a r ke r s a nd gl i a l m a r ke r s ( s uc h a s M A P 2 a nd G F A P r e s pe c t i ve l y) by t he t r a ns pl a nt e d c e l l s ( C he n, S a nbe r g e t a l 2001) S uppor t f o r f unc t i ona l di f f e r e nt i a t i on of H U C B C i nt o C N S c e l l t ype s ha s be e n s ugge s t e d by t he w or k of i nve s t i ga t or s i n ot he r a ni m a l m ode l s of b r a i n i nj u r y ( L u S a nbe r g e t a l 2002) A l t hough t he s e i ni t i a l obs e r v a t i ons s e e m i n c ont r a s t w i t h ou r

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116 f i ndi ngs t he e xpr e s s i on of ne ur a l m a r ke r s doe s not ne c e s s a r i l y m e a n t ha t H U C B C a r e unde r goi ng a pr oc e s s of t r a ns di f f e r e nt i a t i on a nd t h e r e f or e doe s not de m ons t r a t e t ha t t he s e c e l l s onc e i n s i t u c a n r e s t or e b r a i n f unc t i on b y be c om i ng ne w bor n ne u r a l or gl i a l c e l l s T he ot he r t a r ge t o r ga n w a s t he s pl e e n. H e r e a l s o t he H U C B C s e e m e d t o be f ound w i t hi n or ne a r t he va s c ul a t ur e T he p r e s e nc e of t r a ns pl a nt e d c e l l s i n t he s pl e e n c oul d be e vi de nc e of t he s pl e e n r e m ovi ng de a d or f or e i gn c e l l s out of t he va s c ul a t ur e ( a s i s t he c a s e f or s pe nt r e d bl ood c e l l s ) O n t he ot he r ha nd, s i nc e c or d bl ood i s c om pos e d m a i nl y of i m m a t ur e bl ood c e l l s ( T l ym phoc yt e s a nd m ono c yt e s ) t he s pl e e n m a y r e pr e s e nt a n a c t ua l hom i ng s i t e f o r t he H U C B C T hi s i de a i s i n a gr e e m e nt w i t h p r e vi ous f i ndi ngs o f i nve s t i ga t or s t ha t ha ve e m pl oye d H U C B C i n t he a m yot r ophi c l a t e r a l s c l e r os i s m ous e m ode l ( G a r buz ova D a vi s W i l l i ng e t a l 2003 ) I n t hi s s t udy num e r ous c e l l s w e r e f ound i n t he s pl e e n, a nd i nve s t i ga t or s hypot he s i z e d t ha t t he s pl e e n m a y be t he m a i n t a r ge t of i nt r a ve nous l y i nj e c t e d c or d bl ood c e l l s F i ndi ng H U C B C i n t he s pl e e n l e nds c r e de nc e t o t h e hypot he s i s t ha t t he i m m une / i nf l a m m a t or y r e s pons e t ha t f ol l ow s a br a i n i s c he m i c i ns ul t m a y be m odul a t e d by our c e l l t he r a py T hi s i de a a s out l i ne d i n de t a i l i n t he pr e vi ous c ha pt e r s w a s t ha t t he r e m i ght be a pe r i phe r a l a nd c e nt r a l e f f e c t i n duc e d by c or d bl ood t ha t c oul d e xpl a i n t he f unc t i ona l be ne f i t s e e n i n t hi s a nd pr e vi ous s t udi e s W he n w e e xa m i ne d t he e f f e c t o f s t r oke a nd s ubs e que nt H U C B C de l i ve r y on t he r a t s pl e e n, w e obt a i ne d ve r y s t r i ki ng r e s ul t s C or d bl ood i n f us i on r e s c ue d t he s pl e e n w e i ght l os s t ha t oc c ur r e d i n r e s pons e t o t he s t r oke i nj ur y a nd c onc om i t a nt l y r e i ns t a t e d t he nor m a l C D 4/ C D 8 phe not ype p r of i l e t ha t ha d be e n a l t e r e d f ol l ow i ng M C A O P e r ha ps m or e i m por t a nt l y, t he r e duc t i on of

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117 i s c he m i c vol um e c or r e l a t e d i nve r s e l y w i t h t he s pl e e n w e i ght i ndi c a t i ng t ha t t he e f f e c t of H U C B C on t he s pl e e n m a y be of pa t hoge ni c r e l e v a nc e f or t he r e s c ue of t he br a i n hi s t ol ogi c a l l e s i on. F ur t he r m o r e s pl e ni c T c e l l f u nc t i on c ha nge d a f t e r s t r oke a nd a f t e r t r a ns pl a nt a t i on, l e a di ng t o de c r e a s e d p r oduc t i on o f I F N ga m m a a nd i nduc i ng e xpr e s s i on of I L 10 S uc h r e s ul t s c a n be i nt e r pr e t e d i n t he f r a m e of pa s t s t udi e s t ha t ha ve s how n t ha t T c e l l s c a n ha ve a ne u r opr ot e c t i ve e f f e c t ( W ol f F i s he r e t a l 2002; B e c ke r K i ndr i c k e t a l 2003) O ne hypot he s i s m a y be t ha t H U C B C i nduc e t he e ndoge nous T c e l l s t o pr om ot e s ur vi va l of ne ur a l c e l l s but t hi s hypot he s i s a s now r e m a i ns t o be t e s t e d. A not he r a ppr oa c h t ha t w e us e d t o i nve s t i ga t e t he p os s i bl e r ol e of H U C B C i n t he m odul a t i on of t he e ndoge nous pos t s t r oke i m m une / i nf l a m m a t or y a l t e r a t i ons w a s t o e xa m i ne w he t he r H U C B C i nf l ue nc e d t he c e l l ul a r a nd m ol e c ul a r phe nom e na w i t hi n t he br a i n a f t e r s t r oke H U C B C bl oc ke d t he a c ut e i nf i l t r a t i on of i nf l a m m a t o r y c e l l s s uc h a s m i c r ogl i a a nd B c e l l s i n t o t he s t r oke br a i n A t t he s a m e t i m e m R N A a nd p r ot e i n l e ve l s of T N F a l pha w e r e a b r oga t e d by t he i nf us i on o f c o r d bl ood c e l l s w hi l e p r ot e i n o r R N A a m ount s of I L 1be t a o r I L 2 w e r e a l s o de c r e a s e d. T he s e r e s ul t s s t r ongl y s ugge s t t ha t H U C B C m odul a t e e ndoge nous e ve nt s t ha t oc c ur i n t he br a i n a f t e r i s c he m i a A ddi t i ona l l y t he l e ve l s of a nt i i nf l a m m a t or y c yt oki ne s t e nde d t o be hi ghe r a f t e r H U C B C t r e a t m e nt A l t hough t hi s e f f e c t di d not r e a c h s i gni f i c a nc e t hi s phe nom e non c oul d r e pr e s e nt t he e f f e c t of c or d bl ood on t he i nhi bi t i on of pr o i nf l a m m a t or y c yt oki ne s ( s uc h a s T N F a l pha I L 1be t a a nd I L 2) a nd t he s i m ul t a ne ous i nduc t i on of e xpr e s s i on of a nt i i nf l a m m a t o r y m ol e c ul e s T he s e m ol e c ul a r phe nom e nona a r e qui t e t r a ns i e nt a nd t he r e f or e ge ne r a l l y di f f i c ul t t o obs e r ve a t a s i ngl e t i m e poi nt F ut ur e s t udi e s s houl d be di r e c t e d i n f ol l ow i ng t he s e e ve nt s a c r os s t i m e

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118 T a ki ng a l l t he s e f i ndi ng t oge t he r a pos s i bl e m e c ha ni s m by w hi c h c or d bl ood c e l l s ha ve be e n r e pe a t e dl y r e por t e d t o pr o m ot e f unc t i ona l r e c ove r y f r om i s c he m i c i nj ur y m a y be un c ove r e d. F i r s t l y H U C B C m odul a t e s t he s t r oke i nd uc e d i nf l a m m a t or y r e s pons e by bl oc ki ng t he pr oduc t i on of pr o i nf l a m m a t or y c yt ok i ne s a nd by r e duc i n g t he ove r e xpr e s s i on of pr o i n f l a m m a t or y c e l l s w i t hi n t he i s c he m i c br a i n. S e c ondl y, H U C B C ha ve a pe r i phe r a l e f f e c t on t he s pl e e n by r e ve r t i ng t he a l t e r a t i ons on s pl e noc yt e s phe not ype a nd f unc t i on t ha t a r e i nduc e d by s t r oke T he i nt r a c e l l ul a r e ve nt s m e di a t i ng t he s e phe nom e na r e m a i n unknow n, but f ut ur e s t udi e s w i l l be di r e c t e d t o t he unde r s t a ndi ng of t he m ol e c ul a r pa t hw a ys t ha t a r e a c t i va t e d/ s uppr e s s e d dur i ng t he s e e ve nt s F or i ns t a nc e t he t r a ns c r i pt i on f a c t or N F kB t ha t i s i nvol ve d i n t he a c t i va t i on of p r o i n f l a m m a t or y c yt oki ne s s uc h a s T N F a l pha w i l l be s ubj e c t t o a na l ys i s 6. 3 C on c l u s i on C or d bl ood ha s r e c e nt l y e m e r ge d a s a t he r a pe ut i c opt i on f or ne ur ode ge ne r a t i ve di s e a s e s i nc l udi ng s t r oke A s a r e s ul t o f i t s e a s y a va i l a bi l i t y i t s l ow i m m unoge ni c p r o pe r t i e s a nd t he l ow r i s k o f vi r a l t r a ns m i s s i on a s s oc i a t e d w i t h i t s us e i t r e p r e s e nt s a n a t t r a c t i ve t ool f or f ut ur e t he r a pi e s I n a ddi t i on, t he i n t r a ve nous de l i ve r y of t he s e c e l l s m a ke s t hi s t he r a pe ut i c a ppr oa c h e a s i l y a c c e s s i bl e t o m a ny pa t i e nt s D e s pi t e t he s uc c e s s e s i n de m ons t r a t i ng t he be ne f i t of c or d bl ood c e l l s w he n a ppl i e d i nt o s t r oke m ode l s t he m e c ha ni s m s unde r l yi ng i t s e f f e c t ha d not be e n c ha r a c t e r i z e d pr i or t o t he s t udi e s de s c r i be d i n t he di s s e r t a t i on. T hi s w or k ha s pr ovi de d e xt e ns i ve pr og r e s s i n a ddr e s s i ng t hi s bi g que s t i on, f or m i ng a f ounda t i on f or f ur t he r r e s e a r c h.

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119 B e f or e a ppl yi ng t hi s c or d bl ood ba s e d t he r a py i n t he c l i ni c f ur t he r i s s ue s w i l l ne e d t o be a ddr e s s e d. H e r e w e a ddr e s s e d t he dos e of c e l l s ne c e s s a r y t o obt a i n a n opt i m a l be ne f i t O ur be s t dos e ( 10, 000, 000 c e l l s ) t r a ns l a t e s i nt o a l m os t 20 c or ds f or a t r a ns pl a nt i nt o a n or ga ni s m of t he hum a n s i z e I n v i t r o e xpa ns i on t e c hni que s m a y s e r ve a s a t ool t o ove r c om e t hi s i s s ue T he t i m e w i ndow s t i l l r e m a i ns t o be a ddr e s s e d, a l t hough a l l t hi s w or k ha s be e n c onduc t e d by t r a ns pl a nt i ng H U C B C 24 hour s t he s t r oke a s pr e l i m i na r y s t udi e s c onduc t e d i n our l a bor a t or y ha d s how n t ha t t hi s m i ght be t he opt i m a l t i m e t o de l i ve r y. F i na l l y i t i s ve r y i m por t a nt t o t a ke i nt o a c c ount t ha t a l t hough e xpe r i m e nt a l r e s ul t s a r e ve r y pr om i s i ng, t he e f f e c t i ve ne s s of t he s e t r a ns pl a nt s i n hum a ns a r e unknow n. T he e f f i c a c y of t he t r a ns pl a nt w i l l be obvi ous l y i n f l ue nc e d by f a c t or s s uc h a s t he s e ve r i t y, s t a ge a nd s i t e of t he pa t hol ogy of t he ne ur ode ge ne r a t i ve di s or de r a f f e c t i ng t he r e c i pi e nt F or i ns t a nc e t he l oc a t i on a nd t he e xt e nt of t he ne ur ona l da m a ge r e pr e s e nt f a c t or s t ha t ne e d t o be a s s e s s e d be f or e t he t r a ns pl a nt I m por t a nt l y, t he r e w i l l be t he ne e d t o e s t a bl i s h c r i t e r i a ba s e d on c l i ni c a l a nd p a t hol ogi c a l pa r a m e t e r s i n or de r t o s c r e e n w hi c h pa t i e nt s w i l l be good f or t r a ns pl a nt a t i on.

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120 R E F E R E N C E S A boody, K S A B r ow n e t a l ( 2000) N e ur a l s t e m c e l l s di s pl a y e xt e ns i ve t r opi s m f o r pa t hol ogy i n a dul t br a i n: e vi de nc e f r om i nt r a c r a ni a l g l i om a s P r oc N a t l A c a d S c i U S A 97 ( 23) : 12846 51. A H A ( 2004) M a j or ongoi ng s t r oke t r i a l s ( A m e r i c a n H e a r t A s s oc i a t i on) S t r oke 3 5 ( 6) : e 163 73. A z i z i S A D S t oke s e t a l ( 1998) E ngr a f t m e nt a nd m i gr a t i on of hum a n bone m a r r ow s t r om a l c e l l s i m pl a nt e d i n t he br a i ns o f a l bi no r a t s s i m i l a r i t i e s t o a s t r oc yt e g r a f t s P r oc N a t l A c a d S c i U S A 95 ( 7) : 3908 13 B a r ke r J N P L M a r t i n, e t a l ( 2001 ) L ow i nc i de nc e of E ps t e i n B a r r vi r us a s s oc i a t e d pos t t r a ns pl a nt a t i on l ym phopr ol i f e r a t i ve di s or de r s i n 27 2 unr e l a t e d dono r um bi l i c a l c or d bl ood t r a ns pl a nt r e c i pi e nt s B i ol B l ood M a r r ow T r a ns pl a nt 7 ( 7) : 395 9. B e c ke r K D K i ndr i c k e t a l ( 2003) A dopt i ve t r a ns f e r of m ye l i n ba s i c pr ot e i n t ol e r i z e d s pl e noc yt e s t o na i ve a ni m a l s r e duc e s i nf a r c t s i z e : a r ol e f or l ym phoc yt e s i n i s c he m i c br a i n i nj ur y? S t r oke 34 ( 7) : 1809 15. B e c ke r K J R M M c C a r r on, e t a l ( 1997) I m m unol ogi c t ol e r a nc e t o m ye l i n ba s i c pr ot e i n de c r e a s e s s t r oke s i z e a f t e r t r a ns i e nt f oc a l c e r e br a l i s c he m i a P r oc N a t l A c a d S c i U S A 94 ( 20) : 10873 8

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121 B e l a ye v, L O F A l ons o, e t a l ( 1996) M i ddl e c e r e br a l a r t e r y oc c l us i on i n t he r a t by i nt r a l um i na l s ut ur e N e ur ol ogi c a l a nd pa t hol ogi c a l e va l ua t i on of a n i m pr ove d m ode l S t r oke 27 ( 9 ) : 1616 22; di s c us s i on 1623. B e l a ye v, L L K hout or ova e t a l ( 2003) N e ur op r ot e c t i ve e f f e c t o f S ol C D 39 a nove l pl a t e l e t a ggr e ga t i on i nhi bi t o r on t r a ns i e nt m i ddl e c e r e br a l a r t e r y oc c l us i on i n r a t s S t r oke 34 ( 3 ) : 758 63. B e r ge r C W R S c ha bi t z e t a l ( 2004 ) H ypot he r m i a a nd b r a i n de r i ve d ne u r ot r ophi c f a c t or r e duc e gl ut a m a t e s yne r gi s t i c a l l y i n a c ut e s t r oke E xp N e ur o l 185 ( 2 ) : 305 12. B e r ge r C F X i a e t a l ( 2004 ) H i gh dos e a s pi r i n i s ne ur opr ot e c t i ve i n a r a t f oc a l i s c he m i a m ode l B r a i n R e s 998 ( 2) : 237 42 B i c kne s e A R H S G oodw i n, e t a l ( 2002 ) H um a n um bi l i c a l c or d bl ood c e l l s c a n be i nduc e d t o e xpr e s s m a r ke r s f o r ne ur ons a nd gl i a C e l l T r a ns pl a nt 11 ( 3 ) : 261 4 B or l onga n, C V C E S t a hl e t a l ( 1999) C yc l o s por i ne A i nduc e d hype r a c t i vi t y i n r a t s : i s i t m e di a t e d by i m m unos uppr e s s i on, ne u r ot r ophi s m or bot h? C e l l T r a ns pl a nt 8 ( 1) : 153 9. B or l onga n, C V Y T a j i m a e t a l ( 1998) T r a ns pl a nt a t i on of c r yopr e s e r ve d hum a n e m br yona l c a r c i nom a de r i ve d ne u r ons ( N T 2N c e l l s ) pr om ot e s f unc t i ona l r e c ove r y i n i s c he m i c r a t s E xp N e ur ol 149 ( 2 ) : 310 21. B r a z e l t on, T R F M R os s i e t a l ( 2000) F r om m a r r ow t o br a i n: e xpr e s s i on of ne ur ona l phe not ype s i n a dul t m i c e S c i e nc e 290 ( 5 497) : 1775 9 B ut t i ni M K A ppe l e t a l ( 1996) E xpr e s s i on of t um or ne c r os i s f a c t or a l pha a f t e r f oc a l c e r e br a l i s c h a e m i a i n t he r a t N e ur os c i e nc e 71 ( 1) : 1 16.

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122 C a m pa ne l l a M C S c i or a t i e t a l ( 2002 ) F l ow c yt om e t r i c a na l ys i s of i n f l a m m a t or y c e l l s i n i s c he m i c r a t br a i n S t r oke 33 ( 2 ) : 586 92. C he n, H M C hopp e t a l ( 1994) A nt i C D 11b m onoc l ona l a nt i body r e duc e s i s c he m i c c e l l da m a ge a f t e r t r a ns i e nt f oc a l c e r e br a l i s c he m i a i n r a t A nn N e ur ol 35 ( 4) : 458 63 C he n, J Y L i e t a l ( 2001) T he r a pe ut i c be ne f i t of i nt r a c e r e br a l t r a ns pl a nt a t i on o f bone m a r r ow s t r om a l c e l l s a f t e r c e r e b r a l i s c he m i a i n r a t s J N e ur ol S c i 189 ( 1 2) : 49 57 C he n, J P R S a nbe r g, e t a l ( 2001 ) I nt r a ve nous a dm i ni s t r a t i on o f hum a n u m bi l i c a l c or d bl ood r e duc e s be ha vi or a l de f i c i t s a f t e r s t r oke i n r a t s S t r oke 32 ( 11 ) : 2682 8. C he n, J Z G Z ha ng e t a l ( 2003) I nt r a ve nous a dm i ni s t r a t i on of hum a n bone m a r r ow s t r om a l c e l l s i nduc e s a ngi oge ne s i s i n t he i s c he m i c bounda r y z one a f t e r s t r oke i n r a t s C i r c R e s 92 ( 6) : 692 9. C he n, R a nd N E nde ( 2000 ) T he pot e nt i a l f or t h e us e of m ononuc l e a r c e l l s f r om hum a n um bi l i c a l c or d bl ood i n t he t r e a t m e nt o f a m yot r ophi c l a t e r a l s c l e r os i s i n S O D 1 m i c e J M e d 31 ( 1 2) : 21 30 C he n, X M K a t a kow s ki e t a l ( 2002) H um a n b one m a r r ow s t r om a l c e l l c ul t u r e s c ondi t i one d by t r a um a t i c br a i n t i s s ue e xt r a c t s : gr o w t h f a c t or p r oduc t i on. J N e ur os c i R e s 69 ( 5) : 687 91. C he n, Y C R ue t z l e r e t a l ( 2003) M uc os a l t ol e r a nc e t o E s e l e c t i n pr ov i de s c e l l m e di a t e d pr ot e c t i on a ga i ns t i s c he m i c br a i n i nj ur y P r oc N a t l A c a d S c i U S A 100 ( 25 ) : 15107 12. C he ng, B S C hr i s t a kos e t a l ( 1994) T um o r ne c r os i s f a c t or s pr ot e c t ne ur ons a ga i ns t m e t a bol i c e xc i t ot oxi c i ns ul t s a nd pr om ot e m a i nt e n a nc e of c a l c i um hom e os t a s i s N e ur on 12 ( 1) : 139 53.

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123 C ol e D J P M P a t e l e t a l ( 1993) T e m po r a r y f oc a l c e r e br a l i s c he m i a i n s pont a ne ous l y hype r t e ns i ve r a t s : t he e f f e c t of i bup r of e n on i nf a r c t vol um e J P ha r m a c ol E xp T he r 266 ( 3 ) : 1713 7. D e G r a ba T J ( 1998) T he r ol e o f i n f l a m m a t i on a f t e r a c ut e s t r oke : ut i l i t y of pu r s ui ng a nt i a dhe s i on m ol e c ul e t he r a py. N e ur ol ogy 51 ( 3 S uppl 3) : S 62 8 de l Z oppo, G J K J B e c ke r e t a l ( 2001 ) I nf l a m m a t i on a f t e r s t r oke : i s i t ha r m f ul ? A r c h N e ur ol 58 ( 4) : 669 72. D i a r r a A C S he l don, e t a l ( 1999 ) A noxi a e voke d i nt r a c e l l ul a r pH a nd C a 2+ c onc e nt r a t i on c ha nge s i n c ul t ur e d pos t na t a l r a t hi p poc a m pa l ne ur ons N e ur os c i e nc e 93 ( 3) : 1003 16. D i r n a gl U C I a de c ol a e t a l ( 1999) P a t hobi ol o gy of i s c ha e m i c s t r oke : a n i nt e gr a t e d vi e w T r e nds N e ur os c i 22 ( 9 ) : 391 7. E gl i t i s M A a nd E M e z e y ( 1997) H e m a t opoi e t i c c e l l s di f f e r e nt i a t e i nt o bot h m i c r ogl i a a nd m a c r ogl i a i n t he br a i ns o f a dul t m i c e P r oc N a t l A c a d S c i U S A 94 ( 8 ) : 4080 5. E kl of B a nd B K S i e s j o ( 1972) T he e f f e c t of bi l a t e r a l c a r ot i d a r t e r y l i ga t i on upon t he bl ood f l ow a nd t he e ne r gy s t a t e of t he r a t br a i n. A c t a P hys i ol S c a nd 86 ( 2 ) : 155 65. E nc a bo, A E M a t e u, e t a l ( 2003) C D 34+ C D 38 i s a good pr e di c t i ve m a r ke r o f c l oni ng a bi l i t y a nd e xpa ns i on pot e nt i a l of C D 34+ c or d bl ood c e l l s T r a ns f us i on 43 ( 3 ) : 383 9. E nde M a nd N E nde ( 1972 ) H e m a t opoi e t i c t r a n s pl a nt a t i on by m e a ns of f e t a l ( c or d ) bl ood. A ne w m e t hod. V a M e d M on ( 1918) 99 ( 3) : 276 80.

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124 E nde N a nd R C he n ( 2001 ) H um a n um bi l i c a l c or d bl ood c e l l s a m e l i o r a t e H unt i ngt on's di s e a s e i n t r a ns ge ni c m i c e J M e d 32 ( 3 4) : 231 40. E nde N a nd R C he n ( 2002 ) P a r ki ns on's di s e a s e m i c e a nd hum a n um bi l i c a l c or d bl ood. J M e d 3 3 ( 1 4 ) : 173 80. E nde N R C he n, e t a l ( 2001) H um a n um bi l i c a l c or d bl ood c e l l s a m e l i or a t e A l z he i m e r 's di s e a s e i n t r a ns ge ni c m i c e J M e d 32 ( 3 4) : 241 7. E r m e r t M C P a nt a z i s e t a l ( 2003) I n s i t u l oc a l i z a t i on of T N F a l pha / be t a T A C E a nd T N F r e c e p t or s T N F R 1 a nd T N F R 2 i n c ont r ol a n d L P S t r e a t e d l ung t i s s ue C yt oki ne 22 ( 3 4) : 89 100. F or d, A L A L G oods a l l e t a l ( 1995) N or m a l a dul t r a m i f i e d m i c r ogl i a s e pa r a t e d f r om ot he r c e nt r a l ne r vous s ys t e m m a c r opha ge s by f l ow c yt om e t r i c s or t i ng P he no t ypi c di f f e r e nc e s de f i ne d a nd di r e c t e x vi vo a nt i ge n p r e s e nt a t i on t o m ye l i n ba s i c pr ot e i n r e a c t i ve C D 4+ T c e l l s c om pa r e d. J I m m unol 154 ( 9) : 4309 21 F r e nke l D Z H ua ng, e t a l ( 2003) N a s a l va c c i na t i on w i t h m ye l i n ol i gode ndr oc yt e gl yc opr ot e i n r e duc e s s t r oke s i z e by i nduc i ng I L 10 pr oduc i ng C D 4+ T c e l l s J I m m unol 171 ( 12) : 6549 55. F ukuda S C A F i ni e t a l ( 2004 ) F oc a l c e r e br a l i s c he m i a i nduc e s a c t i ve pr ot e a s e s t ha t de gr a de m i c r ova s c ul a r m a t r i x. S t r oke 35 ( 4) : 998 1004. G a r buz ova D a vi s S A E W i l l i ng, e t a l ( 2003 ) I nt r a ve nous a dm i ni s t r a t i on of hum a n um bi l i c a l c or d bl ood c e l l s i n a m ous e m ode l o f a m yot r ophi c l a t e r a l s c l e r os i s : di s t r i but i on, m i gr a t i on, a nd di f f e r e nt i a t i on. J H e m a t ot he r S t e m C e l l R e s 12 ( 3) : 255 70. G a r c i a J H A S L os s i ns ky, e t a l ( 1978) C e r e br a l m i c r ova s c ul a t ur e i n i s c he m i a A dv N e ur ol 20 : 141 9.

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125 G e ndr on, A J T e i t e l ba um e t a l ( 2002) T e m po r a l e f f e c t s of l e f t ve r s us r i ght m i dd l e c e r e br a l a r t e r y oc c l us i on on s pl e e n l ym phoc yt e s u bs e t s a nd m i t oge ni c r e s pons e i n W i s t a r r a t s B r a i n R e s 955 ( 1 2) : 85 97 G oodw i n, H S L M G r unz i nge r e t a l ( 2003) L ong t e r m c r yos t or a ge o f U C bl ood uni t s : a bi l i t y of t he i nt e gr a l s e gm e nt t o c onf i r m bot h i de nt i t y a nd he m a t opoi e t i c pot e nt i a l C yt ot he r a py 5 ( 1 ) : 80 6. G r e e n G P H I L i n G A M oyl a n K C ( 2004 ) T he W a s hi ngt on m a nua l of m e di c a l t he r a pe ut i c s G r ot t a J C L C P e t t i g r e w e t a l ( 1988 ) E f f i c a c y a nd m e c ha ni s m of a c t i on of a c a l c i um c ha nne l bl oc ke r a f t e r gl oba l c e r e b r a l i s c he m i a i n r a t s S t r oke 19 ( 4) : 447 54 H a c ke W G D onna n, e t a l ( 2004) A s s oc i a t i on of out c om e w i t h e a r l y s t r oke t r e a t m e nt : pool e d a na l ys i s of A T L A N T I S E C A S S a nd N I N D S r t P A s t r oke t r i a l s L a nc e t 363 ( 9411) : 768 74 H a r t l R L S c hur e r e t a l ( 1996) E xpe r i m e nt a l a nt i l e ukoc yt e i nt e r ve nt i ons i n c e r e br a l i s c he m i a J C e r e b B l ood F l ow M e t a b 16 ( 6 ) : 1108 19. H D S S ( 2004 U pda t e ) H e a r t D i s e a s e a nd S t r oke S t a t i s t i c A m e r i c a n H e a r t A s s oc i a t i on I nt i s o, D M M Z a r r e l l i e t a l ( 2004 ) T um or ne c r os i s f a c t or a l pha s e r um l e ve l s a nd i nf l a m m a t or y r e s pons e i n a c ut e i s c he m i c s t r oke pa t i e nt s N e ur ol S c i 24 ( 6) : 390 6 J a nde r S M K r a e m e r e t a l ( 1995) L ym phoc yt i c i nf i l t r a t i on a nd e xpr e s s i on of i nt e r c e l l ul a r a dhe s i on m ol e c ul e 1 i n phot oc he m i c a l l y i nduc e d i s c he m i a of t he r a t c or t e x. J C e r e b B l ood F l ow M e t a b 15 ( 1 ) : 42 51. K ondz i ol ka D L W e c hs l e r e t a l ( 2000) T r a ns pl a nt a t i on of c ul t u r e d hum a n ne ur ona l c e l l s f or pa t i e nt s w i t h s t r oke N e ur ol ogy 55 ( 4 ) : 56 5 9.

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126 K ope n, G C D J P r oc kop, e t a l ( 1999 ) M a r r ow s t r om a l c e l l s m i gr a t e t hr oughout f or e br a i n a nd c e r e be l l um a nd t he y di f f e r e nt i a t e i nt o a s t r oc yt e s a f t e r i nj e c t i on i nt o ne ona t a l m ous e br a i ns P r oc N a t l A c a d S c i U S A 96 ( 19) : 10711 6 K or e nkov, A I J P a hnke e t a l ( 2000) T r e a t m e nt w i t h ni m od i pi ne or m a nni t ol r e duc e s pr ogr a m m e d c e l l d e a t h a nd i nf a r c t s i z e f ol l ow i ng f oc a l c e r e br a l i s c he m i a N e ur os ur g R e v 23 ( 3) : 145 50 L a ve r J H M R A bboud, e t a l ( 1995 ) C ha r a c t e r i z a t i on of c ki t e xpr e s s i on by pr i m i t i ve he m a t opoi e t i c pr oge ni t or s i n um bi l i c a l c or d bl ood. E xp H e m a t ol 23 ( 14 ) : 151 5 9. L e w i s I D ( 2002 ) C l i ni c a l a nd e xpe r i m e nt a l us e s of um bi l i c a l c o r d bl ood I nt e r n M e d J 32 ( 12) : 601 9. L i Y J C he n, e t a l ( 2001) A dul t bone m a r r ow t r a ns pl a nt a t i on a f t e r s t r oke i n a dul t r a t s C e l l T r a ns pl a nt 10 ( 1) : 31 40. L i Y M C hopp, e t a l ( 2000) I nt r a s t r i a t a l t r a ns pl a nt a t i on of bone m a r r ow nonhe m a t opoi e t i c c e l l s i m pr ove s f unc t i ona l r e c ove r y a f t e r s t r oke i n a dul t m i c e J C e r e b B l ood F l ow M e t a b 20 ( 9) : 1311 9 L i u, P K R G G r os s m a n, e t a l ( 2001) I s c he m i c i nj ur y a nd f a ul t y ge n e t r a ns c r i pt s i n t he br a i n. T r e nds N e ur os c i 24 ( 10 ) : 581 8. L i u, T R K C l a r k e t a l ( 1994) T um o r ne c r os i s f a c t or a l pha e xp r e s s i on i n i s c he m i c ne ur ons S t r oke 25 ( 7) : 1481 8. L j unggr e n, B R A R a t c he s on, e t a l ( 1974) C e r e br a l m e t a bol i c s t a t e f o l l ow i ng c om pl e t e c om pr e s s i on i s c he m i a B r a i n R e s 73 ( 2) : 291 307.

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