USF Libraries
USF Digital Collections

Differential effects of isoflurane and propofol anesthesia on neurogenesis in young and aged rats

MISSING IMAGE

Material Information

Title:
Differential effects of isoflurane and propofol anesthesia on neurogenesis in young and aged rats
Physical Description:
Book
Language:
English
Creator:
Erasso, Diana Marcela
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
Publication Date:

Subjects

Subjects / Keywords:
Cognitive Impairment
Isoflurane
Postnatal Neurogenesis
Propofol
Dissertations, Academic -- Neurosciences Medicine -- Doctoral -- USF   ( lcsh )
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: Worldwide, millions of young and elderly patients receive procedures that could not be performed without the use of anesthetics. Unfortunately, emerging animal and human data suggest an association between exposure to general anesthesia and impairment of cognitive function in pediatric and geriatric patients. Recent laboratory data have shown that general anesthetics are potentially damaging to the developing and aging brain. However, the mechanism by which this happens is still unknown. General anesthetics affect learning and memory, a brain function involving neural plasticity. An important form of neural plasticity receiving attention is postnatal neurogenesis. This process is highly regulated and involved in hippocampal functions under physiological conditions. This dissertation hypothesizes that anesthetic induced alteration of postnatal neurogenesis may explain the cognitive impairment observed in some pediatric and geriatric patients after anesthesia. In order to accurately address this hypothesis, in the first portion of this dissertation, an animal model is used to examine the effects of two different anesthetics on cognition and new cell proliferation in young and aged rats. Furthermore, the second and third portion of this dissertation emphasizes on the effects of these two widely used anesthetics on each of the various stage of postnatal neurogenesis in young and aged rats.
Thesis:
Disseration (Ph.D.)--University of South Florida, 2011.
Bibliography:
Includes bibliographical references.
System Details:
Mode of access: World Wide Web.
System Details:
System requirements: World Wide Web browser and PDF reader.
Statement of Responsibility:
by Diana Marcela Erasso.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains 119 pages.
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:
usfldc doi - E14-SFE0004967
usfldc handle - e14.4967
System ID:
SFS0028204:00001


This item is only available as the following downloads:


Full Text
xml version 1.0 encoding UTF-8 standalone no
record xmlns http:www.loc.govMARC21slim xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.loc.govstandardsmarcxmlschemaMARC21slim.xsd
leader nam 22 Ka 4500
controlfield tag 007 cr-bnu---uuuuu
008 s2011 flu ob 000 0 eng d
datafield ind1 8 ind2 024
subfield code a E14-SFE0004967
035
(OCoLC)
040
FHM
c FHM
049
FHMM
090
XX9999 (Online)
1 100
Erasso, Diana Marcela.
0 245
Differential effects of isoflurane and propofol anesthesia on neurogenesis in young and aged rats
h [electronic resource] /
by Diana Marcela Erasso.
260
[Tampa, Fla] :
b University of South Florida,
2011.
500
Title from PDF of title page.
Document formatted into pages; contains 119 pages.
Includes vita.
502
Disseration
(Ph.D.)--University of South Florida, 2011.
504
Includes bibliographical references.
516
Text (Electronic dissertation) in PDF format.
3 520
ABSTRACT: Worldwide, millions of young and elderly patients receive procedures that could not be performed without the use of anesthetics. Unfortunately, emerging animal and human data suggest an association between exposure to general anesthesia and impairment of cognitive function in pediatric and geriatric patients. Recent laboratory data have shown that general anesthetics are potentially damaging to the developing and aging brain. However, the mechanism by which this happens is still unknown. General anesthetics affect learning and memory, a brain function involving neural plasticity. An important form of neural plasticity receiving attention is postnatal neurogenesis. This process is highly regulated and involved in hippocampal functions under physiological conditions. This dissertation hypothesizes that anesthetic induced alteration of postnatal neurogenesis may explain the cognitive impairment observed in some pediatric and geriatric patients after anesthesia. In order to accurately address this hypothesis, in the first portion of this dissertation, an animal model is used to examine the effects of two different anesthetics on cognition and new cell proliferation in young and aged rats. Furthermore, the second and third portion of this dissertation emphasizes on the effects of these two widely used anesthetics on each of the various stage of postnatal neurogenesis in young and aged rats.
538
Mode of access: World Wide Web.
System requirements: World Wide Web browser and PDF reader.
590
Advisor:
Saporta Camporesi, Samuel Enrico M.
Advisor:
Saporta Camporesi, Samuel Enrico M.
653
Cognitive Impairment
Isoflurane
Postnatal Neurogenesis
Propofol
690
Dissertations, Academic
z USF
x Neurosciences Medicine
Doctoral.
773
t USF Electronic Theses and Dissertations.
4 856
u http://digital.lib.usf.edu/?e14.4967



PAGE 1

Differential Effects o f Isoflurane a nd Propofol A nesthesia on Neurogenesis i n Young a nd Aged R ats by Diana M. Erasso A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Pathology and Cell Biology College of Medicine University of South Florida Co Majo r Professor: Samuel Saporta, Ph.D. Co -Major Professor: Enrico M. Camporesi, M.D. Paula Bickford, Ph.D. Jay Dean, Ph .D. David Diamond, Ph.D. Date of Approval: April 4 2011 Keywords: postnatal neurogenesis, isoflurane, propofol, cognitive impairment Copyright 2011, Diana M. Erasso

PAGE 2

DEDICATION I dedicate my dissertation to my family and friends. A special feeling of gratitude to my mother Nubia Agudelo, my grandmother Carmen Amira Bustamante, and my aunt Clara Berolo whose words of love, concern, support and encouragement have helped me throughout my graduate student career I also dedicate this work and give special thanks to my fianc C arlos Felipe Chaves who was always encouraging me and stood by me through the good and bad times. His support and encouragement was in the end what made this dissertation possible.

PAGE 3

ACKNOWLEDGMENTS I owe my gratitude to all those people who have made this dissertation possible and because of whom my graduate experience has been one that I will cherish forever I would like to express my deepest gratitude to my two co major professors Dr. Samuel Saporta for his excellent guidance, caring and patience and Dr. Enrico Camporesi, for his patience, encouragement and support. I will also like to thank Dr. Don Cameron for his insightful comments and for kindly allowing me to use his laboratory equipment. I wis h to thank my committee members, Dr Paula Bickford, Dr. Jay Dean, and Dr. David Diamond, who were more than generous with their expertise and precious time. A special thank to Dr. Huafeng Wei, my committee chairman. Many thanks to my laboratory members, Dr. Rafael Chaparro, Dr. Carolina Quiroga Del Rio, and Dr. Rachel Karlnoski for their help and support. My research would not have been possible without their helps.

PAGE 4

i TABLE OF CONTENTS LIST OF FIGURES .............................................................................................................v ABSTRACT .................................................................................................................... vi INTRODUCTION ...............................................................................................................1 Evidence of adverse effects of general anesthetics on the developing brain ..........2 Evidence of adverse effects of general anesthetics on the senescence brain ...........4 Postnatal neurogenesis in the context of learning and memory ...............................7 The process of postnatal neurogenesis .....................................................................7 Regulation of postnatal neurogenesis ......................................................................9 General anesthetics ................................................................................................10 Conclusion .............................................................................................................11 References cited .....................................................................................................12 CHAPTER 1 : QUANTITATIVE ASSESSM ENT OF NEW CELL PROLIFERATION IN THE DENTATE GYRUS AND BE HAVIOR AFTER ISOFLURANE OR PROPOF OL ANESTHESIA IN YOUNG AND AGED RATS .................................................................................................................................19 Abstract ..................................................................................................................19 Introduction ............................................................................................................21 Materials and Methods Animals ......................................................................................................22 Isoflurane anesthesia ..................................................................................23 Propofol anesthesia ....................................................................................23 BrdU injections ..........................................................................................23 Cognitive assessment .................................................................................24 Tissue preparation and BrdU immunohistochemistry ...............................25 Microscopy ................................................................................................25 Statistical analysis ......................................................................................26 Results Physiologic parameters ..............................................................................26 New cell proliferation in the DG of young rats is decreased by

PAGE 5

ii isoflurane anesthesia ..................................................................................26 New cell proliferation in the DG of young rats is decreased by propofol anesthesia ....................................................................................27 The number of BrdU+ cells in the ol factory bulb is not altered by either isof lurane or propofol anesthesia .....................................................27 Isoflurane causes cognitive impairment in aged r at s .................................27 Propofol causes cogni tive impairment in young rats .................................28 Discussion Effect of isoflurane and propofol on new cell proliferation in the brain .................................................................................................28 Effect of isoflurane and propofol ane sthesia on cognitive function ..........30 Conclusion .................................................................................................31 References ci ted .........................................................................................32 CHAPTER 2 : THE INHALATION ANEST HETIC ISOFLURANE AFF ECTS NASCENT CELLS UNDERGOING MATURATION AND SYNAPTIC INTEGRATION IN THE DENTATE GYRUS OF AGED, BUT NOT YOUNG RATS ................................................................................................................................44 Abstract ..................................................................................................................44 Introduction ............................................................................................................46 Materials and Methods Animals ......................................................................................................47 Experiment timeline ...................................................................................47 Preparation and admini stration of thymidine analogs ...............................48 Isoflurane anesthesia ..................................................................................49 Measurement of physiological parameters during isoflurane anesthesia ...................................................................................................49 Tis sue collection and processing ...............................................................49 CldU and IdU immunohistochemistr y .......................................................50 EdU labeling protocol ................................................................................50 Double immunohist ochemistry for linage markers ....................................51 Ki67 immunohistochemistry ......................................................................51 Tunnel assay protocol ................................................................................52 Microscopy and cell counting ....................................................................53 Statistical analysis ......................................................................................53 Results Physiological parameter during isoflurane anesthesia ...............................54 Postnatal proliferation in the dentate gyrus, as assed by the ki67 marker is unaffe cted by isoflurane anesthesia ...........................................54 Isoflurane does not affect nascent cells undergoing differentiation in the DG of t he hippocampus at the time of exposure ..............................55 Isoflurane does not affect nascent cells undergoing migration in the DG of the hippocampus at the time of exposure ..............................55 Isoflurane affect s nascent cells undergoing maturation and synaptic integration in the DG of aged, but not young rats ......................................56

PAGE 6

iii Cell death in the dentate gy rus after i soflurane exposure ..........................57 Discussion Isoflurane has an age dependent eff ect on postnatal neurogenesis ............58 Isoflurane specifically affect s nascent cells undergoing maturation and synaptic I ntegration in the DG of aged rats ........................................59 References cited .....................................................................................................62 CHAPTER 3 : PROPOFOL ANESTHESIA AFFECTS NASCENT CELLS UNDERGOING DIFFERENT IATION, AXON/DENDRIT E TARGETING AND MIGRATION IN THE DENTATE GYRUS OF YOUNG BUT NOT AGED RATS ....................................................................................................................73 Abstract ..................................................................................................................73 Introduction ............................................................................................................75 Materials and Methods Animals ......................................................................................................76 Experiment timeline ...................................................................................76 Preparation and administration of thymidine analogs ...............................77 Propofol anesthesia ....................................................................................78 Measurement of physiological parameters during propofol anesthesia ...................................................................................................78 Tis sue collection and processing ...............................................................78 CldU and IdU immunohistochemistry .......................................................79 EdU labeling protocol ................................................................................79 Double immunohist ochemistry for linage markers ....................................80 Ki67 immunohistochemistry ......................................................................80 Tunnel assay protocol ................................................................................81 Microscopy and cell counting ....................................................................82 Statistical analysis ......................................................................................82 Resu lts Physiological parameter during propofol anesthesia .................................83 Postnatal proliferation in the dentate gyrus, as assed by the ki67 marker is unaf fected by propofol anesthesia .............................................83 Nascent cells undergoing differentiation in the DG of young, but not aged rats, are af fected by propofol anesthesia .....................................84 Nascent cells undergoing migration in the DG are altered by propofol anesthesia in young, but not in aged rats .....................................84 Nascent cells undergoing maturation, in the DG of young and aged rats, are unaf fected by propofol anesthesia .......................................85 Cell death in the DG after propofo l anesthesia ..........................................85 Discussion Propofol anesthesia has a speci fic effec t on the young brain ....................86 Nascent cells undergoing differentiation and migration, in the DG of young rats, at the time of anesthetic exposure are particularly affected by propofol ...................................................................................86 Nascent cells in the DG of young and aged rats seem to be affected

PAGE 7

iv by anesthetics in an agent dependent manner ............................................87 References cited .....................................................................................................89 CONC LUSIONS ................................................................................................................98 Introduction ............................................................................................................98 The process of postnatal neurogenesis ...................................................................99 Age dependent vulnerability to anesthetic in duced cognitive dysfunction ........100 Age nt dependent effects on postnatal neuroge ne sis ............................................101 Conclusion ...........................................................................................................103 References Cited ..................................................................................................104 ABOUT THE AUTHOR ....................................................................................... End Page

PAGE 8

v LIST OF FIGURES Figure 1: Postnatal neurogenesis stages .............................................................18 Figure 2 : Immu nohistochemical labeling of proliferating cells within the DG .....................................................................................37 Figure 3 : New cell proliferation in the DG of young rats is decreased by isoflurane anesthesia ...........................................................................38 Figure 4 : New cell proliferation in the DG of young rats is decreased by propofol anesthesia .............................................................................39 Figure 5 : The number of BrdU+ cells in the olfactory bulb is not altered by isoflurane .......................................................................................40 Figure 6 : The number of BrdU+ cells in the olfactory bulb is not altered by propofol anesthesia ........................................................................41 Figure 7 : Isoflurane causes cogn itive impa irment in aged rats ..........................42 Figure 8 : Propofol causes cogni tive impairment in young rats .........................43 Figure 9: Experimental design ...........................................................................66 Figure 10 : Physiological paramete rs during isoflurane exposure ........................67 Figure 11 : Neural progenitor cell proliferatio n is unaffected by i soflurane ........68 Figure 12 : The number and fate of 4 day old nascent cells in the DG Are unaf fected by isoflurane expos ure ...............................................69 Figure 13 : Cells undergoing maturation, at 8 days after birth, are unaffected by isoflurane ex posure in young and aged rats ................70 Figure 14 : Isoflurane exposure affected cells that underwent maturation And started to integrate in the D G of aged, but not young rats ..........71 Figure 15 : Cell death in the DG after isoflurane exposure ..................................72 Figure 16 : Experimental design ...........................................................................91 Figure 17: Rats physiological parameters during propofol exposure ..................92 Figure 18: Neural progenitor cell proliferation is unaffected by propofol ..........93 Figure 19: The number and fate of 4 days old nascent cells in the DG are altered by propofol anesthesia in young, but not in aged rats ............94 Figure 20: Cells undergoing maturation in the DG at 8 days after birth are altered by prop ofol anesthesia in young, but not in aged rats. ..........95 Figure 21: Propofol anesthesia does not alter nascent cells that underwent maturation and s tarting to integrate in the DG of young or aged rats .........................................................................................96 Figure 22: Cell death in the DG after propofol a nesthesia ..................................97

PAGE 9

vi Figure 23: Summary diagram of the results .......................................................107

PAGE 10

vii ABSTRACT Worldwide, millions of young and elderly patients receive procedures that could not be performed without the use of anesthetics. Unfortunately, emerging animal and human data suggest an association between exposure to general anesthesia and impairment of cognitive function in pediatric and geriatric patients. Recent laboratory data have shown that general anesthetics are potentially damaging to th e developing and aging brain. However, the mechanism by which this happens is still unknown. General anesthetics affect learning and memory, a brain function involving neural plasticity. An important form of neural plasticity receiving attention is postnatal neurogenesis. This process is highly regulated and involved in hippocampal functions under physiological con ditions. This dissertation hypothesize s that anesthetic induced alteration of postnatal neurogenesis may explain the cognitive impairment observe d in some pediatric and geriat ric patients after anesthesia. In order to accurately address this hypot hesis, in the first portion of this dissertation an animal model is used to examine t he effects of two different anesthetics on cognition and new cell proliferation in young and aged rats Furthermore the second and third portion of this dissertation emphasizes on the effects of

PAGE 11

viii these two widely used anesthetics on each of the various stage of postnat al neurogenesis in young and aged rats

PAGE 12

1 INTRODUCTION Thousands of surgeries requiring anesthesia are performed every day in the United States. Anesthetics are among the most potent and rapidly acting drugs in common clinical use, and one of the greatest advances of medici ne allowing complicated surgeries to be performed safely. Unfortunately, recent clinical and animal studies show that exposure to commonly used general anesthetics is associated to the development of co gnitive impairment in pediatric (Loepke and Soriano 2008; Wilder et al. 2009) and geriatric patients (Moller et al. 1998a; Monk et al. 2008) The brain appears to be particularly vulnerable to insults that would go unnoticed at a different age, during development or ag ing. These insults may become evident as impairment of cognitive function. C ognitive changes associated with general anesthesia include i mpaired spatial orientation, learning and memory but the mechanism by which this happens still not known. Spatial lear ning and m emory are considered hippocampal cognitive domains although other brain areas may contribute as well. An important determinant of hippocampal function is the degree to which new neurons are generated from stem cells in the subgranular zone of the dentate gyru s (DG) of the hippocampus (Kempermann 2002; Zhao et al. 2008; Jessberger et al. 2009; Coras et al. 2010) Furthermore postnatal hippocampal neurogenesis has been shown to be altered by factors

PAGE 13

2 such as environmental changes (Kempermann et al. 1997) and modulations of neurotransmitters (Sun et al. 2009) B ecause anesthetics also affect these f actors, it seems reasonable to hypothesize that anesthetics may also affect pos tnatal hippocampal neurogenesis and that its alteration may explain cognitive impairment in pediatric and geriatric patients. Evidence of adverse effects of general anesthetics on the developing brain Emerging human and animal data suggest an association between early exposure to anesthetics and long term impairment of cognitive function, raising concerns regarding the safe use of these drugs in young childre n. Consequently, the prudence of frequent anesthesia exposure of this popul ation is now being scrutinized. Current animal data implicate not only N methyl daspartate ( NMDA) receptor antagonists, but also drugs that potentiate aminobutyric acid ( GABA ) receptors, as potentially neurotoxic to the developing brain (Olney et al. 2000; Young et al. 2005) Drugs that act as (NMDA) receptor an tagonists, like ketamine, and those that act in the (GABA) receptor such as benzodiazepines, induce neuronal injury and neuronal cell death in the brains of young rodents. Drugs that have effects on one or both of these receptors include barbiturates, benzodiazepines, inhaled anesthetics such as isoflurane and nitrous oxide, and intravenous anesthetics such as propofol and ketamine Not all of these drugs have been approved for pediatric use, but the y are commonly used in this young population. In neonatal animal models, isoflurane has been associated with apoptotic neurodegeneration (Yon et al. 2005; Lu et al. 2006) In rats, a 6hour exposure to an anesthetic combination of isoflurane, midazolam, and nitrous oxide a commonly used

PAGE 14

3 combination for long pediatric surgical procedures, has been shown to i nduce widespread a poptotic neurodegeneration in newborn animals, and impairment in learning and memory later in adulthood. Neither nitrous oxide nor midazolam alone produced neuroapoptosis. In contrast, administration of isoflurane alone caused neuroapoptosis (Jevtovic Todorovic et al. 2003) The combination of isoflurane and midazolam produced a substantial increase in the number of apoptotic cells, and the triple combination of isoflurane, midazolam, and nitrous oxide produced the greatest increase in the number of neuroapoptotic cells. Animals treated with the triple combination also demonstrated persistent memory and l earning impairments later in life, which correlated with deficits in hippocampal synaptic function measured electrophysiologically in vitro (Jevtovic Todorovic et al. 2003) Preliminary data in neonatal rats from another research group also suggest that isoflurane, when administered for 4 hours a s a single anesthetic drug, alters fear conditioning and spatial learning in adulthood (Stratmann 2006) Another widely used general anesthetic used for pediatric surgical procedures is the intravenous anesthetic, propofol. However, t he effects of propofol administration on neuronal survival and neurocognitive performance have not been formally studied in young children. A study of the effects of propofol on neuronal structure and neurocognitive performance in mice suggests that propofol exposure during brain development increases neurodegeneratio n, and results in cognitive deficits in adulthood (Fredriksson et al. 2007) Using in vitro preparations of neuronal cell cultures from immature chicks and rats, several investigators noted a dose dependent neuronal structural change after propofol exposure (Honegger and Matthieu 1996; Spahr Schopfer et al. 2000; Vutskits 2005) Supraclinical doses of propofol induced neuronal cell death in

PAGE 15

4 dissociated cell culture models, but failed to demonstrate neurotoxic effects in organotypic slice cultures (Spahr Schopfer et al. 2000) F urthermore exposure of dissociated neurons to propofol in clinically relevant concentrations for up to 3 days did not result in dendritic development changes In conclusi on, no prospective studies have examined the effects of propofol on neuronal structure and neurocognitive outcome in young children, whereas several case reports detail short term neurological abnormalities without long term neurocognitive impairment. However, detailed follow up has not been conducted in this patient population. A study in neonatal mice points to propofols dose dependent neurodegenerative properties, leading to behavioral and learning abnormalities, which are exacerbated by the co administration of ketamine. Animal studi es using in vitro preparations demonstrate only subtle changes, whereas current evidence fails to reveal injurious effects o f propofol on neuronal survival and dendritic development Evidence of adverse effects of general anesthetics on the aging brain According to the 2004 US census, people over eighty years of aged are the fastest growing segment of the population, this population grew 30% over the previous decade (Bureau 2004) Therefore elders are one of the largest group receiving anesthetics. P atients over sixty four years of age have eight fold more procedures per 10000 people tha n patients fifteen years of age or younger and more than twofold more than patients aged 4564 years old. Moreover, the aging brain becomes more suscepti ble to neurodegeneration over time. In other words the aging brain has less reserve and small adverse events may have a greater impact on brain function. In 1998 Moller et al presented the first of a series of multicenter studies on

PAGE 16

5 cognitive dysfunc tion after anesthesia. The study included 1,218 patients, aged 60 yr or older, who underwent major abdominal, noncardiac thoracic, or orthopedic surgery during general anesthesia. Pati ents were tested preoperatively, and at 1 week and 3 months postoperatively Test results were compared with a total of 321 controls recruited from the United Kingdom, 11 centers in Europe, and 2 centers in North America. Patients were classified as experiencing cognitive dysfunction when two Z scores in individual tests declined by 1.96, or the combined average Z score declined greater than 1.96. At 1 week postoperatively, 25.8% of 1,011 pati ents experienced a decline in cognitive function, compared with 3.4% of 176 control subjects. At 3 months postoperatively, 9.9% of 910 patie nts experienced a decline relative to preoperative level of function, compared with 2.8% of controls (Moller et al. 1998b) A number of subsequent studies have described cognitive impairment within the first 10 day s after surgery and anesthesi a ( Dodds and Allison 1998) Williams Russo et a l found a rate of cognitive dysfunction of 5% at 6 months after surgery, although in the absence of a control population, the significance is hard to determine (Williams Russo et al. 1995) A follow up study that evaluated patients at 1 and 2 yr found that the rate of cognitive dysfunction decreased to approximately 1%, which was not statistically significant (Abildstrom et al. 2000) Taken together, it seems that elderly patients manifest measurable deterioration shortly after surgery and anesthesia (25% at 210 days), with gradual resolution such that the incidence declines (10% at 3 months, 5% at 6 months, 1% at 1 yr) to levels nearly indistinguishable from control subject s by approximately 1 yr. In one study less than half of the participants who were classified at having cognitive impairment a t 3 months had detectable decline at 1 week Consequently

PAGE 17

6 cognitive impairment at 1 week did not predict cognitive impairment at 3 months (Rasmussen and Siersma 2004) A large clinical study from D uke University now appears to confirm earlier findings, in the 1998 study (Moller et al. 1998b) showing that significant number of elderly peop le experience long term (three or more months ) changes in cognitive function after anesthesia (Monk et al. 2008) One thousand sixty four patients were classified as young (1839 years old ), middle aged (40 59 years old ) or elderly (60 years old or older). Their cognitive function was assessed with a battery of tests before surgery, at discharge from the hospital, and 3 months after the discharge A t the discharge form the hospital, signs of cognitive impairment were present in 30 41% of patients. At the 3month time point young and middle aged patients had recovered, but 12.7% of elderly patients still showed cognitive impairment. Moreover, patients with cognitive dysfunction were more likely to die in the year after sur gery. In the laboratory, Culley et al. ( 2003) found that 2 hours of anesthesia with 1.2% isoflurane/70% nitrous oxide/30% oxygen produces longlasting, but reversible impairment on a previously learned spatial memory task in 18mo oldrats. Additionally when the same aged animals were exposed to the same concentration of anesthetic for the same period of time, but the spatial memory task performed two weeks after anesthesia, the aged rats subject to general anesthesia were less able than control rats to perform w ell (Culley et al. 2004a) T he aging brain appears to be more susceptible to anesthetic effects than the adult brain T his may be due to the several ways in which they are different, including size, distribution and type of neurotransmitters metabolic function, capacity for plasticity, and

PAGE 18

7 inflammation suggesting that the senescent brain may be more susceptible to anesthetic changes. Postnatal neurogenesis in the context of learning and memory G eneral anesthesia has been i mplicated as a possible cause of cognit ive impairment. L aboratory data shows that spatial memory is impaired by general anesthetics in aged rats (Culley et al. 2003) Spatial memory is considered a hippocampal cognitive domain, even though other areas contribute to spatial learning and memory. A n important determinant of hippocampal function in animals is the degree to which new neurons are generated from stem cells in the subgranular zone of the dentate gyrus (DG) of the hippocampus N eurogenesis in the DG decreases progressively with a ge (Luo et al. 2006; Shruster et al. 2010) but both a decrease in the levels of the stress hormone corticosterone (Mirescu et al. 2004) and environmental enrichmen t (Kempermann et al. 1997) can restore neurogenesis and improve spatial memory function T hus cognitive impairment following anesthetic exposure may be a result of anestheticinduced alteration of postnatal neurogenesis The process of postnatal neurogenesis Neurogenesis is the process of generating functionally integrated neurons from progenitors cells I t was traditionally believed to occur only during the embryonic stag e (Ramon 1913) However, studies of Altman and Das (Altman 1962; Altman and Das 1965) provide d the first indication that new neurons are generated in the postnatal mammalian brain. More recently it has been recognized that postnatal neurogenesis replicates the complex process of neurodevelopment in order to g enerate functional integrated neurons important for learning and memory. Postnatal neurogenesis in

PAGE 19

8 mammals is primarily r estricted to two brain regions: the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampal DG. Neural stem cells proliferate and give rise to new granule cells thro ugh the following developmental stages: p roliferation, differentiation migration, maturation and synaptic integration (Figure 1) (Ming and Song 2005) These stages can be identified on the basis of cell morphology, mitotic capability, electrophysiology characteristics, and expression of developmental regulated markers (Kempermann et al. 2004) Proliferating SVZ cells form a migrating chain and differentiate into ne urons in the olfactory bulb (Alvarez Buylla and GarciaVerdugo 2002; Lazarini and Lledo 2011) Postnatal hippoc ampal progenitor cells divide in the SGZ and develop into neurons in the granular cell layer. Nascent cells become functionally integrated into the DG and have passive membrane properties, ac tion potentials and functional synaptic inputs similar to those f ound in mature dentate granule cells (Snyder et al. 2005) N ewly generated neurons play a significant role in synaptic plasticity (Ming and Song 2005; Snyder et al. 2005) and the reduction in the number of these cells impairs learning and memory (Shors et al. 2002) Neur otransmitters, growth factors, hormones and environmental factors are among the mediators involved in the regulation of adult hippocampal neurogenesis (Kempermann 2002) GABAergic receptor agonists and NMDA receptor antagonists induce dentate gyrus progenitor proliferation with increased newborn neurons in the granule cell layer (Cameron et al. 1995) Enriched environments result in more total granule cell neurons by increasing the survival rate of the progeny of the dividing progenitor cells (Kempermann et al. 1997) On the other hand, impaired hippocampal neurogenesis may

PAGE 20

9 be associated with cognitive decline in aging, major depression and Alzheimers disease (Haughey et al. 2002; Tsuchiya et al. 2003; Raber et al. 2004) Neural Stem Cells (NSCs) generate significant num bers of progenity in young rats, with approximately 9000 new cells or 0.1% of the granula r cell population being replaced daily (Cameron and McKay 2001) Approximately 50% of the neuronal progenity survive, and new granul e cells populate the inner third of the granular cell layer. P ostnatal neurogenesis is not a one step process, as new neurons have to progress through several distinct developmental steps before they fully integrate and eventua lly become indistinguishable fro m granule cells born during embryonic or early postnatal development (Laplagne et al. 2006) Regulation of postnatal neurogenesis The basic rate of neurogenesis in the DG is thought to be genetically determined (Cameron and McKay 2001) but it can be regulated by a variety of factors such as physical activity (van Praag et al. 1999) environmental enrichment (Kempermann et al. 1997), stress (Kempermann et al. 1997; Mirescu et al. 2004) gender, steroids ( Tanapat et al. 1999) inflammation (which reduces the number of newborn cells) (Tanapat et al. 1999; Monje et al. 2003) and aging (Seki and Arai 1995; Kuhn et al. 1996; Tanapat et al. 1999; Luo et al. 2006; Lazarov et al. 2010) T her e is an exponential decrease of neurogenesis throughout the life span in rodents, nonhuman primates and potenti ally humans (Galvan and Jin 2007; Lazarov et al. 2010) With advancing age, the proliferative activity of hippocampal NSCs and neuronal differentiation capacity decline, leading to a dramatic, appro xi mately 10 fold reduction in net neurogenesis between the age of 2 months and 2 years in a rodents life (Kuhn et al. 1996; Kempermann e t al. 2002)

PAGE 21

10 Recently, the amino acid aminobutyric acid ( GABA ) has emerged as a key regulator that controls multiple phases of adult neurogenesis. GABA serves as a feedback regulator of neural production and migration ( Bordey, 2007). GABAergic mechanisms regulate differentiation and the timing of synaptic integration ( Ge et al. 2007a ). General anestheti cs General anesthetics are essential to present day medicine but a detailed understanding of their mechanism of action is still absent. Nevertheless, progress has been made in understanding their mechanism at the molecular and cellular levels. Different molecular targets in various regions of the nervous system are involved in the multiple components of anesthetic action. General anesthetic agents range from inhaled anesthetics, such as isoflurane, desflurane, sevoflurane, nitrous oxide and xenon, to intravenous anesthetics, such as propofol, ketamine, and benzodiazepines. Even though, these agents are chemically different, their proposed mechanism of action is thought to affect synaptic transmission by altering GABA and N methyl daspartate (NMDA) receptors (Campagna 2003) Neurotransmitter gated ion channels, particularly receptors for amino butyric acid (GABA) and glutamate are modulated by most anesthetics at both synaptic and extra synaptic sites. General anesthetics act as either positive or negative allosteric modulators of ligandgated ion channels at clinically effective concentrations (Hemmings et al. 2005) Most inhaled anesthetics enhance GABA receptor function, increasing channel opening to enhance inhibition at both synaptic and extra synaptic receptor. I n addition they appear to depress excitatory synapt ic transmission presynaptically, where their principal action seems to be a reduc tion in glutamate release. M ost intravenous

PAGE 22

11 anesthetics, including propofol, selectively modulate GABA receptor function by enhancing gating of the receptors by GABA Because GABA and NMDA mediated neuronal regulation is essential for postnatal neurogenesis, exposure to general anesthetics could potentially interfere with the neurogenic process and ultimately result in cognitive impairment. Conclusion In summary, increasing clinical and laboratory data have raised significant concerns regarding the safety of general anesthetics used on young and old individuals. General anesthetics have been implicated as a possible cause of cognitive impairment, becaus e surgery in pediatric and geriatric patients often involves general anesthesia. General anesthesia has also been shown to impair spatial memory in rats (Culley et al. 2004a; Culley et al. 2004b) O ne of the factors involved in spatial m emory is postnatal neurogenesis. A l teration of neurogenesis may be a possible reason for the cognitive impairment experienced in some pediatric and geriatric patients after general anesthesia. Moreover, neurogenesis is a multistep process and alteration of one or more of these stages may r esult in subtle but adverse consequences. The effect of anesthesia on the different stages of neurogenesis is not known. We hypothesize that alteration of neurogenesis by anesthesia may explain the cognitive impairment observed in some pediatric and geriatric patients.

PAGE 23

12 References Abildstrom H, Rasmussen LS, Rentowl P, Hanning CD, Rasmussen H, Kristensen PA, Moller JT (2000) Cognitive dysfunction 12 years after non cardiac surgery in the elderly. ISPOCD group. International Study of Post Operative Cognitive Dysfunction. Acta Anaesthesiol Scand 44: 12461251 Altman J (1962) Are new neurons formed in the brains of adult mammals? Science 135: 11271128 Altman J, Das GD (1965) Autoradiographic and histological evidence of postnatal hi ppocampal neurogenesis in rats. J Comp Neurol 124: 319335 Alvarez Buylla A, Garcia Verdugo JM (2002) Neurogenesis in adult subventricular zone. J Neurosci 22: 629634 Bureau USC (2004) U.S. Interim Projections by Age and Sex, Race, and Hispanic Origin. In : Cameron HA, McEwen BS, Gould E (1995) Regulation of adult neurogenesis by excitatory input and NMDA receptor activation in the dentate gyrus. J Neurosci 15: 46874692 Cameron HA, McKay RD (2001) Adult neurogenesis produces a large pool of new granule cel ls in the dentate gyrus. J Comp Neurol 435: 406417 Campagna MK, Forman SA (2003) Mechanisms of actions of inhaled anesthetics. N Engl J Med: 21102124 Coras R, Siebzehnrubl FA, Pauli E, Huttner HB, Njunting M, Kobow K, Villmann C, Hahnen E, Neuhuber W, W eigel D, Buchfelder M, Stefan H, Beck H, Steindler DA, Blumcke I (2010) Low proliferation and differentiation capacities of adult hippocampal stem cells correlate with memory dysfunction in humans. Brain 133: 33593372

PAGE 24

13 Culley DJ, Baxter M, Yukhananov R, Cr osby G (2003) The memory effects of general anesthesia persist for weeks in young and aged rats. Anesth Analg 96: 10041009, table of contents Culley DJ, Baxter MG, Crosby CA, Yukhananov R, Crosby G (2004a) Impaired acquisition of spatial memory 2 weeks af ter isoflurane and isoflurane nitrous oxide anesthesia in aged rats. Anesth Analg 99: 13931397; table of contents Culley DJ, Baxter MG, Yukhananov R, Crosby G (2004b) Long term impairment of acquisition of a spatial memory task following isoflurane nitrous oxide anesthesia in rats. Anesthesiology 100: 309314 Dodds C, Allison J (1998) Postoperative cognitive deficit in the elderly surgical patient. Br J Anaesth 81: 449462 Fredriksson A, Ponten E, Gordh T, Eriksson P (2007) Neonatal exposure to a combinati on of N methyl D aspartate and gammaaminobutyric acid type A receptor anesthetic agents potentiates apoptotic neurodegeneration and persistent behavioral deficits. Anesthesiology 107: 427436 Galvan V, Jin K (2007) Neurogenesis in the aging brain. Clin Interv Aging 2: 605610 Haughey NJ, Nath A, Chan SL, Borchard AC, Rao MS, Mattson MP (2002) Disruption of neurogenesis by amyloid beta peptide, and perturbed neural progenitor cell homeostasis, in models of Alzheimer's disease. J Neurochem 83: 15091524 He mmings HC, Jr., Akabas MH, Goldstein PA, Trudell JR, Orser BA, Harrison NL (2005) Emerging molecular mechanisms of general anesthetic action. Trends Pharmacol Sci 26: 503510 Honegger P, Matthieu JM (1996) Selective toxicity of the general anesthetic propofol for GABAergic neurons in rat brain cell cultures. J Neurosci Res 45: 631636 Jessberger S, Clark RE, Broadbent NJ, Clemenson GD, Jr., Consiglio A, Lie DC, Squire LR, Gage FH (2009) Dentate gyrus specific knockdown of adult neurogenesis impairs spatial and object recognition memory in adult rats. Learn Mem 16: 147154

PAGE 25

14 Jevtovic Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF (2003) Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci 23: 876882 Kempermann G (2002) Why new neurons? Possible functions for adult hippocampal neurogenesis. J Neurosci 22: 635638 Kempermann G, Gast D, Gage FH (2002) Neuroplasticity in old age: sustained fivefold induction of hippocampal neurogenesis by longterm environmental enrichment. Ann Neurol 52: 135143 Kempermann G, Jessberger S, Steiner B, Kronenberg G (2004) Milestones of neuronal development in the adult hippocampus. Trends N eurosci 27: 447452 Kempermann G, Kuhn HG, Gage FH (1997) More hippocampal neurons in adult mice living in an enriched environment. Nature 386: 493495 Kuhn HG, DickinsonAnson H, Gage FH (1996) Neurogenesis in the dentate gyrus of the adult rat: age relat ed decrease of neuronal progenitor proliferation. J Neurosci 16: 20272033 Laplagne DA, Esposito MS, Piatti VC, Morgenstern NA, Zhao C, van Praag H, Gage FH, Schinder AF (2006) Functional convergence of neurons generated in the developing and adult hippoca mpus. PLoS Biol 4: e409 Lazarini F, Lledo PM (2011) Is adult neurogenesis essential for olfaction? Trends Neurosci 34: 2030 Lazarov O, Mattson MP, Peterson DA, Pimplikar SW, van Praag H (2010) When neurogenesis encounters aging and disease. Trends Neurosc i 33: 569579 Loepke AW, Soriano SG (2008) An assessment of the effects of general anesthetics on developing brain structure and neurocognitive function. Anesth Analg 106: 16811707 Lu LX, Yon JH, Carter LB, Jevtovic Todorovic V (2006) General anesthesia a ctivates BDNF dependent neuroapoptosis in the developing rat brain. Apoptosis 11: 16031615

PAGE 26

15 Luo J, Daniels SB, Lennington JB, Notti RQ, Conover JC (2006) The aging neurogenic subventricular zone. Aging Cell 5: 139152 Ming GL, Song H (2005) Adult neurogene sis in the mammalian central nervous system. Annu Rev Neurosci 28: 223250 Mirescu C, Peters JD, Gould E (2004) Early life experience alters response of adult neurogenesis to stress. Nat Neurosci 7: 841846 Moller HJ, Gagiano CA, Addington DE, Von Knorring L, Torres Plank JF, Gaussares C (1998a) Long term treatment of chronic schizophrenia with risperidone: an openlabel, multicenter study of 386 patients. Int Clin Psychopharmacol 13: 99106 Moller JT, Cluitmans P, Rasmussen LS, Houx P, Rasmussen H, Canet J Rabbitt P, Jolles J, Larsen K, Hanning CD, Langeron O, Johnson T, Lauven PM, Kristensen PA, Biedler A, van Beem H, Fraidakis O, Silverstein JH, Beneken JE, Gravenstein JS (1998b) Long term postoperative cognitive dysfunction in the elderly ISPOCD1 study. ISPOCD investigators. International Study of Post Operative Cognitive Dysfunction. Lancet 351: 857861 Monje ML, Toda H, Palmer TD (2003) Inflammatory blockade restores adult hippocampal neurogenesis. Science 302: 17601765 Monk TG, Weldon BC, Garvan CW, Dede DE, van der Aa MT, Heilman KM, Gravenstein JS (2008) Predictors of cognitive dysfunction after major noncardiac surgery. Anesthesiology 108: 1830 Olney JW, Farber NB, Wozniak DF, Jevtovic Todorovic V, Ikonomidou C (2000) Environmental agents that have the potential to trigger massive apoptotic neurodegeneration in the developing brain. Environ Health Perspect 108 Suppl 3: 383388 Raber J, Fan Y, Matsumori Y, Liu Z, Weinstein PR, Fike JR, Liu J (2004) Irradiation attenuates neurogenesis and exacerbates ischemia induced deficits. Ann Neurol 55: 381389 Ramon Cy (1913) Degeneration and Regeneration of the Nervous sytem. London: Oxford Univ. Press

PAGE 27

16 Rasmussen LS, Siersma VD (2004) Postoperative cognitive dysfunction: true deterioration versus random variation. Acta Anaesthesiol Scand 48: 11371143 Seki T, Arai Y (1995) Age related production of new granule cells in the adult dentate gyrus. Neuroreport 6: 24792482 Shors TJ, Townsend DA, Zhao M, Kozorovitskiy Y, Gould E (2002) Neurogenesis may relate t o some but not all types of hippocampal dependent learning. Hippocampus 12: 578584 Shruster A, Melamed E, Offen D (2010) Neurogenesis in the aged and neurodegenerative brain. Apoptosis Nov;15(11):141521 Snyder JS, Hong NS, McDonald RJ, Wojtowicz JM (2005) A role for adult neurogenesis in spatial long term memory. Neuroscience 130: 843852 Spahr Schopfer I, Vutskits L, Toni N, Buchs PA, Parisi L, Muller D (2000) Differential neurotoxic effects of propofol on dissociated cortical cells and organotypic hippocampal cultures. Anesthesiology 92: 14081417 Stratmann G, Bell JD, Bickler P, Alvi R, Ku B, Magnusson KR, Liu J. (2006) Neonatal Isoflurane anesthesia causes a permanent neurocognitive deficit in rats. Society for Neurosciences Sun B, Halabisky B, Zhou Y Palop JJ, Yu G, Mucke L, Gan L (2009) Imbalance between GABAergic and Glutamatergic Transmission Impairs Adult Neurogenesis in an Animal Model of Alzheimer's Disease. Cell Stem Cell 5: 624 633 Tanapat P, Hastings NB, Reeves AJ, Gould E (1999) Estrogen st imulates a transient increase in the number of new neurons in the dentate gyrus of the adult female rat. J Neurosci 19: 57925801 Tsuchiya D, Hong S, Kayama T, Panter SS, Weinstein PR (2003) Effect of suture size and carotid clip application upon blood flow and infarct volume after permanent and temporary middle cerebral artery occlusion in mice. Brain Res 970: 131139

PAGE 28

17 van Praag H, Kempermann G, Gage FH (1999) Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat Neuros ci 2: 266270 Vutskits GE, Tassony E, Kiss JZ (2005) Clinically relevant concentrations of propofol but not midazolam alter in vitro dendritic development of isolated gamma aminobutyric acid positive interneurons. Anesthesiology 102: 970976 Wilder RT, Fl ick RP, Sprung J, Katusic SK, Barbaresi WJ, Mickelson C, Gleich SJ, Schroeder DR, Weaver AL, Warner DO (2009) Early exposure to anesthesia and learning disabilities in a population based birth cohort. Anesthesiology 110: 796804 Williams Russo P, Sharrock NE, Mattis S, Szatrowski TP, Charlson ME (1995) Cognitive effects after epidural vs general anesthesia in older adults. A randomized trial. JAMA 274: 4450 Yon JH, Daniel Johnson J, Carter LB, Jevtovic Todorovic V (2005) Anesthesia induces neuronal cell de ath in the developing rat brain via the intrinsic and extrinsic apoptotic pathways. Neuroscience 135: 815827 Young C, Jevtovic Todorovic V, Qin YQ, Tenkova T, Wang H, Labruyere J, Olney JW (2005) Potential of ketamine and midazolam, individually or in com bination, to induce apoptotic neurodegeneration in the infant mouse brain. Br J Pharmacol 146: 189197 Zhao N, Zhong C, Wang Y, Zhao Y, Gong N, Zhou G, Xu T, Hong Z (2008) Impaired hippocampal neurogenesis is involved in cognitive dysfunction induced by th iamine deficiency at early prepathological lesion stage. Neurobiol Dis 29: 176185

PAGE 29

18 POSTNATAL NEUROGENESIS STAGES Figure 1 Postnatal neurogenesis a developmental process that includes proliferation and fate speci differentiation, maturation, migration and incorporation into the existing neural circuitry of their progeny in the mature nervous system. Modified from Ming GL, Song H (2005) Adult neurogenesis in the mammalian central nervous system. Annu Rev Neurosci 28: 223 250

PAGE 30

19 CHAPTER 1 QUANTITATIVE ASSESSM ENT OF NEW CELL PROLIFERATION IN THE DENTATE GYRUS AND BE HAVIOR AFTER ISOFLURANE OR PROPOFOL ANESTHESIA IN YOUNG AND AGED RATS Abstract Backgroun d : There is a growing body of evidence showing that a statistically significant number of people experience long term changes in cognition after anesthesia. We hypothesize that this cognitive impairment may result from an anesthetic induced alteration of adul t hippocampal cell proliferation. Methods: To test this hypothesis, we investigated the effects of isoflurane and propofol on new cell proliferation and cognition of young (4 monthold) and aged (21 monthold) rats that were anesthetized for 3 hours with either 1.5% isoflurane or 35mg/kg/hr propofol. An additional group of young and aged rats exposed to room air or intralipid, served as co ntrols. All rats were injected intraperitoneally (IP) with 50 mg/Kg of 5bromo2deoxyuridine ( BrdU) immediately after anesthesia. A novel appetitive olfactory learning test was used to assess learning and memory two days after anesthesia. One week after anesthesia, rats were euthanize d and the brains analyzed for new cell

PAGE 31

20 proliferation in the dentate gyrus, and migration of newly formed cells in the subventricular zone to the olfactory bulb. Results: We found that exposure to either isoflurane (p=0.017) or propofol (p=0.006) decreased hippocampal cell proliferation in young, but not in aged rats. This anestheticinduced decrease was specific to new cell proliferation in the hippocampus, as new cell proliferation and migration to the olfactory bulb was unaffected. Isoflurane anesthesia produced cognitive impairment in aged rats (p=0.044) but not in young rats. Conversely, propofol anesthesia resulted in cognitive impairment in young (p=0.01) but not in aged rats Conclusions: These resu lts indicate that isoflurane and propofol anesthesia affect postnatal hippocampal cell proliferation and cognition in an age dependent manner. Keywords: isoflurane, propofol, cognitive impairment, new cell proliferation, cognition.

PAGE 32

21 Introduction Since its introduction to medical practice, anesthetic agents have enabled surgeries to be performed with ease, ensuring analgesia, unconsciousness and amnesia. Nonetheless, growing clinical and laboratory data suggest that anesthetic agents have long last ing consequences that may lead to cognitive impairment (Moller et al. 1998a; Monk et al. 2008) particularly in elderly and pediatric patients. Ten to fifteen percent of elderly patients suffer from difficulties with concentration and attention after anesthesia (Moller et al. 1998b) Furthermore, recent studies suggest an association between early exposure to general anesthesia and long term impairment of cognitive function in pediatric patients (Wilder et al. 2009) This phenomenon has been observed in animal models. Culley and colleagues (2004a, 2004b) reported enduring deficits in spatial working memory of aged rats after exposure to isoflurane, nitrous oxide, or isoflurane nitrous oxide anesthesia (Culley et al. 2004a; Culley et al. 2004b) Similarly, young rats exposed to isoflurane show persistent memory and lear ning deficits (Jevtovic Todorovic et al. 2003; Culley et al. 2004a; Zhang et al. 2008) Postnatal generation of neurons occurs throughout life and has been clearly demonstrated in t wo brain regions, the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus and the subventricular zone (SVZ) of the lateral ventricle where new neurons migrate into the olfactory bulb (OB) (Altman and Das 1965; Eriksson et al. 1998; Gage 2002; Ming and Song 2005; Taupin 2006a; Taupin 2006b) This process is more pronounced during early postnatal life and decreases with age (Galvan and Jin

PAGE 33

22 2007; Lazarov et al. 2010) which may make new cell proliferation at these time periods more susceptible to insults. Postnatal neurogenesis in the DG has been shown to be involved in learning and memory (Kempermann 2002; Shors et al. 2002; Zhao et al. 2008; Jessberger et al. 2009) Howeve r, the effects of anesthetics on neurogenesis are not well understood, raising concerns regarding the effect of certain anesthetic agents on cognition. In this study, we investigated whether isoflurane and/or propofol affected learning and new cell prolif eration in the brain of young and aged rats shortly after administration of anesthesia. We hypothesized that cognitive impairment following anesthesia may result from an anestheticinduced alteration of hippocampal new cell proliferation. Evaluation of new cell proliferation immediately following anesthesia constitutes the first step for a more extensive study of the effects of anesthesia on neurogenesis. Materials and Methods All experiments were conducted in accordance with the National Institute of Health Guide and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use committee of the University of South Florida, College of Medicine, Tampa, Fl. Animals : t o evaluate the effects isoflurane and propofol on the developing and aging brain, rats of two different ages were used for this study: 4month old (young) and 21month old (aged) male Fischer 344 rats (Harlan Sprague Dawley, Indianapolis, IN). All rats wer e acclimated and handled for two weeks prior to the beginning of the experimental procedures.

PAGE 34

23 Isoflurane Anesthesia : one cohort of rats was randomly assigned to four different groups (n=12 per group): young rats exposed to 1.5% isoflurane delivered in 2 L /min O2 via nose cone for 3 hours, young rats exposed to a mixture of air and 2 L/min O2 in the cage for 3 hours, aged rats exposed to 1.5% isoflurane delivered in 2 L/min O2 via nose cone for 3 hours, and aged rats exposed to a mixture of air and 2 L/min O2 in the cage for 3 hours. I soflurane (Forane, Ohmeda Caribe, NJ, USA) was delivered via a standard anesthesia vaporizer. Perioperiative parameters such as body temperature, oxygen saturation and heart rate were measured throughout the anesthesia period. Body temperature of rats was maintained at 37C with a thermostatically controlled heating pad. Propofol Anesthesia : a separate cohort of rats was randomly assigned to four different groups breathing air (n=10 per group): young rats that received 35 mg/kg/hr of propofol or 10% intralipid (control) for 3 hrs, and aged rats that received 35 mg/kg/hr of propofol or 10% intra lipid for 3 hours. Unanesthetized rats were placed in a restrainer and a tail vein catheter was implanted and attached to a syringe pump (model 11 Plus; Harvard Apparatus). Propofol (Disoprivan; AstraZeneca) or 10 % intralipid (Fresenius Kabi; Sweden) that served as control was continuously administered for 3 hours. Body temperature of rats was maintained at 37C with a thermostatically controlled heating pad. BrdU Injections : t o evaluate the effects of isoflurane and propofol on new cell proliferation in the brain, 5Bromo 2deoxyuridine (BrdU; Sigma Aldrich Munich, Germany) was injected intraperitoneally at a dose of 50 mg/kg in all groups immediately following the anesthesia or control procedure and 18 hours after the first injection.

PAGE 35

24 Cognitive assessme nt: c ognitive function was assessed using a task that requires the rats to make odor reward associations using foraging for palatable food, which results in rapid acquisition with minimal stress and fear (Eich enbaum 1998) Rats performed the novel odor discrimination task two days after exposure to either isoflurane or propofol anesthesia. Rats were initially habituated to a 97 cm2 black Plexiglas box, and shaped to receive treats in the box. Rats were food deprived the night prior to behavioral testing. Two odors were presented in adjacent corners of the box. A treat was placed next to one odor while the second odor was not paired to a reward (Fortin et al. 2002) The rat was placed in the middle of the box facing away from the odors and reward and all owed to explore the novel odors and find the reward. The position of the odor/reward pair was randomized between trials. Each rat received ten learning trials (120 seconds cut off). Each rat received one test trial, which assessed accuracy of memory and ti me to reach the reward. An Etho Vision computerized tracking and movement analysis system (Noldus Information Technology) was used to record and collect behavioral data. Due to technical difficulties w ith the tracking system, number of errors and time spent on each corner during the olfactory task was only recorded for young rats exposed to either propofol or intralipid. A blinded individual performed the behavioral experiment and all equipment used for behavioral testing was wiped with 70% ethanol bet ween animal uses to eliminate olfactory trails. Tissue p reparation and BrdU immunohistochemistry : one week after anesthesia, rats were deeply anesthetized with pentobarbital (50 mg/kg, i.p.) and transcardially perfused with 100 ml saline. Brains were removed, and immersion fixed in 4% paraformaldehyde for 5 days, and then placed in 20% sucrose. Frozen sagittal

PAGE 36

25 with 50% formamide/2X Saline Sodium Citrate (SSC) at 65C f or 2 hours, rinsed in 2X (SSC), incubated in 2N HCL for 30 minutes at 37C, and then washed with borate buffer (pH 8.5), followed by phosphate buffered saline (PBS). Subsequently, endogenous peroxidase activity was quenched with 3% H2O2 solution in PBS. Se ctions were blocked for 1 hour in 3% normal horse serum and 0.25% Triton X 100 in PBS (PBS TS). Sections were incubated overnight with mouse anti rat BrdU (1:100; Roche) in PBS TS. The following day, sections were washed in PBS, incubated for one hour in biotinylated secondary antibody (horse anti mouse IgG rat adsorbed 1:200; Vector Laboratories, Burlingame, CA) in PBS TS, and washed in PBS before incubation for 1 hour in avidinbiotin substrate (ABC kit, Vector Laboratories, Burlingame, CA). Sections were washed in PBS for 10 minutes and reacted with 3,3 diaminobenzidine tetrahydrochloride ( DAB) solution (Thermo Scientific, Rockford, IL). Sections were then mounted onto glass slides, dehydrated, and coverslipped with mounting medium ( Cytoseal 60,Stephens Scientific, Riverdale) (Bachstetter et al. 2010) Microscopy : unbiased stereological methods were used to estimate the numbers of BrdU labeled cells (BrdU+) in the subgranular zone of the DG. Twelve equally spaced sections throughout the medial lateral extent of the DG were collected. Because BrdU+ cells are a rare event, th e number of BrdU+ cells in each DG examined was summed for individual animals and the sum from each animal was then multiplied by the section spacing to estimate the total number of BrdU+ cells (Mouton 2002) These data were then used to calculate group means for estimates of total BrdU+ cells. Labeled cells that were greater than 1 cell diameter away from the subgranular layer were not included in the

PAGE 37

26 count (Figure 2 ). Onl y cells that had a clearly defined nuclear outline, with speckled or solid labeling over the nucleus, were considered BrdU positive. BrdU+ cells were visualized using a bright field microscope by an individual blinded to the treatment of the animals. Stati stical a nalysis : s tatistical analysis was performed using a two way analysis of variance (ANOVA) for repeated measurements followed by a Tukeys post hoc test. All results are presented as the mean SEM. Statistical comparison of the data was performed using GraphPad Prism version 5.00 for Mac (GraphPad Software, San Diego California USA, www.graphpad.com ) Results Physiologic al p arameters : perioperiative variables were recorded every thirty minutes during the anesthesia procedure. Body temperature of the rats was maintained at 37C. Oxygen saturation averaged 85% and 86%, respectively, for aged and young rats exposed to propofol, and 94% and 95%, respectively, for age d and young rats exposed to isoflurane. Heart rate averaged 327 and 340 beats per minute, respectively, for aged and young rats exposed to propofol, and 297 and 328 beats per minute, respectively, for aged and young rats exposed to isoflurane. New cell proliferation in the DG of young rats is decreased by isoflurane anesthesia : f igure 3 shows new cell proliferation assessed immediately after isoflurane anesthesia. A two way Analysis of Variance ANOVA (F3, 27= 67.39; p= 0.017) revealed that, when compared to the control group, isoflurane anesthesia significantly decreased the number of BrdU+ cells in the subgranular zone (SGZ) of the DG of young rats. There

PAGE 38

27 was not a statistical difference in the number of BrdU+ cells between aged rats exposed to isoflurane and aged control rats (p= 0.084). As previously reported (Seki and Arai 1995; Garcia et al. 2004; Luo et al. 2006; Lazarov et al. 2010) we found a decr ease in new cell proliferation in aged, as compared to young, rats (p< 0.0001). New cell proliferation in the DG of young rats is decreased by propofol anesthesia : new cell proliferation was assessed immediately after propofol anesthesia (Figure 4) A two way ANOVA (F3, 30= 51.13; p= 0.006) revealed that, when compared to the group exposed to intralipid, propofol anesthesia significantly decreased the number of BrdU+ cells in the DG of young rats. There was not a statistical difference in the number of BrdU+ cells between aged rats exposed to propofol and aged rats exposed to intralipid (p= 0.181). We also found a decrease in new cell proliferation in aged, as compared to young, rats (p< 0.0001) as expected. The number of BrdU+ cells in the olfactory bulb is not altered by either isoflurane or propofol anesthesia: t o assess whether anesthesia produced an overall decline in cell proliferation, the number of BrdU+ cells was counted in the granular and glomerular cell layer of the olfactory bulb (Figure 5 and Figure 6). No significant difference was found in the number of BrdU+ cells in the glomerular or granule cell layers in rats exposed to either propofol or isoflurane anesthesia when compared with controls. However, a decrease in new cell proliferatio n in aged, as compared to young rats (p< 0.0001) was found. Isoflurane causes cognitive impairment in aged rats : c ognitive function was assessed two days after rats were exposed to isoflurane (Figure 7 ). Data were analyzed using a twoway ANOVA with repea ted measures and revealed that young rats learned

PAGE 39

28 the task (p<0.0001). Aged rats also acquired the task (p<0.0001), though not as rapidly as young rats. Aged rats exposed to isoflurane showed a deficit in learning, as compared to the aged rats that served as controls exposed to room air (F3, 38= 21.11; p= 0.044). Learning in young rats exposed to isoflurane was not impaired as compared to the young control group (p= 0.495). Propofol causes cognitive impairment in young rats : c ognitive function was assessed two days after rats were exposed to propofol (Figure 8). Data were analyzed using a twoway ANOVA with repeated measures and revealed that young rats learned the task (p<0.0001). Aged rats also acquired the task (p<0.0001), though not as rapid as young ra ts. Young rats exposed to propofol showed a deficit in learning, as compared to other groups (F3, 40= 17.58; p= 0.01). Learning in the aged group exposed to propofol was not impaired when compared to the aged control group (p= 0.067) (Figure 8A) Moreover, young rats exposed to propofol were making significantly more errors than young controls (F1, 12= 6.49; p= 0.026) (Figure 8 B). When examining the amount of time spent in the correct v s. the incorrect corner (Figure 8C), it was found that young rats exposed to propofol were spending significantly more time in the incorrect corner as compared to young controls (F3, 296= 4.71; p= 0.0038) Discussion Effect of isoflurane and p ropofol on new cell proliferation in the brain : our study reveals three inter esting results. First, as previously reported (Seki and Arai 1995; Garcia et al. 2004; Luo et al. 2006; Lazarov et al. 2010) we found a decrease in new cell proliferation in aged rats compared to young rats, in both neurogenic regions. With

PAGE 40

29 increasing age, there is a dramatic decline in subventricular zone and DG cell proliferation. Second, thr ee hours of isoflurane or propofol anesthesia affect DG cell proliferation immediately after anesthesia in young rats, while new cell proliferation in the DG of aged rats is unaffected, suggesting an age dependent susceptibility. Cell proliferation in the young DG, although occurring at the higher rate, seems more susceptible to propofol and isoflurane anesthesia than DG cell proliferation in the aged brain (Shruster et al. 2010) Similar studies by other groups have shown an alteration of DG cell proliferation in P60 and P7 rats after 4 hours of isoflurane anesthesia (Stratmann et al. 2009) Third, we found that while isoflurane or propofol anesthesia resulted in a decreas e in the number of Brdu+ cells in the DG of young rats, the number of Brdu+ cells in the in the granular and glomerular cell layer of the olfactory bulb (OB) was unaffected. In this study, we wanted to examine whether the anesthetic agent effect was confin ed to the DG or if there was a generalized effect on progenitor cell proliferation in the brain with anesthesia. We found that isoflurane and propofol seem to have a specific effect on the DG. New cell proliferation in the DG may be more susceptible to dis ruption by anesthetics than new cell proliferation in the SVZ. For example, a reduction in new cell proliferation in the SGZ, but not in the SVZ has been reported after low dose irradiation of the heads of adult rodents (Tada et al. 2000; Mizumatsu et al. 2003; Snyder et al. 2005) Few studies have focused on DG cell proliferation with propofol anesthesia. Studies report no alterations in cell proliferation after 6 hours of propofol via jugular vein

PAGE 41

30 catheter and 100 mg/kg of BrdU given intraperitoneally (Lasarzik 2006) Similarly, a study in which young and middle aged Sprague Dawley rats received one of four anesthetics (propofol, isoflurane, dexmedetomidine, and ketamine) for 8 hrs and received 200 mg/Kg BrdU during the anesthetic period found no effect of the anesthetics on DG cell proliferation (Tung et al. 2008) The discrepancies between these studies and ours may be due to differences in the rat strain (F344 vs. Sprague Dawley), the timing of the anesthetic (6 hrs vs. 3 or 4 hrs), the dose of BrdU (100, 200 or 50 mg/Kg), or timing of the BrdU injection (before, during or following anesthesia). Effect of isoflurane and propofol a nesthesia on cognitive function: c ognitive impairment has been observed in aged rats (1820 month old) following exposure to anesthesia using the 12 arm radial arm maze. When studying the effect of anesthesia on old memory retrieval, aged rats were impaired while adult rats improved their performance (Culley et al. 2003) On the other hand, when animals were tested for new memory formation 2 weeks after exposure to anesthesia, aged animals were impaired (Culley et al. 2004a) while adult animals (6 month old) showed no impairment (Crosby et al. 2005) In our study, aged animals in the control group took less time to find the reward than aged animals exposed to isoflurane even though both groups learned the task. Aged animals in the isoflurane group took more time to reach the reward and made more errors during the task than aged control animals. The refore, learning was delayed by the anesthetic. When looking at the effect of isoflurane on cognition on young animals under the same testing conditions, we found that cognitive function is unaffected by isoflurane exposure. Our study, supports previous fi ndings of cognitive impairment after isoflurane anesthesia (Culley et al. 2003; Culley et al. 2004a; Culley et al. 2004b)

PAGE 42

31 Pr opofol is widely used among pediatric and geriatric patients. However, there are few studies on its effect on cognitive function in an animal model. In one study, propofol anesthesia in aged rats (18 month old) did not affect spatial memory assessed by the 12arm radial maze (Lee et al. 2008) Similarly, in o ur study, propofol anesthesia had no effect on cognition in aged rats using the olfactory association task. However, we found that, similar to isoflurane, propofol had an age dependent effect, though opposite to that found with isoflurane. Propofol caused cognitive impairment in young animals. Conclusion : i n summary, we found that isoflurane and propofol anesthesia produce cognitive impairments as assessed by our olfactory behavioral paradigm that are age, and agent dependent. The effect on progenitor cell proliferation is age dependent and selective to the dentate gyrus. Furthermore, propofol in young animals impairs learning and decreases progenitor cell proliferation. This study is the basis for more detailed studies on the effect of these two anesthetic agents on the process of neurogenesis

PAGE 43

32 References Altman J, Das GD (1965) Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J Comp Neurol 124: 319335 Bachstetter AD, Jernberg J, Schlunk A, Vila JL, Hudson C, Cole MJ, Shytle RD, Tan J, Sanberg PR, Sanberg CD, Borlongan C, Kaneko Y, Tajiri N, Gemma C, Bickford PC (2010) Spirulina promotes stem cell genesis and protects against LPS induced declines in neural stem cell proliferation. PLoS One 5: e10496 Baudier J, Glasser N, Gerard D (1986) Ions binding to S100 proteins. I. Calcium and zinc binding properties of bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein: Zn2+ regulates Ca2+ binding on S100b protei n. J Biol Chem 261: 81928203 Coleman GL, Barthold W, Osbaldiston GW, Foster SJ, Jonas AM (1977) Pathological changes during aging in barrier reared Fischer 344 male rats. J Gerontol 32: 258278 Couillard Despres S, Winner B, Schaubeck S, Aigner R, Vroemen M, Weidner N, Bogdahn U, Winkler J, Kuhn HG, Aigner L (2005) Doublecortin expression levels in adult brain reflect neurogenesis. Eur J Neurosci 21: 114 Crosby C, Culley DJ, Baxter MG, Yukhananov R, Crosby G (2005) Spatial memory performance 2 weeks after general anesthesia in adult rats. Anesth Analg 101: 13891392 Culley DJ, Baxter M, Yukhananov R, Crosby G (2003) The memory effects of general anesthesia persist for weeks in young and aged rats. Anesth Analg 96: 10041009, table of contents Culley DJ, Ba xter MG, Crosby CA, Yukhananov R, Crosby G (2004a) Impaired acquisition of spatial memory 2 weeks after isoflurane and isoflurane nitrous oxide anesthesia in aged rats. Anesth Analg 99: 13931397; table of contents Culley DJ, Baxter MG, Yukhananov R, Crosby G (2004b) Long term impairment of acquisition of a spatial memory task following isoflurane nitrous oxide anesthesia in rats. Anesthesiology 100: 309314

PAGE 44

33 Duan X, Kang E, Liu CY, Ming GL, Song H (2008) Development of neural stem cell in the adult brain. C urr Opin Neurobiol 18: 108115 Eichenbaum H (1998) Using olfaction to study memory. Ann N Y Acad Sci 855: 657669 Eriksson PS, Perfilieva E, Bjork Eriksson T, Alborn AM, Nordborg C, Peterson DA, Gage FH (1998) Neurogenesis in the adult human hippocampus. N at Med 4: 13131317 Fortin NJ, Agster KL, Eichenbaum HB (2002) Critical role of the hippocampus in memory for sequences of events. Nat Neurosci 5: 458462 Gage FH (2002) Neurogenesis in the adult brain. J Neurosci 22: 612613 Galvan V, Jin K (2007) Neuroge nesis in the aging brain. Clin Interv Aging 2: 605610 Garcia A, Steiner B, Kronenberg G, BickSander A, Kempermann G (2004) Age dependent expression of glucocorticoidand mineralocorticoid receptors on neural precursor cell populations in the adult murine hippocampus. Aging Cell 3: 363371 Hemmings HC, Jr., Akabas MH, Goldstein PA, Trudell JR, Orser BA, Harrison NL (2005) Emerging molecular mechanisms of general anesthetic action. Trends Pharmacol Sci 26: 503510 Jessberger S, Clark RE, Broadbent NJ, Clem enson GD, Jr., Consiglio A, Lie DC, Squire LR, Gage FH (2009) Dentate gyrus specific knockdown of adult neurogenesis impairs spatial and object recognition memory in adult rats. Learn Mem 16: 147154 Jevtovic Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF (2003) Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci 23: 876882 Johnson T, Monk T, Rasmussen LS, Abildstrom H, Houx P, Korttila K, Kuipers HM, Hanning CD, Siersma VD, Kristensen D, Canet J, Ibanaz MT, Moller JT (2002)

PAGE 45

34 Postoperative cognitive dysfunction in middle aged patients. Anesthesiology 96: 13511357 Kempermann G (2002) Why new neurons? Possible functions for adult hippocampal neurogenesis. J Neurosci 22: 635638 Lasarzik IE, K; Orth, C; Kornes, F; Werner, C (2006) Influence of Propofol on Endogenous Stem Cell Proliferation in the Dentate Gyrus of Adult Rats. Journal of Neurosurgical Anesthesiology 18: 287288 Lazarov O, Mattson MP, Peterson DA, Pimplikar SW, van Praag H (2010) When neurogenesis encounters aging and disease. Trends Neurosci 33: 569579 Lee IH, Culley DJ, Baxter MG, Xie Z, Tanzi RE, Crosby G (2008) Spatial memory is intact in aged rats after propofol anesthesia. Anesth Analg 107: 1211 1215 Luo J, Daniels SB, Lennington JB, Notti RQ, Conover JC (2006) The aging neurogenic subventricular zone. Aging Cell 5: 139152 Ming GL, Song H (2005) Adult neurogenesis in the mammalian central ner vous system. Annu Rev Neurosci 28: 223250 Mizumatsu S, Monje ML, Morhardt DR, Rola R, Palmer TD, Fike JR (2003) Extreme sensitivity of adult neurogenesis to low doses of X irradiation. Cancer Res 63: 40214027 Moller HJ, Gagiano CA, Addington DE, Von Knor ring L, Torres Plank JF, Gaussares C (1998a) Long term treatment of chronic schizophrenia with risperidone: an openlabel, multicenter study of 386 patients. Int Clin Psychopharmacol 13: 99106 Moller JT, Cluitmans P, Rasmussen LS, Houx P, Rasmussen H, Can et J, Rabbitt P, Jolles J, Larsen K, Hanning CD, Langeron O, Johnson T, Lauven PM, Kristensen PA, Biedler A, van Beem H, Fraidakis O, Silverstein JH, Beneken JE, Gravenstein JS (1998b) Long term postoperative cognitive dysfunction in the elderly ISPOCD1 st udy. ISPOCD investigators. International Study of Post Operative Cognitive Dysfunction. Lancet 351: 857861

PAGE 46

35 Monk TG, Weldon BC, Garvan CW, Dede DE, van der Aa MT, Heilman KM, Gravenstein JS (2008) Predictors of cognitive dysfunction after major noncardiac surgery. Anesthesiology 108: 1830 Mouton PR (2002) Principles and Practices of Unbiased Stereology: An introduction for bioscientistss. The Johns Hopkins University Press, Baltimore, Maryland Sarnat HB, Nochlin D, Born DE (1998) Neuronal nuclear antigen ( NeuN): a marker of neuronal maturation in early human fetal nervous system. Brain Dev 20: 8894 Scholzen T, Gerdes J (2000) The Ki 67 protein: from the known and the unknown. J Cell Physiol 182: 311322 Seki T, Arai Y (1995) Age related production of new granule cells in the adult dentate gyrus. Neuroreport 6: 24792482 Shors TJ, Townsend DA, Zhao M, Kozorovitskiy Y, Gould E (2002) Neurogenesis may relate to some but not all types of hippocampal dependent learning. Hippocampus 12: 578584 Shruster A, Melamed E, Offen D (2010) Neurogenesis in the aged and neurodegenerative brain. Apoptosis Snyder JS, Hong NS, McDonald RJ, Wojtowicz JM (2005) A role for adult neurogenesis in spatial long term memory. Neuroscience 130: 843852 Stratmann G, Sall JW, May LD, Bel l JS, Magnusson KR, Rau V, Visrodia KH, Alvi RS, Ku B, Lee MT, Dai R (2009) Isoflurane differentially affects neurogenesis and long term neurocognitive function in 60day old and 7day old rats. Anesthesiology 110: 834 848 Tada E, Parent JM, Lowenstein DH, Fike JR (2000) X irradiation causes a prolonged reduction in cell proliferation in the dentate gyrus of adult rats. Neuroscience 99: 3341 Taupin P (2006a) Adult neural stem cells, neurogenic niches, and cellular therapy. Stem Cell Rev 2: 213219

PAGE 47

36 Taupin P (2006b) Neural progenitor and stem cells in the adult central nervous system. Ann Acad Med Singapore 35: 814820 Tung A, Herrera S, Fornal CA, Jacobs BL (2008) The effect of prolonged anesthesia with isoflurane, propofol, dexmedetomidine, or ketamine on neural cell proliferation in the adult rat. Anesth Analg 106: 17721777 Vega CJ, Peterson DA (2005) Stem cell proliferative history in tissue revealed by temporal halogenated thymidine analog discrimination. Nat Methods 2: 167169 Wilder RT, Flick RP, Sprung J, Katusic SK, Barbaresi WJ, Mickelson C, Gleich SJ, Schroeder DR, Weaver AL, Warner DO (2009) Early exposure to anesthesia and learning disabilities in a population based birth cohort. Anesthesiology 110: 796804 Zhang G, Dong Y, Zhang B, Ichinose F, Wu X, Culley DJ, Crosby G, Tanzi RE, Xie Z (2008) Isoflurane induced caspase 3 activation is dependent on cytosolic calcium and can be attenuated by memantine. J Neurosci 28: 45514560 Zhao C, Deng W, Gage FH (2008) Mechanisms and functional implications of adult neurogenesis. Cell 132: 645660

PAGE 48

37 Figure 2. Immunohistochemical labeling of proliferating cells within the dentate gyrus (DG) with bromodeoxyuridine (A) 4 month old propofol (B) 4 monthold intralipid (C) 21 month old propofol (D) 21 month old intralipid.

PAGE 49

38 Figure 3. New cell proliferation in the DG of young rats is decreased by isoflurane anesthesia Number of BrdU+ cells in the dentate gyrus (DG) of young (4 month old) and aged (21 monthold) rats, after 3 hours of 1.5% isoflurane anesthesia (isoflurane) or no isoflurane (control). There is a statistically significant difference between young control and young isoflurane exposed rats (*p= 0.0171), but not a statistically significant difference in the aged groups.

PAGE 50

39 Figure 4. New cell proliferation in the DG of young rats is decreased by propofol anesthesia Number of BrdU+ cells in the dentate gyrus (DG) of young (4 month old) and aged (21 monthold) rats, after 3 hours of 35 mg/Kg/hr of propofol anesthesia (propofol) or 35 mg/kg/hr *p= 0.0171.of 10% intralipid (intralipid). There is a statistical ly significant difference between young intralipid and young propofol infused rats (*p=0.0062), but there is not difference in the age d groups.

PAGE 51

40 Figure 5. The number of BrdU+ cells in the o lfactory bulb is not altered by isoflurane. Number of BrdU+ cells in the granular and glomerular cell layer of the olfactory bulb (OB) of young (4 month old) and aged (21 month old) rats, after 3 hours of 1.5% isoflurane anesthesia (isoflurane) or no isoflurane (control). There was not a statistica l ly significant difference between the controls and the isoflurane exposed rats.

PAGE 52

41 Figure 6. The number of BrdU+ cells in the olfactory bulb is not altered by propofol anesthesia. Number of BrdU+ cells in the granular and glomerular cell layer of the olfactory bulb (OB) of young (4 monthold) and aged (21 month old) rats, after 3 hours of 35 mg/Kg/hr of propofol anesthesia (propofol) or 35 mg/kg/hr of 10% intralipid (intralipid). There was not a statistically significant difference between the controls and the isoflurane exposed rats.

PAGE 53

42 Figure 7. Isoflurane causes cognitive impairment in aged rats. This graph shows the a verage time to find reward during the olfactory learning test of young (4 month old) and aged (21 monthold) rats 7 days after 3 hours of 1.5% isoflurane anesthesia (isoflurane) or no isoflurane (control). Animals were tested on the behavioral paradigm two days following isoflurane anesthesia. Aged rats also acquired the behavioral task (p<0.0001), though not as rapid as young rats. Learning in aged animals was impaired by isoflurane (*p= 0.0443).

PAGE 54

43 Figure 8. Propofol causes cognitive impairment in young rats. Rats were tested on the behavioral parad igm two days following propofol anesthesia. (A) This graph shows the a verage time to find reward during the olfactory learning test of young (4 month old) and aged (21 monthold) rats 7 days a fter 3 hours of 35 mg/Kg/hr of p ropofol anesthesia (Propofol) or 35 mg/kg/hr of 10% intralipid (intralipid). Aged rats also acquired the behavioral task (p<0.0001), though not as rapid as young rats. Learning in young rats was impaired by propofol (*p=0.0099). (B) This graph shows the n umber of errors made by young rat s exposed to either propofol or intralipid during the olfactory learning test. Young rats exposed to propofol made significantly more errors than young controls (p=0.026). (C) Time spent in correct vs. incorrect corner of young rats exposed to either propofol or intralipid. Young rats exposed to propofol spent significantly more time in the incorrect corner than young rats exposed to intralipid (*p= 0.0038). In addition, young rats exposed to propofol spent significantly more time in the incorrect vs. the correct corner (**p=0.0167)

PAGE 55

44 CHAPTER 2 THE INHALATION ANEST HETIC ISOFLURANE AFF ECTS NASCENT CELLS UNDERGOING MATURATIO N AND SYNAPTIC INTEG RATION IN THE DENTATE GYRUS OF AGE D, BUT NOT YOUNG RAT S Abstract Background: Worldwide volatile anesthetics are used in millions of young and elderly patients Nevertheless recent studies on human and animal s s uggest that anesthesia early or late in life may cause cognitive impairment, the mechanism of which is still unknown. Postnatal hippocampal neurogenesis is involved in learning and memory (Culley et al. 2004a; Coras et al. 2010) and an anestheticinduc ed alteration of postnatal neurogenesis may, in part, explain the cognitive impairment observed after anesthesia. This study asserts the effects of isoflurane on neurogenesis Even though, the majority of studies focus on the effects of isoflurane on postnatal neurogenesis after the exposure, this study concentrate s on the effects of isoflurane on the undergoing neurogenesis at the time of exposure. Methods: 1.5% isoflurane in 2L/min O2 was administered via nose cone for 3 hours to 3monthold and 20 monthold F344 rats. Three different thymidine analogs (CldU, IdU, and EdU) were intraperitoneally injected at three different time points (21 days, 8 days, and 4 days respectively ) prior to isoflurane or contr ol exposure in order to

PAGE 56

45 assess integration, maturation and differentiation of nascent cells in the dentate gyrus (DG) at the time of isoflurane anesthesia. Moreover, phenotype identification at each time point was assessed by co localiz ation of each thymidine analog with the corresponding histological marker for each stage of neurogenesis. TUNEL assay was also performed to assess cell death in the DG after isoflurane. Results: In this study, isoflurane did not affect any stage of postna tal neurogenesis in young rats. Even though, no differences were detected on hippocampal progenitor proliferation, nascent cell differentiation, or migration and axon dendrite targeting of nascent cells in the hippocampus of aged rats isoflurane significantly decreased the number of nascent cells that were in the process of integration at the time of exposure (p=0.022) only in the aged rats. Additionally it was found that in aged rats ; isoflurane significant ly reduced neuronal produc tion (p=0.008), while astrocyte production was significantly increased (p=0.003). A significant effect of isoflurane on cell death was not found. Conclusion: These results suggest an age and stage dependent effect of isoflurane Because isoflurane anesthesia appears to be particularly harmful to the aged brain, especially to nascent cells undergoing maturation and synaptic integration in the dentate gyrus of the hippocampus Keywords : isoflurane, cognitive impairment, postnatal neurogenesis, cogni tion

PAGE 57

46 Introduction I ncreasing clinical and laboratory findings suggest long term or even permanent impairment of cognitive function following the administration of an anesthetic drug (Moller et al. 1998; Loepke and Soriano 2008) A commonly used agent to induce and maintain general anest hesia is the inhaled anesthetic isoflurane which is widely used in various types of surgery in pedi atric and geriatric patients. Isoflurane is thought to produce its effects by acting as a aminobutyric acid ( GABA ) agonist and a N methyl d aspartate ( NMDA ) receptor antagonist. Recent laboratory data indicate that isoflurane exposure induces neurodegeneration in newborns (Jevtovic Todorovic et al. 2003; Li et al. 2007) and that anesthesia exposure during development is a significant risk factor for impairment of cognitive function later in life (Wilder et al. 2009) Additionally Culle y et al. found that in 18mo old rats, anesthesia for 2h with 1.2% isoflurane/ nitrous oxide/ oxygen produces long lasting, but reversible impairment on a new and on previously learned spatial memory task (Culley et al. 2003; Culley et al. 2004a) Spatial memory is considered to be a hippocampal cognitive domain (Snyder et al. 2005) An important determinant of hippocampal function is postnatal generation of neurons throughout life in certain areas of the brain, particularly the dentate gyrus (DG) of the hippocampus, and the subventric ular zone (SVZ) of the lateral ventricle (E riksson et al. 1998; Gage 2002; Coras et al. 2010) Postnatal neurogenesis i s more pronounced early in life and decreases with age (Luo et al. 2006; Galvan and Jin 2007; Lazarov et al. 2010) Even though prior studies label neurogenic cells after isoflurane exposure (Tung et al. 2008; Stratmann et al. 2010) this study considers it important to label neurogenic cells prior to isoflurane exposure, in order to study its effects on each stage of postnatal

PAGE 58

47 neurogenesis P ostnatal neurogenesis is not a one step but a multi step process that involves the proliferation of progenitors, differentiation and maturation of the nascent cells, and the incorporation of these cells into the existing circuit. And an alteration of one or more of these stages may result in cognitive dys function. Material and Methods A nimals : a ll experiments were conducted in accordance with the National Institute of Health Guide and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use committee of the University of South Florida, College of Medicine. Male Fisher 344 (F344) rats (Harlan Sprague Dawley, Indianapolis, IN), were pair housed in environmentally controlled conditions (12:12h light: dark cycle at 211C) and provided food and water ad lib. Rats of two age groups were used in this study, young (3 months old) and aged (20 mont hs old). Rats were excluded from the study if they became jaundiced, or had abdominal tumors. Experiment al timeline : t hree different thymidine analogs (CldU, IdU, and EdU) were intraperitoneally injected at three different time points (21 days, 8 days, and 4 days respectively ) prior to the anesthetic or control exposure in order to assessed integration, maturation and differentiation of nascent cells in the dentate gyrus (DG) at the time of isoflurane anesthesia. Moreover, phenotype i dentification at each time point was assessed by co localization of each thymidine analog with the corresponding histological marker for each neurogenic stage. Quantification of co localization of CldU and the mature neuronal marker NeuN or the astrocytic marker S100 assessed nascent cells undergoing synaptic integration at the time of isoflurane anesthesia. Quantification of co expression

PAGE 59

48 of IdU and the immature neuronal marker DCX or the astrocytic marker S100 assessed nascent cells undergoing migration and axon/dendrite targeting. Furthermore, b y determining co localization of EdU with the immature neuronal marker DCX or the astrocytic marker S100 nascent cells undergoing differentiation were assessed. Lastly, nascent cells proliferation was assessed u sing the endogenous marker Ki67. Labeling of each neurogenic stage was performed in the same animal (Figure 9). Preparation and administration of thymidine a nalogs : s olutions of 5Chloro2 deoxyuridine (CldU) and 5Iodo2 Deoxyuridine (IdU) were prepared at equimolar concentrations. CldU (Sigma #C6891) was prepared at 17 mg/ml and IdU (MP#100357) at 23 mg/ml. Injections at 2.5 ml/Kg resulted in an equimolar final effective delivery of 42.5 mg/Kg CldU or 57.5 mg/Kg IdU as previously described (Vega and Peterson 2005) Both halogenated thymidine analog solutions were prepared in sterile saline and administered by intraperitoneal (IP) injection to ensure precise delivery to facilitate quantitative comparisons Because cell division is a rare event in the subgranular zone of elderly rats and because m any of these newborn cells do not survive, we were concerned that a short thymidine analog labeling protocol would not yield a new cell number sufficient for a meaningful analysis. Therefore, three times the thymidine analog corresponding to a specific time point was administered; three IP injections at 4 hour intervals in one day were given. As shown in Figure 9, animals in all groups were injected with CldU, 21 days before control or isoflurane exposure ; and IdU, 8 days before control or isof lurane exposure A solution of 5ethynyl 2 deoxyuridine (EdU) was prepared at a concentration of 60 mg/ml in sterile saline. IP injections of EdU (cat #. E10187, Invitrogen, Carlsbad, CA)

PAGE 60

49 resulted in a final effective delivery of 160 mg/Kg., A nimals in a ll groups were injected only once with EdU, 4 days before isoflurane anesthesia (Fig ure 9) Isoflurane anesthesia : a fter a two week acclimation period to the vivarium under standard conditions, rats were randomly ass igned to four different groups (n=8 per group): young rats exposed to 1.5% isoflurane delivered in 2 L/min O2 via nose cone for 3 hours, young rats exposed to a mixture of air and 2 L/min O2 in the cage for 3 hours, aged rats exposed to 1.5% isoflurane del ivered in 2 L/min O2 via nose cone for 3 hours, and aged rats exposed to a mixture of air and 2 L/min O2 in the cage for 3 hours. I soflurane (Forane, Ohmeda Caribe, NJ, USA) was delivered via a standard anesthesia vaporizer. Body temperature of rats was maintained at 37C throughout the anesthesia period with a thermostatically controlled heating pad. Measurement of p hysiological p arameters during isoflurane anesthesia : r e ctal temperature was monitored throughout the exposure and recorded every 30 minutes using a thermaletmonitoring thermometer ( Physitemp instrument Inc, Clifton, NJ, USA). Moreover, hemoglobin oxygen saturation (SpO2) and heart rate (HR) were recorded every 30 minutes using the SurgiVet multiparameter monitor (S miths medical, Dublin, OH, USA). Diastolic, systolic and mean a rterial pressures were measured every 30 minutes throughout the anesthesia period by tail cuff using the CODA noninvasive blood pressure system for rats (CODA2, Kent Scientific Corporation). Tissue collection and processing : twenty four hours after control or isoflurane exposure rats were deeply anesthetized with pentobarbital (50 mg/ Kg, i.p) and transcardially perfused with 100 ml saline f ollowed by ice cold 4% paraformaldehyde in 0.1M PBS. Brains were removed, postfixed in the same fixative solution at 4 C

PAGE 61

50 overnight, transferred to 20% sucrose in PBS until equilibrated, and the n frozen in Tissue Tek OCT before cryostat sectioning. Sagittal cryosections (30 m thick) through the entir e DG were collected serially, transferred to a cryoprotect ant solution, and stored at 20C until used. These sections were used to assess proliferation and phenotypic determination of nascent cells in the DG. All standard staining procedures were conducted on free floating sections using every twelfth section through the entire hippocampus beginning with a random start and including sections before and after the hippocampus to ensure that the entire structure was sampled. CldU and IdU i mmunohistochemistry : s ections were washed three times for 10 min in PBS before being treated with 0.2N HCL for 10 min at 37C for DNA denature to facilitate antibody access, sections were then washed with borate buffer (pH 8.5), followed by three washes of PBS. Sections were then incubated in 5% normal goat serum and 0.25% Triton X 100 in PBS (PBS TS) for 60 min, and stained at 4 C. Detection of halogenated thymidine analogs was accomplished using rat anti BrdU (Accurate cat# OBT 0030; clone BU1/75) at 1:250 for CldU and mouse anti BrdU (Becton Dickinson cat# 347580; clone B44) at 1:500 for IdU. All secondary antibodies were used at 1:300 and were conjugated to the fluorophore Alexa 594 or Alexa 488. EdU labeling protocol : Ed U labeling was performed using Click iTTM EdU imaging kit cat # C10339 (Invitrogen, Carlsbad, CA) according to the manufacturers protocol. This protocol is normally intended for use in cell culture, but was adapted for histological staining of brain tissue. Sagittal cryosections (30 m thick) were w ashed three times for 10 min in PBS. After washing twice with 3% bovine serum albumin (BSA) in PBS sections were permeablized with 0.5% Triton X 100 in PBS for 20 min.

PAGE 62

51 S ections were then washed t wice with 3% BSA in PBS and incubated with the Click iTTM re action cocktail ( CuSO4, Alexa Fluor 594 Azide, and manufactures reaction buffer additive ) for 30 min while protect ed from light. S ections were washed on e more time with 3% BSA in PBS, mounted onto glass slides, and coverslipped with Vectashield mounting medium for fluorescence (cat# H 1000 Vector Laboratories, Burlingame, CA, USA). Double immunohistochemistry for lineage markers : t he phenotypes of CldU, IdU and EdU labeled cells were determined using double immunofluorescent staining. Antibodies against doublecortin ( DCX ) neuronal nuclei ( NeuN ) and S100 were used to detect immature neurons, mature neurons and astrocytes, respectively. DNA denaturation, followed by incubation of the primary antibodies for CldU and IdU w as performed as described above. A rabbit polyclonal goat antibody raised against human DCX (1:200 cat# 4604 Cell Signaling technology, Inc. Danvers, MA, USA) was used in an antibody cocktail with the IdU antibody to assess immature neurons. A mouse monoclonal antibody against neuronal nucl ei, NeuN (MAB377 Millipore, Billerica, MA, USA) was used at a concentration of 1:500 in an antibody cocktail with the CldU antibody to assess mature neurons. A rabbit monoclonal goat antibody raised against S100 (ab52642 abcam, Cambridge, MA, USA) was use d at a concentration of 1:3000 in an antibody cocktail with CldU or IdU to assess astrocytes. For double labeling of EdU/DCX and EdU/S100 EdU staining was performed first, followed by incubation with DCX or S100 respectively. Ki67 i mmunohistochemistry : Ki67 is expressed in cells G1 through M phase of the cell c ycle (Scholz en and Gerdes 2000) For detection of Ki67, s ections were

PAGE 63

52 pretreated with 1X S aline Sodium Citrate (SSC) at 80 C for 40 minutes. Subsequently, endogenous peroxidase activity was quenched with 0.6 % H2O2 solution in PBS for 20 min Sections were block ed f or 1 hour in 2% normal goat serum and 0.25% Triton X 100 in PBS (PBSTS). Sections were incubated overnight at 4 C with a rabbit polyclonal antibody against human Ki67 (NCL Ki67p; Novocastra Laboratories/Vision BioSystems, Newcastle upon Tyne, UK) at a dilution of 1:2000 in PBS TS. The following day, sections were washed in PBS, incubated for one hour in biotin ylated secondary antibody (goat anti rabbit IgG rat adsorbed 1:1000; BA 1000 Vector Laboratories, Burlingame, CA) in PBS TS, and washed in PBS before incubation for 1 hour in avidinbiotin substrate (ABC kit cat no. PK 6100, Vector Laboratories, Burlingame, CA) Sections were washed in PBS for 10 minutes and reacted with 3,3 diaminobenzidine tetrahydrochloride ( DAB) solution ( cat no.1856090, Thermo Scientific, Rockford, IL). Sections were then mounted onto glass slides, dehydrated, and coverslipped with mounting medium ( Cytoseal 60,Stephens Scientific, Riverdale) Tunel assay protocol : i n order to determine if isoflurane had an effect on cell death t he in situ cell death detection kit, Fluorescein (cat no. 11684795910, Roche, Mannheim, Germany ) was used according to t he manufacturer's instructions. Sections were incubated with 3% bovine serum albumin in 0.1 mol/L Tris HCL (pH 7.5) for 30 mins followed by 50 L of TUNEL reaction mixture on each sample for 60 mins at 37C. After washing, slides were incubated with 0.3% H2O2 in methanol for 10 mins, followed by 3% bovine serum albumin in 0.1 mol/L Tris HCL (pH 7.5) for 30 mins at room temperature. Incubation with peroxidase conjugated anti fluorescein (diluted 1:5) at 37C for 30 mins was performed.

PAGE 64

53 Microscopy and cell co unting : unbiased stereological methods were used to estimate the numbers of positive labeled cells ( + labeled cells ) in the subgranular zone of the DG. Twelve equally spaced sections throughout the medial lateral extent of the DG were collected. Because + labeled cells are a rare event, the number of + labeled cells in each DG examined was summed for individual animals and the sum from each animal was then multiplied by the section spacing to e stimate the total number of + labeled cells (Mouton 2002) These data were used to calculate group m eans for estimates of total + labeled cells. +Labeled cells that were greater than 1 cell diameter away from the subgranular layer w ere not included in the count Sections were imaged on an Olympus FV1000 laser scanning microscope with four laser sources: 405 Diode, multi line Argon (for Alexa 488), green HeNe (for Alexa 594), and red HeNe was used for all i mmunofluorescence photomicro graphs. When quantification of percentage of positive cells was deter m ined, Z stacks were created at 0.5 m intervals throughout the 30 m of the sections with a guard region of 2 m excluded form top and bottom of the Z stack. The Z stacks were rotated in all planes to verify double labeling. Images of positive staining were adjusted to make optimal use of the dynamic range of detection. Statistical analyses : data are presented as mean SEM. Analysis of variance (ANOVA) with Tukeys post hoc or a t test w as used to compare the differences from the control group. P values less than 0.05 were considered statistically significant, the significance testing was one tailed and GraphPad Prism version 5.00 for Mac (GraphPad Software, San Diego California USA, www.graphpad.com ) was used to analyze the data.

PAGE 65

54 Results Physiological p arameters during i soflurane anesthesia : r ectal temperature was recorded every 30 minutes duri ng the isoflurane exposure in young (n=8) and aged (n=8) rats (Figure 10A). The body temperature was between 36 and 37C during the first hour, and then it increased almost 1 C and remained stable for the rest of the isoflurane exposure, with an average of 37 0.23 and 37 0.13 for young and aged rats respectively. Mean arterial blood pressure throughout the isoflurane exposure averaged 89.45 2.74 and 84.8 3.50 for young and aged rats respectively (Figure 10 B). Heart rate and hemoglobin oxygen saturation also remained stable during the 3 hours exposure (Figure 10C). An age difference in most of the measured parameters was detected. Postnatal p roliferation in the Dentate Gyrus, as assessed by the Ki67 marker, is u naffected by isoflurane anesthesia : t he effects of isoflurane exposure (1.5% for 3 hours) on neuronal cell proliferation in the SGZ in young (3 month old) and aged (20 monthold) F344 rats was determined by analyzing Ki 67 labeling, 24 hours after exposure. Figure 11A shows that Ki67 positive cells in both young and aged rats were primarily located on the border of the granule cell layer. Isoflurane does not appear to have an effect on newborn cells generated during the 24 hours after exposure (Figure 11B) The results were subjected to a 2 (a ge: young, aged) X 2 (treatment: control, isoflurane) factorial ANOVA. There was a significant effect of age (F (3, 7) = 8.57, p <0.001), but no significant effect of treatment (F (3, 7) = 0.51, p = 0.818). Isoflurane exposure did not result in a statistically significant effect in the young (p= 0.248) nor in the aged rats (p=0.212).

PAGE 66

55 Isoflurane does not affect nascent cells undergoing differentiation in the DG of the hippocampus at the time of ex posure : w e quantified the number of newborn cells undergoing differentiation at the time of the isoflurane exposure by estimating the number of EdU positive cells. And determined the phenotype of these newly born cells using double labeling of EdU and the immature neuronal marker DCX (Figure 12C) and EdU and the astrocytic marker S100 (Figure 2. 4A B). The resu lts were subject to a 2 (age: young, aged) X 2 (treatment: control, isoflurane) factorial ANOVA. There was a significant effect of age (F (2,11)= 16.61, p<0.0001), but no significant effect of treatment (F (2,11)= 6.61, p= 0.06). We found that there was no difference in the number of EdU+ cells and no difference in the proportion of EdU labeled cells starting to express neuronal or glial phenotype at the time of isoflurane exposure in young or aged rats (Figure 12A B ) Our analysis revealed that 57% of the EdU labeled cells in the young control rats expressed DCX, indicating neuronal differentiation, while only 4.2% of the EdU labeled cells express S100 indicating that they have differentiated into mature astrocytes When the percentage of each p henotype was summed, it revealed a number less than 100%, indicating that either some of these cells were already going through maturation and were expressing th e mature neuronal marker NeuN ( not quantified at this time point) or they were still undefined. From a detailed study by Palmer et al. (Palmer et al. 2000) it is known that at early time points proliferating endothelial cells account for a large percentage (more than 35% in the rat model of that study) of the dividing cells. Isoflurane does not affect nascent cells undergoing migration in the DG of the hippocampus at the time of exposure : w e quantified the number of newborn cells undergoing maturation at the time of the isoflurane exposure by estimating the number of

PAGE 67

56 IdU positive cells. And determined the phenotype of these newly born cells using double labeling of IdU and the immature neuronal marker DCX (Figure 13C) and IdU and the astrocytic marker S100 (Figure 2.5A B) The res ults were subject to a 2 (age: young, aged) X 2 (treatment: control, isoflurane) factorial ANOVA. There was a significant effect of age (F (7,11)= 70.18, p<0.0001), but no significant effect of treatment (F (7,11)= 1.87, p=0.086). We found that there was no difference in the number of IdU+ cells and no difference in the proportion of IdU labeled cells maturating into neurons or astrocytes at the time of isoflurane exposure in young or aged rats (Figure 13A B ). Our analysis revealed that 70% of the IdU labe led cells in the young control rats expressed DCX, indicating neuronal maturation, while only 15% of the IdU labeled cells express S100 indicating that they have differentiated into mature astrocytes. When the percentage of each phenotype was summed, it revealed a number less than 100%, indicating that either some of these cells were already going through maturation and were expressing the mature neuronal marker NeuN (not quantified at this time point). Isoflurane affects nascent cells undergoing maturation and integration in the DG of aged, but not young rats : w e estimated the number of newborn cells undergoing synaptic integration at the time of isoflurane exposure by estimating th e number of CldU positive cells, a nd determined the phenotype of t hese newly born cells (Figure 14A B) using double labeling of: CldU and the mature neuronal marker NeuN (Figure 14C) and CldU and the astrocytic marker S100 (Figure 2. 6D ). There was a significant effect of age (F (7,11)= 16.48, p<0.0001), and a significant effect of treatment (F (7,11)= 1.52, p=0.04). Isoflurane significantly decreased the number of CldU+ cells (*p=0.02), and the number of CldU+/NeuN+ cells (**p=0.008), but increased the number of

PAGE 68

57 CldU+/S100 + cells (***p=0.033). We found that i soflurane decreased the number of CldU labeled cells, neurogenesis, and astrocyte formation in the DG of aged, but not young rats The number of CldU labeled cells in aged rats was 36% lower in isoflurane exposed rats ( F (5,32)=5.74 p= 0.022) but unaffected in young rats (Figure 14C D). Isoflurane exposure reduced neuronal production, as judged by CldU+/NeuN+ colocalization, significantly in aged rats ( *p= 0.008) Conversely, the number of newborn astrocytes increased significantly in aged rats (***p=0.033) after isoflurane exposure. Cell death in the Dentate Gyrus after isoflurane exposure: c ell death positive label in the DG was assessed by the DNA strand breaks (TUNEL Assay) in young and aged rats 24 hours aft er isoflurane exposure (Figure 15). The results were subject to a 2 (age: young, aged) X 2 (treatment: control, isoflurane) factorial ANOVA. There was not a significant effect of age (F (2,3)= 3.90, p=0.07), and no significant effect of treatment (F (2,3)= 0.47, p=0.64). Isoflurane exposure did not result in a statistically significant effect in the young (p=0.06) nor in the aged rats (p=0.27). The number of cells positive for TUNEL was not different between isoflurane exposed or control rats, neither in the young n or in the aged groups (Figure 15) This indicates that isoflurane exposure has no effect on cell death in the DG. Discussion The rate of neurogenesis found in this study is consistent with previous reports of reduced neurogenesis in aged compared with young animals (Luo et al. 2006; Shruster et al. 2010) The initial hypothesis of this study was that isoflurane induced alteration of one or more stages of postnatal neurogenesis could in part explain the cognitive

PAGE 69

58 dysfunction experienced in some pati ents after anesthesia. This hypothesis was based on recent laboratory data showing that isoflurane exposure early in life results in subsequent behavioral impairment (Jevtovic Todorovic et al. 2003; Lu et al. 2006; Li et al. 2007; Loepke and Soriano 2008) a nd that isoflurane exposure late in life also produces enduring cognitive i mpairment (Culley et al. 2003; Culley et al. 2004a; Culley et al. 2004b) The main findings of this study are that isoflurane causes an alteration in nascent cells undergoing maturation and synaptic integration in the dentate gyrus (DG) of aged, but not young rats. Therefore, the ef fect s of isoflurane on postnatal neurogenesis are stage and age dependent. Isoflurane has an age dependent effect on postnatal neurogenesis : on this study isoflurane was found to be harmful to nascent cells exclusively in the DG of aged rats. Th i s finding may be attributed to the fact that in general the aged brain appears to be particularly vulnerable to insults that would go unnoticed at a different age, and to the fact that with age postnatal neurogenesis exhibits a decline. T he aged brain is u ndergoing the involuntary changes of senesce, and becoming more susceptible to neurodegeneration, with Alzheimers disease affecting nearly 50% of elders of 80 years (Xie et al. 2006) (Xie et al. 2007) Moreover, it has being shown that in the aged brain, NMDA receptor antagonists, such as nitrous oxide and ketamine, produce mitochondria swelling in cerebrocortical neurons of adult and aged rats, with the aged brain being more sensitive than the adult brain (Jevtovic Todorovic and Carter 2005) In addition, isoflurane has been implicated in Alzheimers disease (Xie et al. 2006; Xie et al. 2008; Zhang et al. 2008) In fact, a number of studies have demonstrated enduring cognitive

PAGE 70

59 dysfunction in aged, but not young rats anesthet ized previously with isoflurane (Culley et al. 2004a; Culley et al. 2004b) Postnatal neurogenesis exhibit s an agerelated decline (Seki and Arai 1995; Luo et al. 2006; Galvan and Jin 2007; Shruster et al. 2010) which has been associated with decline in hippocampal dependent spatial memory (Snyder et al. 2005; Zhao et al. 2008a; Zhao et al. 2008b; Coras et al. 2010) Desp ite an age related reduction in the formation of nascent hippocampal neurons, the neurons that are added appear functionally equival ent to those in young brain (Toni et al. 2008) Another study suggest that the number of neural stem cells does not decline with age, but that these cells rather have a decrease in the proliferating rate (Lugert et al. 2010) that could be due to a decreased volume of the vascular niche (Hattiangady and Shetty 2008) Besides the presence or absence of neural stem cells, the decline of neurogenesis in the aged brain might be due to a loss of extrinsic signals, a reduced responsiveness of the aging neural stem cells to normal signaling, or both. Since postnatal neurogenesis requires a specific environment, niche, that provides the signals needed to maintain and control the developmental stages of nascent cells (Ming an d Song 2005) The neurogenic niche and the nascent cells form a functional unit that is regulated by signaling of neurotransmitters, neurotrophic factors, and grow factors (Palmer et al. 2000) Therefore, an age related alteration in any of these signaling may account for the particularly susceptibility to isoflurane in the aged rat brain. Isoflurane specifically affects nascent cells undergoing maturation and synaptic integration in the DG of aged rats : t o the best of our knowledge this is the first study examining the effects of isoflurane anesthesia on each one of the stage s of

PAGE 71

60 postnatal neurogenesis at the time of exposure By using this experimental model, we were able to conclude that isoflurane specif ically affects nascent cells undergoing the last neurogenic stage, maturation and synaptic integration. Even though on this current study, cognitive function was not assessed it may be suggested that disappearance of these cells has an immediate effect on cognitive function. Culley et al have consistently found cognitive dysfunction in aged rats up t o three weeks after exposure to nitrous oxide, isoflurane or both (Culley et al. 2004a) However, they were not able to demonstrate cognitive dysfunction eight weeks after anesthetic ex posure. Furthermore Stratmann et al using a similar paradigm did not find cognitive dysfunction sixteen weeks after the anesthetic exposure (Stratmann et al. 2010) A possible explanation is that consistently with human data showing resolution of cog nitive dysfunction over time. In the majority of patients diagnose one week after surgery, there is no evidence of cognitive dysfunction three months after surgery and the majority of patient diagnose at three month after do not suffer from cognitive dysfunction one or two years after. This is consistent with our findings, isoflurane decreased of nascent cells undergoing maturation and synaptic integration so disappearance of these cells has an immediate effect on cognitive function as seen on the clinical setting. In addition, postnatal neurogenesis after the exposure may not be affected and the normal cycle of proliferation may be restored so that cognitive function is restored. Taking together, these findings indicate that depending on the age of the individual, isoflurane can be either harmful or harmless. Future studies, correlating immunohistochemistry and behavioral assessment may clarify how isoflurane anesthesia

PAGE 72

61 may result in cognitive dysfunction in some patients and from that a way of preventin g may be implemented.

PAGE 73

62 References Coras R, Siebzehnrubl FA, Pauli E, Huttner HB, Njunting M, Kobow K, Villmann C, Hahnen E, Neuhuber W, Weigel D, Buchfelder M, Stefan H, Beck H, Steindler DA, Blumcke I (2010) Low proliferation and differentiation capacities of adult hippocampal stem cells correlate with memory dysfunction in humans. Brain 133: 33593372 Culley DJ, Baxter M, Yukhananov R, Crosby G (2003) The memory effects of general anesthesia persist for weeks in young and aged rats. Anesth Analg 96: 10041009, table of contents Culley DJ, Baxter MG, Crosby CA, Yukhananov R, Crosby G (2004a) Impaired acquisition of spatial memory 2 weeks after isoflurane and isoflurane nitrous oxide anesthesia in aged rats. Anesth Analg 99: 13931397; table of contents Culley DJ, Baxter MG, Yukhananov R, Crosby G (2004b) Long term impairment of acquisition of a spatial memory task following isoflurane nitrous oxide anesthesia in rats. Anesthesiology 100: 309314 Eriksson PS, Perfilieva E, Bjork Eriksson T, Alborn AM, Nordborg C, Peterson DA, Gage FH (1998) Neurogenesis in the adult human hippocampus. Nat Med 4: 13131317 Gage FH (2002) Neurogenesis in the adult brain. J Neurosci 22: 612613 Galvan V, Jin K (2007) Neurogenesis in the aging brain. Clin Interv Aging 2: 605610 Hattiangady B, Shetty AK (2008) Aging does not alter the number or phenotype of putative stem/progenitor cells in the neurogenic region of the hippocampus. Neurobiol Aging 29: 129147 Jevtovic Todorovic V, Carter LB (2005) The anesthetics nitrous oxide and ketamine are more neurotoxic to old than to young rat brain. Neurobiol Aging 26: 947956 Jevtovic Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF (2003) Early exposure to common anesthetic agents

PAGE 74

63 causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci 23: 876882 Lazarov O, Mattson MP, Peterson DA, Pimplikar SW, van Praag H (2010) When neurogenesis encounters aging and disease. Trends Neurosci 33: 569579 Li Y, Liang G, Wang S, Meng Q, Wang Q, Wei H (2007) Effects of fetal exposure to isoflurane on postnatal memory and learning in rats. Neuropharmacology 53: 942950 Loepke AW, Soriano SG (2008) An assessment of the effects of general anesthetics on developing brain structure and neurocognitive function. Anesth Analg 106: 16811707 Lu LX, Yon JH, Carter LB, Jevtovic Todorovic V (2006) General anesthesia activates BDNF dependent neuroapoptosis in the developing rat brain. Apoptosis 11: 16031615 Lugert S, Basak O, Knuckles P, Haussler U, Fabel K, Gotz M, Haas CA, Kempermann G, Taylor V, Giachino C (2010) Quiescent and active hippocampal neural stem cells with distinct morphologies respond se lectively to physiological and pathological stimuli and aging. Cell Stem Cell 6: 445 456 Luo J, Daniels SB, Lennington JB, Notti RQ, Conover JC (2006) The aging neurogenic subventricular zone. Aging Cell 5: 139152 Ming GL, Song H (2005) Adult neurogenesis in the mammalian central nervous system. Annu Rev Neurosci 28: 223250 Moller JT, Cluitmans P, Rasmussen LS, Houx P, Rasmussen H, Canet J, Rabbitt P, Jolles J, Larsen K, Hanning CD, Langeron O, Johnson T, Lauven PM, Kristensen PA, Biedler A, van Beem H, F raidakis O, Silverstein JH, Beneken JE, Gravenstein JS (1998) Long term postoperative cognitive dysfunction in the elderly ISPOCD1 study. ISPOCD investigators. International Study of Post Operative Cognitive Dysfunction. Lancet 351: 857861 Mouton PR (2002) Principles and Practices of Unbiased Stereology: An introduction for bioscientistss. The Johns Hopkins University Press, Baltimore, Maryland

PAGE 75

64 Palmer TD, Willhoite AR, Gage FH (2000) Vascular niche for adult hippocampal neurogenesis. J Comp Neurol 425: 479494 Scholzen T, Gerdes J (2000) The Ki 67 protein: from the known and the unknown. J Cell Physiol 182: 311322 Seki T, Arai Y (1995) Age related production of new granule cells in the adult dentate gyrus. Neuroreport 6: 24792482 Shruster A, Melamed E, Of fen D (2010) Neurogenesis in the aged and neurodegenerative brain. Apoptosis Nov;15(11):141521 Snyder JS, Hong NS, McDonald RJ, Wojtowicz JM (2005) A role for adult neurogenesis in spatial long term memory. Neuroscience 130: 843852 Stratmann G, Sall JW, Bell JS, Alvi RS, May LV, Ku B, Dowlatshahi M, Dai R, Bickler PE, Russell I, Lee MT, Hrubos MW, Chiu C (2010) Isoflurane does not affect brain cell death, hippocampal neurogenesis, or long term neurocognitive outcome in aged rats. Anesthesiology 112: 305315 Toni N, Laplagne DA, Zhao C, Lombardi G, Ribak CE, Gage FH, Schinder AF (2008) Neurons born in the adult dentate gyrus form functional synapses with target cells. Nat Neurosci 11: 901907 Tung A, Herrera S, Fornal CA, Jacobs BL (2008) The effect of prol onged anesthesia with isoflurane, propofol, dexmedetomidine, or ketamine on neural cell proliferation in the adult rat. Anesth Analg 106: 17721777 Vega CJ, Peterson DA (2005) Stem cell proliferative history in tissue revealed by temporal halogenated thymi dine analog discrimination. Nat Methods 2: 167169 Wilder RT, Flick RP, Sprung J, Katusic SK, Barbaresi WJ, Mickelson C, Gleich SJ, Schroeder DR, Weaver AL, Warner DO (2009) Early exposure to anesthesia and learning disabilities in a population based birth cohort. Anesthesiology 110: 796804 Xie Z, Culley DJ, Dong Y, Zhang G, Zhang B, Moir RD, Frosch MP, Crosby G, Tanzi RE (2008) The common inhalation anesthetic isoflurane induces caspase

PAGE 76

65 activation and increases amyloid betaprotein level in vivo. Ann Neur ol 64: 618627 Xie Z, Dong Y, Maeda U, Moir R, Inouye SK, Culley DJ, Crosby G, Tanzi RE (2006) Isoflurane induced apoptosis: a potential pathogenic link between delirium and dementia. J Gerontol A Biol Sci Med Sci 61: 13001306 Xie Z, Dong Y, Maeda U, Moir RD, Xia W, Culley DJ, Crosby G, Tanzi RE (2007) The inhalation anesthetic isoflurane induces a vicious cycle of apoptosis and amyloid betaprotein accumulation. J Neurosci 27: 12471254 Zhang G, Dong Y, Zhang B, Ichinose F, Wu X, Culley DJ, Crosby G, Tanz i RE, Xie Z (2008) Isoflurane induced caspase 3 activation is dependent on cytosolic calcium and can be attenuated by memantine. J Neurosci 28: 45514560 Zhao C, Deng W, Gage FH (2008a) Mechanisms and functional implications of adult neurogenesis. Cell 132: 645660 Zhao N, Zhong C, Wang Y, Zhao Y, Gong N, Zhou G, Xu T, Hong Z (2008b) Impaired hippocampal neurogenesis is involved in cognitive dysfunction induced by thiamine deficiency at early pre pathological lesion stage. Neurobiol Dis 29: 176185

PAGE 77

66 Figure 9. Experimental d esign. Rats in all groups were injected 21, 8, and 4 days before isoflurane exposure, with CldU, IdU and EdU respectively. In order to study the effect s of isoflurane on integration, maturation and differentiation of neuronal cell proliferation in the DG of young and aged rats

PAGE 78

67 Figure 10. Physiological Parameters D uring Isoflurane Exposure (A) Rectal tem perature was recorde d 30 mins after isoflurane induction in youn g (n=8) and aged (n=8) rats. T emperature was between 36 and 37C during the first hour, and then it increased almost 1 C, and remained stable for the rest of the isoflurane exposure. (B) Systolic, diastolic and me an arterial blood pressure s (mmHg) were also recorded every 30 mins after isoflurane induction in young and aged rats. The blood pressure was stable dur ing isoflurane inhalation. Blood pressure in the aged rats was slightly less as compared to the young ra ts (C) H emoglobin oxygen saturation and heart rate were also recorded every 30 minutes after isoflurane induction in young and aged rats and were within normal limits Data are shown as mean SEM.

PAGE 79

68 Figure 11. Neural Progenitor Cell Proliferation is Unaffected by Isoflurane A nesthesia (A ) Shows the estimated number of Ki67 positive cells in the DG 24 hours after isoflurane anesthesia in young and aged rats. The results were subjected to a 2 (age: young, aged) X 2 (treatment: control, isoflurane) factorial ANOVA. There was a significant effect of age (F (3, 7) = 8.57, p <0.001), but no significant effect of treatment (F (3, 7) = 0.51, p = 0.818). Isoflurane ex posure did not result in a statistically significant effect in the young (p = 0.248) nor in the aged rats ( p=0.212) (B) Immunohistochemical staining for Ki67 positive cells (20X upper image ) and (40X lower image ) in DG 24 hours after isoflurane exposure in young and aged rats.

PAGE 80

69 Figure 12. The Number and Fate of 4 days old Newborn Cells in the DG are Unaffected by Isoflurane Exposure (A and B) (A) Quantificatio n of EdU+ cells (right column), EdU+/DCX+ cells (middle column), and EdU+/S100 + cells (left column) in the DG of young rats after isoflurane exposure. (B) Quantification of EdU+ cells (right column), EdU+/DCX+ cells (middle column), and EdU+/S100 + cells (left column) in the DG of aged rats after isoflurane exposure The results were subject to a 2 (age: young, aged) X 2 (treatment: control, isoflurane) factorial ANOVA. There was a significant effect of age (F (2,11)= 16.61, p<0.0001), but no significant effect of treatment (F (2,11)= 6.61, p=0.06) (C) Confocal plane of the same cell showing colocalization of EdU+ (red), DCX+ (green) and EdU+/DCX+ (orange) indicating differentiation into neuronal phenotype. N ewly formed double labeled cells were visualized using concofal microscopy in an orthogonal projection composed of 25 optical z planes (0.5mm thick).

PAGE 81

70 Figure 13. Cells undergoing maturation at 8 days after birth, are unaffected by isoflurane exposure in young and aged rats (A and B) (A) Quantification of IdU+ cells (right column), IdU+/DCX+ cells (middle column), and IdU+/S100 + cells (left column) in the DG of young rats after isoflurane exposure. (B) Quantification of IdU+ cells (right column), IdU+/DCX+ cells (middle column), and IdU+/S100 + cells (left column) in the DG of aged rats after isoflurane exposure. The results were subject to a 2 (age: young, aged) X 2 (treatment: control, isoflurane) factorial ANOVA. There was a significant effect of age (F (7,11)= 70.18, p<0.0001), but no significant effect of treatment (F (7,11)= 1.87, p=0.086) (C ) Confocal plane of the same cell s showing co localization of IdU+ (red), DCX+ (green) and IdU+/DCX+ (orange) indicating neuronal phenotype. Cells undergoing maturation at 8 days were visualized using concofal microscopy in an orthogonal projection composed of 25 optical z planes (0.5mm thick).

PAGE 82

71 Figure 14. Isoflurane exposure affected cells that underwent maturation and started to integrate in the DG of aged, but not young rats. (A and B) Quantification of CldU+ cells (right column), CldU+/NeuN+ cells (middle column), and Cl dU+/S100 + cells (left column) in the DG of young (A ) and aged (B ) rats after isoflurane. The results were subject to a 2 (age: young, aged) X 2 (treatment: control, isoflurane) factorial ANOVA. There was a significant effect of age (F (7,11)= 16.48, p<0.0001), and a significant effect of treatment (F (7,11)= 1.52, p=0.04). Isoflurane significantly decreased the number of CldU+ cells (*p=0.02) and the number of CldU+/NeuN+ cells (**p=0.008), but increased the number of CldU+ /S100 + cells (***p=0.033) (C) Confocal plane of the same cell, showing co localization of CldU+ (green ), NeuN+ (red) and CldU+/NeuN + (orange) indicating neuronal phenotype ( D ) Si ngle Confocal plane of the same ce ll, showing co localization of CldU ( red ), S100 + (green) and Cl dU+/S100 + (orange) indicating astrocytic phenotype. Newly formed double labeled cells undergoing maturation were visualized using concofal microscopy in an orthogonal projection composed of 25 optical z planes (0.5mm thick).

PAGE 83

72 Figure 15. Cell death in the DG after isoflurane exposure. Quantification of the TUNEL+ labeling in the DG of young and aged rats, 24 hours after isoflurane exposure. Isoflurane exposure has no effect on cell death in the DG The results were subject to a 2 (age: young, aged) X 2 (treatment: control, isoflurane) factorial ANOVA. There was not a significant effect of age (F (2,3)= 3.90, p=0.07), and no significant effect of treatment (F (2,3)= 0.47, p=0.64). Isoflurane exposure did not result in a statistically significant effect in the young (p=0.06) nor in the aged rats (p=0.27).

PAGE 84

73 CHAPTER 3 PROPOFOL ANESTHESIA AFFECTS NASCENT CELL S UNDERGOING DIFFERENTIATION, AXO N/DENDRITE TARGETING AND MIGRATION IN THE DENTATE GYRUS OF YOUNG BUT NOT AGED RATS Abstract Background : Propofol is a widely used agent for anesthetic induction and long term sedation due to its favorable profile that ensures a rapid onset of action and a rapid recovery However, r ecent clinical and laboratory studies suggest an association between commonly used general anesthetics and the impairment of cognitive function. In vitro studies suggest that propofol may be toxic to developing neurons (Honegger and Matthieu 1996; Vutskits 2005; Al Jahdari et al. 2006) Therefore, w e hypothesize; that anestheticinduced alteration of one or more stages of postnatal neurogenesis may explain the impairment of cognitive function experi enced by some patients. Methods: 35 mg/kg/hr of propofol or 10% intralipid (control) were continuously infused via tail vein catheter for 3 hours to 3 monthold and 20 monthold F344 rats. Th ree different thymidine analogs (CldU, IdU, and EdU) were intraperitoneally injected at three different time points (21 days, 8 days, and 4 days) prior to the anesthetic or intralipid infusion in order to assessed integration, maturation and differentiation of

PAGE 85

74 nascent cells in the dentate gyrus (DG) at the time o f propofol anesthesia. Moreover, phenotype identification at each time point was assessed by colocalization of each thymidine analog with the corresponding histological marker for each stage of neurogenesis. TUNEL assay was also performed to assess cell d eath in the DG after propofol. Results: no differences were found in hippocampal progenitor proliferation, or neuronal synaptic integration. Nevertheless propofol infusion significantly decreased the number of nascent cell s that were in the process of di fferentiation, migration (p=0.034) and axon/dendrite targeting ( p=0.034) only in young, but not in aged rats In addition, we found that in these young rats, propofol significantly reduced neuronal differentiation, as judged by EdU+/ DCX + co localization (p=0 .023). P ropofol also significantly reduced the number of cells undergoing migration and axon/dendrite targeting as assessed by IdU+/ DCX+ co localization (p=0.047) C onversely, astrocyte production was significantly increased (p<0.0001). A significant increased on cell death after propofol infusion was not found. Conclusions: These results suggest that propofol has an age and stage dependent effect. Because, p ropofol anesthesia appears to be particularly harmful to the young brain, es pecially to nascent cells undergoing differentiation, migration and axon/dendrite targeting. Keywords: propofol, cognitive impairment, postnatal neurogenesis

PAGE 86

75 Introduction Due to the amount of unavoidable surgeries that pediatric and geriatric patients undergo daily, and to recently found evidence that suggest a relationship between anesthetic exposure and the development of cognitive dysfunction early and late in life. It is imper ative to study the mechanisms by which this occurs. Even though, clinically propofol has recently been related to cognitive dysfunction in pediatric patients (Mellon et al. 2007) in the laboratory setting not much attention has been given. Propofol is an intravenous anesthetic characterized by a rapid onset, clear emergence, and lack of cumulative effects observed in the clinical use, which makes it w idely used for induction and maintenance of anesthesia. Despite its remarkable safety profile, there is emerging interest concern ing side effects of propofol particularity in pediatric patients. Propofol has been involved in neurological sequelae in infants (Meyer et al. 2010) and e xperimental in vitro studies suggest that propofol may be toxic to developing neurons (Honegger and Matthieu 1996; Vutskits 2005) In vivo studies are needed in order to properly confirm the in vitro findings allowing us to better understand the clinical situation. Because cognitive function is in part dependent on the generation of neurons throughout life, a process known as postnatal neurogenesis which is a multistep process including proliferation, differentiation, and maturat ion of the nascent cells A n alteration of one or more stages of postnatal neurogenesis may results in impairment of cognitive function (Kempermann 2002; Dupret et al. 2008; Coras et al. 2010) Thus, it is reasonable to assume that an anestheticinduce alteration of postnatal neurogenesis may explain the cognitive dysfunction experienced by some patients. I n this study we examined the

PAGE 87

76 effects of propofol anesthesia on each one of stages of the postnatal neurogenesis in the dentate gyrus of young and aged rats. Material and Methods A nimals : a ll experiments were conducted in accordance with the National Institute of Health Guide and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use committee of the University of South Florida, College of Medicine. Male Fisher 344 (F344) rats (Harlan Sprague Dawley, Indianapolis, IN), were pair housed in environmentally controlled conditions (12:12h light: dark cycle at 211C) and provided food and water ad lib. Two age gr oups of animals young (3 months old) and aged (20 mont hs old) were used in this study. Animals were excluded from the study if they became jaundiced, or had pituitary tumors. Experiment al time line : t hree different thymidine analogs (CldU, IdU, and EdU) were intraperitoneally injected at three different time p oints (21 days, 8 days, and 4 days respectively ) prior to the anesthetic or control infusion in order to assessed integration, maturation and differentiation of nascent cells in the dentate gyrus (DG) at the time of propofol anesthesia. Moreover, phenotype identification at each time point was assessed by co localization of each thymidine analog with the corresponding histological marker for each neurogenic stage. Quantification of co localization of CldU and the mature neuronal marker NeuN or the astrocyt ic marker S100 assessed nascent cells undergoing synaptic integration at the time of propofol anesthesia. Quantification of co expression of IdU and the immature neuronal marker DCX or the astrocytic marker S100 assessed nascent cells undergoing migration and axon/dendrite targeting. Furthermore, b y

PAGE 88

77 determining co localization of EdU with the immature neuronal marker DCX or the astrocytic marker S100 nascent cells undergoing differentiation were assessed. Lastl y, nascent cells proliferation was assessed using the endogenous marker Ki67. Labeling of each neurogenic stage was performed in the same animal (Figure 1 6). Preparation and administration of thymidine a nalogs : s olutions of 5Chloro2 deoxyuridine (CldU) and 5Iodo2 Deoxyuridine (IdU) were prepared at equimolar concentrations. CldU (Sigma #C6891) was prepared at 17 mg/ml and IdU (MP#100357) at 23 mg/ml. Injections at 2.5 ml/Kg resulted in an equimolar final effective delivery of 42.5 mg/Kg CldU or 57.5 mg/Kg IdU as previously described (Vega and Peterson 2005) Both halogenated thymidine analog solutions were prepared in sterile saline and administered by intraperitoneal (IP) injection to ensure precise delivery to facilitate quantitative comparis ons Because cell division is a rare event in the subgranular zone of elderly rats and because many of these newborn cells do not survive, we were concerned that a short thymidine analog labeling protocol would not yield a new cell number sufficient for a meaningful analysis. Therefore, three times the thymidine analog corresponding to a specific time point was administered; three IP injections at 4 hour intervals in one day were given. As shown in Figure 16, animals in all groups were injected with CldU, 2 1 days before intralipid or propofol infusion; and IdU, 8 days before intralipid or propofol infusion. A solution of 5ethynyl 2 deoxyuridine (EdU) was prepared at a concentration of 60 mg/ml in sterile saline. IP injections of EdU (cat # E10187, Invitr ogen, Carlsbad, CA) resulted in a final effective delivery of 160 mg/Kg. Due to money constrains, animals in

PAGE 89

78 all groups were injected only once with EdU, 4 days before intralipid or propofol infusion (Figure 1 6 ). Propofol anesthesia : a cohort of rats was randomly assigned to four different groups breathing air (n=10 per group): young rats that received 35 mg/kg/hr of propofol or 10% intralipid (control) for 3 hrs, and aged rats that received 35 mg/kg/hr of propofol or 10% intralipid for 3 hours. Unanesthet ized rats were placed in a restrainer and a tail vein catheter was implanted and attached to a syringe pump (model 11 Plus; Harvard Apparatus). Propofol (Disoprivan; AstraZeneca) or 10 % intralipid (Fresenius Kabi; Sweden) that served as control was contin uously administered for 3 hours. Body temperature of rats was maintained at 37C with a thermostatically controlled heating pad. Measurement of Perioperiative Parameters during propofol anesthesia : r e ctal temperature was monitored using a thermalet monito ring thermometer ( Physitemp instrument Inc, Clifton, NJ, USA). Moreover, hemoglobin oxygen saturation (SpO2) and heart rate (HR) were recorded during anesthesia period using the SurgiVet multi parameter monitor (Smiths medical, Dublin, OH, USA). Diastolic, systolic and mean a rterial pressures were measured every 30 minutes throughout the anesthesia period by tail cuff using the CODA noninvasive blood pressure system for rats (CODA2, Kent Scientific Corporation). Tissue collection and processing : twenty four hours after intralipid or propofol infusion, rats were deeply anesthetized with pentobarbital (50 mg/ Kg, i.p) and transcardially perfused with 100 ml saline f ollowed by ice cold 4% paraformaldehyde in 0.1M PBS. Brains were removed, postfixed in the same fixative solution at 4 C

PAGE 90

79 overnight, transferred to 20% sucrose in PBS until equilibrated, and then embedded in Tissue Tek OCT before cryostat sectioning. Sagittal cryosections (30 m thick) through the entire DG were collected serially, transf erred to a cryoprotectant solution, and stored at 20C until used. These sections were used to assess proliferation and phenotypic determination of nascent cells in the DG. All standard staining procedures were conducted on free floating sections using every twelfth section for the entire hippocampus beginning with a random start and including sections before and after the hippocampus to ensure that the entire structure was sampled. CldU and IdU Immunohistochemistry : f or immunohistochemistry sections were washed three times for 10 min in PBS before being treated with 0.2N HCL for 10 min at 37 C for DNA denature to facilitate antibody access, sections were then washed with borate buffer (pH 8.5), followed by three washes of PBS. Sections were then incubated in 5% normal goat serum and 0.25% Triton X 100 in PBS (PBS TS) for 60 min, and stained at 4C. Detection of halogenated thymidine analogs was accomplished using rat anti BrdU (Accurate cat# OBT 0030; clone BU1/75) at 1:250 for CldU and mouse anti BrdU (Be cton Dickinson cat# 347580; clone B44) at 1:500 for IdU. All secondary antibodies were used at 1:300 and were conjugated to the fluorophore Alexa594. EdU labeling protocol : Ed U labeling was performed using Click iTTM EdU imaging kit cat # C10339 (Invitrog en, Carlsbad, CA) according to the manufacturers protocol. This protocol is normally intended for use in cell culture, but it was adapted for histological staining of brain tissue. Sagittal cryosections (30 m thick) were washed three times for 10 min in PBS. After washing twice with 3% bovine serum albumin

PAGE 91

80 (BSA) in PBS the sections were permeablized with 0.5% Triton X 100 in PBS for 20 min. The sections were washed twice with 3% BSA in PBS and then incubated with the Click iTTM reaction buffer, CuSO4, Ale xa Fluor 594 Azide, and the reaction buffer additive for 30 min while protected from light. The sections were washed one more time with 3% BSA in PBS. Sections were then mounted onto glass slides, and coverslipped with Vectashield mounting medium for fluo rescence (cat# H 1000 Vector Laboratories, Burlingame, CA, USA). Double immunohistochemistry for lineage markers : t he phenotypes of CldU, IdU and EdU labeled cells were determined using double immunofluorescent staining. Antibodies against DCX, NeuN and S 100 were used to detect immature neurons, mature neurons and astrocytes, respectively. DNA denaturation, followed by incubation of the primary antibodies for CldU and IdU was performed as described above, together with a rabbit polyclonal goat antibody ra ised against human DCX (1:200 cat# 4604 Cell Signaling technology, Inc. Danvers, MA, USA) was used in an antibody cocktail with the IdU antibody, a mouse monoclonal antibody against neuronal nuclei, NeuN (MAB377 Millipore, Billerica, MA, USA) was used at a concentration of 1:500 in an antibody cocktail with the CldU antibody, and a rabbit monoclonal goat antibody raised against S100 (ab52642 abcam, Cambridge, MA, USA) was used at a concentration of 1:3000 in an antibody cocktail with CldU and IdU. For double labeling of EdU/DCX and EdU/S100 EdU staining was performed first, followed by incubation in with a rabbit polyclonal goat antibody against human DCX or in and a rabbit monoclonal goat antibody against S100 respectively. Ki67 Immunohistochemistry: Ki67 is expressed in cells G1 through M phase of

PAGE 92

81 the cell c ycle (Scholz en and Gerdes 2000) For detection of Ki67, s ections were pretreated with 1X S aline Sodium Citrate (SSC) at 80 C for 40 minutes. Subsequently, endogenous peroxidase activity was quenched with 0.6% H2O2 solution in PBS for 20 min. Sections were block ed f or 1 hour in 2% normal goat serum and 0.25% Triton X 100 in PBS (PBS TS). Sections were incubated overnight at 4 C with a rabbit polyclonal antibody against human Ki67 (NCL Ki67p; Novocastra Laboratories/Vision BioSystems, Newcastle upon Tyne, UK) at a dil ution of 1:2000 in PBS TS. The following day, sections were washed in PBS, incubated for one hour in biotin ylated secondary antibody (goat anti rabbit IgG rat adsorbed 1:1000; BA 1000 Vector Laboratories, Burlingame, CA) in PBS TS, and washed in PBS before incubation for 1 hour in avidinbiotin substrate (ABC kit cat no. PK 6100, Vector Laboratories, Burlingame, CA). Sections were washed in PBS for 10 minutes and reacted with 3,3 diaminobenzidine tetrahydrochloride ( DAB) solution ( cat no.1856090, Thermo S cientific, Rockford, IL). Sections were then mounted onto glass slides, dehydrated, and coverslipped with mounting medium ( Cytoseal 60,Stephens Scientific, Riverdale) Tunel assay protocol : in order identify which of the labeled cells were dying, t he in situ cell death detection kit, Fluorescein (cat no. 11684795910, Roche, Mannheim, Germany ) was used according to t he manufacturer's instructions. Sections were incubated with 3% bovine serum albumin in 0.1 mol/L Tris HCL (pH 7.5) for 30 mins followed b y 50 L of TUNEL reaction mixture on each sample for 60 mins at 37C. After washing, slides were incubated with 0.3% H2O2 in methanol for 10 mins, followed by 3% bovine serum albumin in 0.1 mol/L Tris HCL (pH 7.5) for 30 mins at room temperature.

PAGE 93

82 Incubatio n with peroxidase conjugated anti fluorescein (diluted 1:5) at 37 C for 30 mins was performed. Micro scopy and cell counting : unbiased stereological methods were used to estimate the numbers of positive labeled cells ( + labeled cells ) in the subgranular zone of the DG. Twelve equally spaced sections throughout the medial lateral extent of the DG were collected. Because + labeled cells are a rare event, the number of +labeled cells in each DG examined was summed for individual animals and the sum from each animal was then multiplied by the section spacing to e stimate the total number of + labeled cells (Mouton 2002) These data were then used to calculate group m eans for estimates of total + labeled cells. Labeled cells that were greater than 1 cell diameter away from the subgranular layer were not included in the count Sections were imaged on an Olympus FV1000 MPE multiphoton laser scanning microscope with four laser sources: 405 Diode, multi line Argon (for Alexa 488), green HeNe (for Alexa 594), and red HeNe was used for all immunofluorescence photomicrographs. When quantification of percentage of positive cells was determined, Z stacks were created at 1 m intervals throughout the 30 m of the sections with a guard region of 2 m excluded form top and bottom of the Z stack. The Z stacks were rotated in all planes to verify double labeling. Images of positive staining were adjusted to make optimal use of the dynamic range of detection. Statistical analyses : data are presented as mean SEM. Analysis of variance (ANOVA) with Tukeys post hoc or a t test was used to compare the differences from the control group. P values less than 0.05 were considered statist ically significant, the

PAGE 94

83 significant testing was one tailed and GraphPad Prism version 5.00 for Mac (GraphPad Software, San Diego California USA, www.graphpad.com ) was used to analyze the data. Results Physiological parameter during propofol anesthesia : r ectal tem perature was recorded every 30 mins after propofol induction in young (n=8) and aged (n=8) rats. Temperature was stable through the anesthesia period in both young and aged rats, with temperature in the aged rats being approximately 1 C less than in young rats. Systolic, diastolic and mean arterial blood pressure (mmHg) were recorded every 30 mins after propofol induction in young and aged rats. The blood pressure was stable during the exposure with a small de cline in the aged rats as compared to the young rats. Moreover, hemoglobin oxygen saturation and heart rate were also r ecorded every 30 minutes after propofol induction in young and aged rats. Data are shown as mean SEM (Figure 17 ). Postnatal proliferation in the Dentate Gyrus, as assessed by the Ki67 marker, is unaffected by propofol exposure : t he effects of propofol anesthesia (35 mg/Kg/hr for 3 hours) on neuronal cell proliferation in the SGZ in young (3 month old) and aged (20 month old) F3 44 rats was determined by analyzing Ki 67 labeling, 24 hours after exposure. Figure 18B shows that Ki67 positive cells in both young and aged rats were primarily located on the border of the granule cell layer. Propofol does not appear to have an effect on newborn cells generated during the 24 hours after exposure (Figure 18A). The results were subjected to a 2 (age: young, aged) X 2 (treatment: control, propofol) factorial ANOVA. There was a significant effect of age (F (3, 7) = 28.93, p < 0.0001), but no significant effect of treatment (F (3, 7) = 0.98, p = 0.47). Propofol anesthesia did not result in a statistically significant effect in the young (p= 0.130) nor in the aged rats (p=0.400).

PAGE 95

84 Nascent c ells undergoing differentiation in the DG of young, but not aged rats, are affected by propofol anesthesia: w e look at the number of newborn cells undergoing differentiation at the time of propofol infusion by estimating the number of EdU positive cells. And determined the phenotype of these ne wly born cells using double labeling of EdU and the immatu re neuronal marker DCX (Figure 19 C) and EdU and the astrocytic marker S100 (Figure 19A B). The results were subject to a 2 (age: young, aged) X 2 (treatment: intralipid, propofol ) factorial ANOVA. There was a significant effect of age (F (2,11)= 16.61, p<0.0001), and a significant effect of treatment (F (2,11)= 6.61, p< 0.05). Propofol anesthesia resulted in a statistically significant decrease in the number of EdU+ cells (*p=0.034), in the number of EdU+/DCX+ cells (**p=0.023), but not in the number of EdU+/S100B cells (***p=0.260) Nascent c ells undergoing migration in the DG are altered by propofol anesthesia in young, but not in aged rats: w e counted at the numbe r of newborn cells undergoing migration at the time of the propofol, anesthesia by estimating the number of IdU positive cells. And determined the phenotype of these newly born cells using double labeling of IdU and the immatu re neuronal marker DCX (Figure 20C) and IdU and the astrocytic marker S100 (Figure 20 D ). The results were subject to a 2 (age: young, aged) X 2 (treatment: intralipid, propofol) factorial ANOVA. There was a significant effect of age (F (7,11)= 73.02, p<0.0001), and a significant effect of treatment (F (7,11)= 1.47, p<0.05). Propofol significantly decreased the number of IdU+ cells (*p=0.034), IdU+/DCX+ cells (**p=0.047), and increased the number of I dU+/S100 + cells (***p<0.0001) in the DG of young, but not aged rats

PAGE 96

85 Nascent cel ls undergoing maturation in the DG of young and aged rats, are unaffected by propofol anesthesia: w e estimated the number of newborn cells undergoing synaptic integration at the time of propofol exposure by estimating the number of CldU positive cells, and determined the phenotype of these newly born cells (Figure 21 A B) using double labeling of: CldU and the matur e neuronal marker NeuN (Figure 21 C) and CldU and the astrocytic marker S100 Ther e was a significant effect of age (F (7,11)= 52.65, p<0.0001), but not a significant effect of treatment (F (7,11)= 1.94, p=0.07). Cell death in the DG after propofol anesthesia: c ell death positive label in the DG was assessed by the DNA strand breaks (TUNEL Assay) in young and aged rats 24 hours aft er propofol anesthesia (Figure 22). The results were subject to a 2 (age: young, aged) X 2 (treatment: intralipid, propofol) factorial ANOVA. There was not s significant effect of age (F (2,3)= 3.53, p=0.08) and no significant effect of treatment (F (2,3)= 0.42, p=0.67). Propofol anesthesia did not result in a statistically significant effect in the young (p=0.19) nor in the aged rats (p=0.12). Discussion The findings of this study are: first the expected agerelated decrease in the rate of postnatal neurogenesis in aged as compared to young rats. Second, the age dependent effects of propofol on the young but not on the aged rat brain. Third, the stage dependent effect of propofol on nascent cells undergoing differentiation, migration and axon/dendrite targeting but not on nascent cells undergoing maturation and synaptic integration. And lastly it was found that the cognitive dysfuncti on experienced by some

PAGE 97

86 patients after anesthesia and whi ch may be explained by an anesthetic induced alteration of postnatal neurogenesis appears to be agent dependent, with isoflurane being particularly harmful to the aged brain, and propofol being particularly harmful to the young brain. Propofol anesthesia has a specific effect on the young brain : human and animal data suggest that propofol is harmful to developing neurons in the young brain that result in cognitive sequelae (Takamatsu et al. 2005; Vutskits 2005; Mellon et al. 2007; Meyer et al. 2010) A study that examines neurogenesis as an explanation for cognitive dysfunction, did not find and impairment of cognitive f unction in eighteenmonth old rats exposed to two hours of propofol anesthesia (Lee et al. 2008) However, they did not examine the effect of propofol on the entire process of postnatal neurogenesis. In this study it was found that propofol particularly affects nascent cells in the DG of young rat s, but not aged rats. T he neurogenic niche is regulated by signaling from growth factors, trophic factors and neurotransmitters among other s (Palmer et al. 2000; Taupin 2006) du ring development many of the s e signals are altered, and the neurogenic process itself its occurring at a higher rate. For all this and more, the young brain is particularly vulnerable to insult s in this case partic ularly vulnerable to ane sthetic toxicity. Nascent cells undergoing differentiation and migration in the DG of young rats, at the time of anesthetic exposure are particularly affected by propofol : e vidence suggesting that propofol may be toxic to developing neurons (Spahr Schopfer et al. 2000; Fredriksson et al. 2007) and increasing clinical and laboratory data suggest an anesthetic induced impairment of cognitive function (Loepke and Soriano 2008) is

PAGE 98

87 currently raising concerns in pediatric anesthesia. A study of the effects of propofol on neuronal s tructure and neurocognitive performance in mice suggests that propofol increases neurodegeneration, leading to adult, behavioral, and learning impairment in a dose dependent manner (Fredriksson et al. 2007) Moreover, an in vitro study of neuronal cell cultures from immatu re chicks and rats showed that s upracl inical doses of propofol induce neuronal cell death in dissociated cell culture models, but it fails to demonstrate neurotoxic effects in organotypic slice culture (Spahr Schopfer et al. 2000) Even though, no prospective studies have examined the propofol effects on neuronal structure and neurocognitive outcome in chil dren, several case reports detail shortterm neurological abnormalities In this study, a similar effect of propofol on the brain of young rats was found. This effect was found to be specific to the nascent cells underg oing the first stages of postnatal neurogenesis suggesting a specific susceptibility of immature nascent cells in the DG. Nascent cells in the DG of young and aged rats seem to be affected by anesthetics in agent dependent manner : t his study shows that a anesthetic, i.e. propofol, produces cognitive effects on one age population, i.e. young, whereas the other does not i.e. isoflurane, as previously reported. The most obvious explanation is these two anesthetics have different receptor mechanisms of actio n. Propofol inhibits synaptic transmission primarily by enhancing GABA receptors while having smaller effects on NMDA receptors. On the other hand, isoflurane acts less selectively on GABA and NMDA receptors. These differences in affinity for specific receptors are also associated with differences in hippocampal electrophysiology, with isoflurane blocking induction of long term potentiation (LTP) and long t erm depression (LTD) (Stevens 1998) while

PAGE 99

88 propofol only partially inhibits LTP and ha s no effect on LTD (Nagashima et al. 2005) Consequently, it appears that there are import ant functional differences among anesthetics with respect to memory process. Due to these inherent differences in the molecular actions, not all agents that produce general anesthesia result in cognitive impairments. In conclusion, these results add to the growing body of evidence indicating that exposure to general anesthetics early and late in life cause cognitive dysfunction, and suggest that vulnerability to these deleterious effects may be age and agent dependent.

PAGE 100

89 References Al Jahdari WS, Saito S, Nakano T, Goto F (2006) Propofol induces growth cone collapse and neurite retractions in chick explant culture. Can J Anaesth 53: 10781085 Coras R, Siebzehnrubl FA, Pauli E, Huttner HB, Njunting M, Kobow K, Villma nn C, Hahnen E, Neuhuber W, Weigel D, Buchfelder M, Stefan H, Beck H, Steindler DA, Blumcke I (2010) Low proliferation and differentiation capacities of adult hippocampal stem cells correlate with memory dysfunction in humans. Brain 133: 33593372 Dupret D Revest JM, Koehl M, Ichas F, De Giorgi F, Costet P, Abrous DN, Piazza PV (2008) Spatial relational memory requires hippocampal adult neurogenesis. PLoS One 3: e1959 Fredriksson A, Ponten E, Gordh T, Eriksson P (2007) Neonatal exposure to a combination of N methyl D aspartate and gammaaminobutyric acid type A receptor anesthetic agents potentiates apoptotic neurodegeneration and persistent behavioral deficits. Anesthesiology 107: 427436 Honegger P, Matthieu JM (1996) Selective toxicity of the general anesthetic propofol for GABAergic neurons in rat brain cell cultures. J Neurosci Res 45: 631636 Kempermann G (2002) Why new neurons? Possible functions for adult hippocampal neurogenesis. J Neurosci 22: 635638 Lee IH, Culley DJ, Baxter MG, Xie Z, Tanzi R E, Crosby G (2008) Spatial memory is intact in aged rats after propofol anesthesia. Anesth Analg 107: 1211 1215 Loepke AW, Soriano SG (2008) An assessment of the effects of general anesthetics on developing brain structure and neurocognitive function. Anes th Analg 106: 16811707 Mellon RD, Simone AF, Rappaport BA (2007) Use of anesthetic agents in neonates and young children. Anesth Analg 104: 509 520

PAGE 101

90 Meyer P, Langlois C, Soete S, Leydet J, Echenne B, Rivier F, Bonafe A, Roubertie A (2010) Unexpected neurol ogical sequelae following propofol anesthesia in infants: Three case reports. Brain Dev 32: 872878 Mouton PR (2002) Principles and Practices of Unbiased Stereology: An introduction for bioscientistss. The Johns Hopkins University Press, Baltimore, Maryland Nagashima K, Zorumski CF, Izumi Y (2005) Propofol inhibits long term potentiation but not long term depression in rat hippocampal slices. Anesthesiology 103: 318326 Palmer TD, Willhoite AR, Gage FH (2000) Vascular niche for adult hippocampal neurogenesi s. J Comp Neurol 425: 479494 Scholzen T, Gerdes J (2000) The Ki 67 protein: from the known and the unknown. J Cell Physiol 182: 311322 Spahr Schopfer I, Vutskits L, Toni N, Buchs PA, Parisi L, Muller D (2000) Differential neurotoxic effects of propofol on dissociated cortical cells and organotypic hippocampal cultures. Anesthesiology 92: 14081417 Stevens CF (1998) A million dollar question: does LTP = memory? Neuron 20: 12 Takamatsu I, Sekiguchi M, Wada K, Sato T, Ozaki M (2005) Propofol mediated impair ment of CA1 long term potentiation in mouse hippocampal slices. Neurosci Lett 389: 129132 Taupin P (2006) Adult neural stem cells, neurogenic niches, and cellular therapy. Stem Cell Rev 2: 213219 Vega CJ, Peterson DA (2005) Stem cell proliferative history in tissue revealed by temporal halogenated thymidine analog discrimination. Nat Methods 2: 167169 Vutskits GE, Tassony E, Kiss JZ (2005) Clinically relevant concentrations of propofol but not midazolam alter in vitro dendritic development of isol ated gamma aminobutyric acid positive interneurons. Anesthesiology 102: 970976

PAGE 102

91 Figure 1 6. Experimental design. Rats in all groups were injected 21, 8, and 4 days before propofol exposure, with CldU, IdU and EdU respectively. In order to study the effect s of propofol on integration, maturation and differentiation of neuronal cell proliferation in the DG of young and aged rats

PAGE 103

92 Figure 17 Rats Physiological Parameters during Propofol Exposure (A) Rectal tem perature was recorded every 30 mins after propofol induction in youn g (n=8) and aged (n=8) rats. T emperature was stable through the anesthesia period in both young and aged rats with temperature in the aged rats being approximately 1 C less than in young rats (B) Sys tolic, diastolic and mean arterial blood pressure (mmHg) were recorded every 30 mins after propofol induction in young and aged rats. The blood pressure was stable during the exposure with a small decline in the aged rats as compared to the young rats. (C) Hemoglobin oxygen saturation and heart rate were also r ecorded every 30 minutes after propofol induction in young and aged rats. Data are shown as mean SEM.

PAGE 104

93 Figure 18. Neural Progenitor Cell Proliferation is Unaffected by Propofol Anesthesia (A) Shows the estimated number of Ki67 positive cells in the DG 24 hours after propofol anesthesia in young and aged rats. The results were subjected to a 2 (age: young, aged) X 2 (treatment: control, propofol ) factorial ANOVA. There was a signi fican t effect of age (F (3, 7) = 28.93, p <0.0001), but no significant effe ct of treatment (F (3, 7) = 0.98, p = 0.47). Propofol anesthesia did not result in a statistically significant effect in the young (p = 0.130) nor in the aged rats ( p=0.400). (B) Immunohistochemical staining for Ki67 positive cells (20X upper image ) and (40X lower image ) in DG 24 hours after propofol exposure in young and aged rats.

PAGE 105

94 Figure 19 The number and f ate of 4 days old nascent cells in the DG are altered by propofol anesthesia in young, but not in aged rats (A and B) (A) Quantificatio n of EdU+ cells (right column), EdU+/DCX+ cells (middle column), and EdU+/S100 + cells (left column) in the DG of young rats after propofol (B) Quantification of EdU+ cells (right column), EdU+/DCX+ cells (middle column), and EdU+/S100 + cells (left column) in the DG of aged rats after propofol The results were subject to a 2 (age: youn g, aged) X 2 (treatment: intralipid propofol ) factorial ANOVA. There was a significant effect of age (F (2,11)= 16.61, p<0.0001), and a significant effect of treatment (F (2,11)= 6.61, p< 0.05). Propofol anesthesia resulted in a statistically significant decrease in the number of EdU+ cells (*p=0.034), in the number of EdU+/DCX+ cells (**p=0.023), but not in the number of EdU+/S100B cells ( p=0.260) (C) Confocal plane of the same cell, showing colocalization of EdU+ (red), DCX+ (green) and EdU+/DCX+ (orange) indicating differentiation into neuronal phenotype. N ewly formed double labeled cells were visualized using concofal microscopy in an orthogonal projection composed of 25 optical z planes (0.5mm thick).

PAGE 106

95 Figure 20 Cells undergoing maturation in the DG at 8 days after birth are altered by propofol anesthes ia in young, but not in aged rats. (A and B) Quantification of IdU+ cells (right column), IdU+/DCX+ cells (middle column), and IdU+/S100 + cells (left column) in the DG of young rats (A) and aged rats (B) after propofol The results were subject to a 2 (age: young, aged) X 2 (treatment: intralipid, propofol ) factorial ANOVA. There was a significant effect of age (F (7,11)= 73.02, p<0.0001), and a significant effect of treatment (F (7,11)= 1.47, p<0.05) Propofol significantly decreased the number of IdU + cells (*p=0.034), and IdU+/DCX+ cells (**p=0.047), but increased IdU+/S100 + cells (***p<0.0001) in the DG of young, but not aged rats (C) Confocal plane of the same cells, showing co localization of IdU+ (red), DCX+ (green) and IdU+/DCX+ (orange) indica ting neuronal phenotype. (D) Confocal plane of the same cell, showing colocalization of IdU+ (red), S100 + (green) and IdU+/S100 + (orange) indicating astrocytic phenotype. Double labeled cells were visualized using concofal microscopy in an orthogonal projection composed of 25 optical z planes (0.5mm thick).

PAGE 107

96 Figure 21. Propofol anesthesia does not alter nascent cells that underwent m aturation and Starting to integrate in the DG of young or aged rats Quantification of CldU+ cells (right column), CldU+/NeuN+ cells (middle column), and CldU+/S100 + cells (left column) in the DG of young (A) and aged (B) rats after propofol. The results were subject to a 2 (age: youn g, aged) X 2 (treatment: intralipid propofol) factorial ANOVA. There was a significant effect of age (F (7,11)= 52.65, p<0.0001), but not a significant effect of treatment (F (7,11)= 1.94, p=0.07). (C) Confocal plane of the same cell, showing co localization of CldU+ (green), NeuN+ (r ed) and CldU+/NeuN+ (orange) indicating neuronal phenotype. Newly formed double labeled cells undergoing integration were visualized using concofal microscopy in an orthogonal projection composed of 25 optical z planes (0.5mm thick).

PAGE 108

97 Figure 22. Cell death in the DG after propofol a nesthesia Quantification of the TUNEL+ labeling in the DG of young and aged rats, 24 hours after propofol. Propofol anesthesia has no effect on cell death in the DG of young or aged rats The results were subject to a 2 (a ge: young, aged) X 2 (treatment: intralipid, propofol) factorial ANOVA. There was not s significant effect of age (F (2,3)= 3.53, p=0.08), and no significant effect of treatment (F (2,3)= 0.42, p=0.67). Propofol anesthesia did not result in a statisticall y significant effect in the young (p=0.19) nor in the aged rats (p=0.12).

PAGE 109

98 CONCLUSIONS : ANESTHETIC INDUCED ALTERATION O F POSTNATAL NEUROGENESIS, A POSSIBLE CAUSE OF TH E COGNITIVE DYSFUNCTIO N OBSERVED IN SOME PEDIATRIC AND G ERIATRIC PATIENTS Introduction The use of general anesthetics involving millions of pediatric and geriatric patient s is widespread today Emerging, clinical and laboratory findings suggest that general anesthesia early or late in life may cause impairment of cognitive function, the mechanism of which is still unknown (Dodds and Allison 1998; Culley et al. 2003; Stratmann 2006; Loepke and Soriano 2008) Because cognitive function is in part dependent on postnatal neurogenesis (Shors et al. 2001; Snyder et al. 2005; Coras et al. 2010) It is reasonable to infer that an anesthetic induced altera tion of postnatal neurogenesis may in part explain the cognitive impairment observe d after anesthesia in pediatric and geriatric patients This dissertation discusses the effects of two different kinds of general anesthetics, isoflurane and propofol, on postnatal neurogenesis. F irst it focuses on the ir effects on the entire process of postnatal neurogenesis. Second, it examines these effects on the two populations that seem to be mor e vulnerable to anestheticinduced cognitive impairment. Third, it studies two kinds of a nesthetics that are widely used, but have different mechanisms of action.

PAGE 110

99 The process of postnatal n eurogenesis One of the major objectives of thi s dissertation is to provide an analysis of the effects of isoflurane and propofol anesthesia on the entire process of postnatal neurogenesis, which is a complex process that involves multiple stages. Postnatal neurogenesis is a multistep process that involves a series of developmental stages; t he final result is that few nascent cells are added to the existing hippocampal neuronal circuitry. The neurogenic process starts with proliferation of the neural/stem progenitor cells producing a pool of immature ce lls, the majority of which will become neurons if they survive. The percentage of surviving neurons varies depending on the strain of animal used; it c an be as great as 75% or as little as 25% of the proliferating cells (Kempermann et al., 1997c) Subsequently, nascent cells that had differentiated and survived go through a migratory stage that occurs while the cells a re becoming functionally mature (Kempermann et al., 2003b). Nascent neurons become morphologically and physiologically fully mature 6 to 8 week s after birth in the young rodent (van Praag et al., 2002; Esposito et al., 2005; Zhao et al., 2006; Toni et al., 2008). A lteration of one or more stages of postnatal neurogenesis may explain the cognitive dysfunction observed in some pediatri c and geriatric patients following general anesthesia. Indeed in this dissertation by examining the effects of isoflu rane and propofol on nascent cell number and phenotype at each developmental stage of neurogenesis a n anestheticinduced alteration of one or more developmental stages was found. A summary of the results is presented on figure 23. Cognitive function following anesthesia was assessed i n the fir st p ortion of this dissertation. However, emerging data show that newly generated neurons in the dentate gyrus of the hippocampus play a significant role

PAGE 111

100 in spatial learning and memory, and that the reduction in the number of these cells impairs cognitive function (Shors et al. 2001; Dupret et al. 2008; Zhao et al. 2008; Coras et al. 2010) In addition, t he role of postnatal neurogenesis in hippocampal dependent learning and memory appears to be affected by different factors For instance, environmental enrichment (Kempermann et al. 1997) an d exercise (van Praag et al. 1999) results in increased neurogenesis. Conversely, stress (Mirescu et al. 200 4) irradiation (Tada et al. 2000; Mizumatsu et al. 2003) and age (Klempin and Kempermann 2007; Shruster et al. 2010) decrease neurogenesis and impair different aspects of hippocampal dependent memory (Dupret et al. 2008) For that reason, it is reasonable to hypothesize that general anesthetics may have an effect on cognition. Age dependent vulnerability to anesthetic induced cognitive dysfunction Impairment of cognitive function in pediatric and geriatric patients who received sedation or anesthesia has raised concern regarding their relevance and potential implications for the practice of anesthesia. T his dissertation particularly focuses on the effects of anesthetics on these two populations. Previous studies suggest an age dependent vulnerability to the development of cognitive dysfunction following anesthesia (Culley et al. 2003; Jevtovic Todorovic et al. 2003; Culley et al. 2004; Stratmann 2006; Meyer et al. 2010) Certainly, the data presented in this dissertation supports those findings The hippocampus appears to be particularly sus ceptible to general anesthetics, during development and aging. N eurogenesis, gliogenesis and synaptogenesis occur at a high rate during development During aging there is a decline in cell proliferation in the SGL, which is associated with decline in hippocampal dependent spatial memory (Coras et al. 2010) Despite an age related reduction in the formation of new hippocampal

PAGE 112

101 neurons, the neurons that are added appear functionally equivalent to those in young brain. This suggest s that neurogenesis in the aged brain is not aberrant, but simply down regulated with reduced cell proliferation and retarded neuronal maturation (Rao et al. 2005) T his dissertation examined the two previously mentioned age groups because they are the two populations that show cognitive decline. Moreover, neurogenesis particularly in these two populations occurs at a different rate than in other populations. D uring development and aging neurogenesis occurs at a higher and lower rate respectively In addition to the age dependent effect suggested by the results on this dissertation, an agent dependent ef fect was also found. I soflurane appears to be harmful to the aged but not the young brain. On the other hand, propofol seems to be harmful to the young but not the aged brain Agent dependent effects on postnatal neurogenesis The final o bjective of this dissertation was to identify the effects of two kinds of general anesthetics As reported here one general anesthetic, isoflurane, produces cellular effects in one age group, aged animals, while the other propofol, does not. A possible explanation is tha t these general anesthetics have different receptor mechanisms of action. Isoflurane inhibits synaptic transmission in the central nervous system by acting on various systems including those implicated in aminobutyric acid ( GABA ) and N methyldaspartate ( NMDA) receptors (Orser et al. 1994; Solt and Forman 2007) P ropofol a cts primarily by enhancing GABA and having sm aller effect s on NMDA receptors (Nishikawa and MacIver 2000) These diff erences in receptor affinity are also associated with differences in hippocampal electrophysiology, with isoflurane blocking

PAGE 113

102 induction of long term potentiation (LTP) and long term depression (LTD) (Stevens 1998) while propofol only partially inhibits LTP and has no effect on LTD (Nagashima et al. 2005) Another factor, is the regulation of postnatal neurogenesis by neurotransmitters (Ge et al. 2007) For instance, GABA causes depolarization of immature neurons (Nagashima et al. 2005; B ordey 2006; Platel et al. 2007) Moreover, GABA antagonism increases proliferation of neural progenitor cells, whereas GABA agonism exhibits t he opposite effects (Tozuka et al. 2005) Interestingly, a lack of GABA induced depolarization in newborn neurons leads to significant defects in their dendritic development and synapse formation. Taken together, GABA induced depolarization promotes the differentiation, maturation, and functional integration of newly gene r ated cells in the postnatal dentate gyrus (DG). Similar ly glutamate release from astrocytes and neurons may activate the immature neuron receptors and regulate the development of immature neurons (Montana et al. 2004) Mor eover, recent studies of electrophysiology have shown that newborn neurons in the DG begin to receive functional glutamatergic inputs two weeks after they are born (Esposito et al 2005, GE e t al 2006, Carleton et al 2003), and NMDA receptors antagonists have been shown to increase the number of new immature neurons (Tashi ro et al 2006). Thus, G lutamate has a direct regulatory ro l e on the stages of postnatal neurogenesis. Consequently GABA and NMDA signaling are neurogenic trophic factors and excessive stimulation or under stimulation of them in the young or aged brain may cause behavioral consequences by altering neural connectivity (Manent et al. 2007) So it appears that there are important functional differences among anesthetics with respect to

PAGE 114

103 memory process, and due to these inherent differences in the molecular actions, not all agents that produce general anesthesia result in cognitive impair ment in a particular age group Conclusion F or many years, general anesthetics have been considered to be safe since there were not reports indicating otherwise. However emerging clinical and laboratory data suggest that pediatric and geriatric patients are at risk of cognitive dysfunction following general anesthesia. Current st udies and this dissertation suggest that an anestheticinduced alteration of postnatal neurogenesis may result in later impairment of cognitive function. B ecause neurogenesi s is p articularly vulnerable during developing and aging, the two main affected groups are at the extremes of age. The use of anesthetics is a necessity that cannot be avoided in pediatric and geriatric patients when lifet hreatening conditions require surgery Therefore, it is urgent that we improve our unde rstanding of the mechanisms underlying the cognitive function impairment induced by anesthetics.

PAGE 115

104 References Bordey A (2006) Adult neurogenesis : basic concepts of signaling. Cell Cycle 5: 722728 Coras R, Siebzehnrubl FA, Pauli E, Huttner HB, Njunting M, Kobow K, Villmann C, Hahnen E, Neuhuber W, Weigel D, Buchfelder M, Stefan H, Beck H, Steindler DA, Blumcke I (2010) Low proliferation and differ entiation capacities of adult hippocampal stem cells correlate with memory dysfunction in humans. Brain 133: 33593372 Culley DJ, Baxter M, Yukhananov R, Crosby G (2003) The memory effects of general anesthesia persist for weeks in young and aged rats. Ane sth Analg 96: 10041009, table of contents Culley DJ, Baxter MG, Crosby CA, Yukhananov R, Crosby G (2004) Impaired acquisition of spatial memory 2 weeks after isoflurane and isoflurane nitrous oxide anesthesia in aged rats. Anesth Analg 99: 13931397; table of contents Dodds C, Allison J (1998) Postoperative cognitive deficit in the elderly surgical patient. Br J Anaesth 81: 449462 Dupret D, Revest JM, Koehl M, Ichas F, De Giorgi F, Costet P, Abrous DN, Piazza PV (2008) Spatial relational memory requires hippocampal adult neurogenesis. PLoS One 3: e1959 Ge S, Pradhan DA, Ming GL, Song H (2007) GABA sets the tempo for activity dependent adult neurogenesis. Trends Neurosci 30: 18 Jevtovic Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF (2003) Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci 23: 876882 Kempermann G, Kuhn HG, Gage FH (1997) More hippocam pal neurons in adult mice living in an enriched environment. Nature 386: 493495 Klempin F, Kempermann G (2007) Adult hippocampal neurogenesis and aging. Eur Arch Psychiatry Clin Neurosci 257: 271280

PAGE 116

105 Loepke AW, Soriano SG (2008) An assessment of the effec ts of general anesthetics on developing brain structure and neurocognitive function. Anesth Analg 106: 16811707 Manent JB, Jorquera I, Mazzucchelli I, Depaulis A, Perucca E, Ben Ari Y, Represa A (2007) Fetal exposure to GABA acting antiepileptic drugs gen erates hippocampal and cortical dysplasias. Epilepsia 48: 684693 Meyer P, Langlois C, Soete S, Leydet J, Echenne B, Rivier F, Bonafe A, Roubertie A (2010) Unexpected neurological sequelae following propofol anesthesia in infants: Three case reports. Brain Dev 32: 872878 Mirescu C, Peters JD, Gould E (2004) Early life experience alters response of adult neurogenesis to stress. Nat Neurosci 7: 841846 Mizumatsu S, Monje ML, Morhardt DR, Rola R, Palmer TD, Fike JR (2003) Extreme sensitivity of adult neurogen esis to low doses of X irradiation. Cancer Res 63: 40214027 Montana V, Ni Y, Sunjara V, Hua X, Parpura V (2004) Vesicular glutamate transporter dependent glutamate release from astrocytes. J Neurosci 24: 26332642 Nagashima K, Zorumski CF, Izumi Y (2005) Propofol inhibits long term potentiation but not long term depression in rat hippocampal slices. Anesthesiology 103: 318326 Nishikawa K, MacIver MB (2000) Excitatory synaptic transmission mediated by NMDA receptors is more sensitive to isoflurane than are nonNMDA receptor mediated responses. Anesthesiology 92: 228236 Orser BA, Wang LY, Pennefather PS, MacDonald JF (1994) Propofol modulates activation and desensitization of GABAA receptors in cultured murine hippocampal neurons. J Neurosci 14: 77477760 P latel JC, Lacar B, Bordey A (2007) GABA and glutamate signaling: homeostatic control of adult forebrain neurogenesis. J Mol Histol 38: 303311 Rao MS, Hattiangady B, Abdel Rahman A, Stanley DP, Shetty AK (2005) Newly born cells in the ageing dentate gyrus display normal migration, survival and neuronal fate choice but endure retarded early maturation. Eur J Neurosci 21: 464476

PAGE 117

106 Shors TJ, Miesegaes G, Beylin A, Zhao M, Rydel T, Gould E (2001) Neurogenesis in the adult is involved in the formation of trace me mories. Nature 410: 372376 Shruster A, Melamed E, Offen D (2010) Neurogenesis in the aged and neurodegenerative brain. Apoptosis Nov;15(11):141521 Snyder JS, Hong NS, McDonald RJ, Wojtowicz JM (2005) A role for adult neurogenesis in spatial long term mem ory. Neuroscience 130: 843852 Solt K, Forman SA (2007) Correlating the clinical actions and molecular mechanisms of general anesthetics. Curr Opin Anaesthesiol 20: 300306 Stevens CF (1998) A million dollar question: does LTP = memory? Neuron 20: 12 Stra tmann G, Bell JD, Bickler P, Alvi R, Ku B, Magnusson KR, Liu J. (2006) Neonatal Isoflurane anesthesia causes a permanent neurocognitive deficit in rats. Society for Neurosciences Tada E, Parent JM, Lowenstein DH, Fike JR (2000) X irradiation causes a prol onged reduction in cell proliferation in the dentate gyrus of adult rats. Neuroscience 99: 3341 Tozuka Y, Fukuda S, Namba T, Seki T, Hisatsune T (2005) GABAergic excitation promotes neuronal differentiation in adult hippocampal progenitor cells. Neuron 47: 803815 van Praag H, Kempermann G, Gage FH (1999) Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat Neurosci 2: 266270 Zhao N, Zhong C, Wang Y, Zhao Y, Gong N, Zhou G, Xu T, Hong Z (2008) Impaired hippocampal neurogenesis is involved in cognitive dysfunction induced by thiamine deficiency at early pre pathological lesion stage. Neurobiol Dis 29: 176185

PAGE 118

107 Figure 23. Summary diagram of the results presented in this dissertation

PAGE 119

ABOUT THE AUTHOR Diana M Erasso received her Bachelors degree in Biology and Chemistry from the University of South Florida, Tampa Florida in 2005. Aft er receiving her undergraduate degree, she began graduate school at the University of South Florida in 2006. Diana joined the laboratory of Dr. Samuel Saporta and Dr. Enrico Campore si, where she completed her Ph .D w ork looking at the effects of general anesthetics on neurogenesis. Diana successfully defended her Doctoral dissertation in spring 2011 at the University of South Florida.