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Neuroprotection with anesthetics in two models of cerebral ischemia

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Title:
Neuroprotection with anesthetics in two models of cerebral ischemia
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Book
Language:
English
Creator:
Chaparro-Buitrago, Rafael
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University of South Florida
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Tampa, Fla
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Subjects / Keywords:
Hypotension
Caspase
Stroke
Hypoxia
Anesthesia
Dissertations, Academic -- Molecular Pharmacology and Physiology -- Doctoral -- USF   ( lcsh )
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non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: Neuroprotection with anesthetics has been studied for many decades; important advances in this field have modified the way Anesthesiologists treat patients in the operating room. Animal models have played an important role in the study of ischemia in the operating room. Recent studies have demonstrated that the effect of anesthetics seems to be different in different animal models. We decided to evaluate anesthetics in a well known model of cerebral ischemia and also in hypotensive models designed by us. We used a model of cerebral ischemia (MCAO) to test anesthetics neuroprotective effect in a two-week period. Then, we used a model of hypotension to characterize the damage caused by this type of insult. Finally we characterized a model of hypotension plus hypoxia that can mimic real situations in the OR. We found that anesthetics alone do not have a neuroprotective effect after two weeks in the MCAO model; but the combination of anesthetics with caspase vii inhibitors can decrease the damage caused by ischemia. The caspase inhibitor by itself did not show a significant neuroprotective effect. We also found that repetitive periods of profound hypotension can cause important damage in the hippocampus but no memory or neurological changes were seen. The induction of only one episode of hypotension plus hypoxia did not alter the morphology of the hippocampus although induced memory changes that were reverted by the use of anesthetics.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2010.
Bibliography:
Includes bibliographical references.
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by Rafael Chaparro-Buitrago.
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Title from PDF of title page.
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Document formatted into pages; contains X pages.
General Note:
Includes vita.

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University of South Florida
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usfldc doi - E14-SFE0003362
usfldc handle - e14.3362
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Neuroprotection With Anesthetics In T w o Models O f Cerebral Ischemia by Rafael Eduardo Chaparro Buitrago M.D. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular Pharmacology and Physiology College of Medicine University of South Florida Co Major Professor: Enrico Camporesi, M.D. Co Major Professor: Dave Morgan, Ph.D. Marcia N. Gordon, Ph.D. Hans Schweiger, M.D. Nagwa Dajani, Ph.D. Date of Approval: April 16, 2010 Keywords: hypotension, caspase, stroke, hypoxia, anesthesia Copyright 2010 Rafael E duardo Chaparro Buitrago, M.D.

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! ! DEDICATION This work is dedicated to my family, especially to my daughter Danna, my wife Carolina, my parents Gladys and Rafael and my g rand mother La Mama Bertha". Without them this journey would not be possible.

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ACKNOWLEDGEMENTS I especially want to thank ; Dr. Enrico Camporesi my major professor and also my friend, his guidance took me to finish this process with worldwide recognition. Dr. Dave Morgan who was the first person that opened his doors and introduced me into the research world. Dr. Samuel Saporta for his h elp and guidance. My laboratory; Carolina Quroga, Diana Erasso, Chrystal Price a nd R a chel Karlnoski for their help and support. Franjesca Jackson for her friendship and help and Margaret Baldwing for all the long hours in the operating room.

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! Note to the Reader : The original document contains color t hat is necessary for understanding the data. T he original dissertation is on file with the USF library in Tampa, Florida.

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! i TABLE OF CONTENTS LIST OF TABLES iii LIST OF FIGURES i v ABSTRACT v i INTRODUCTION Perioperative neuronal injury 1 Pathophysiology of ischemic cerebral injury 2 Hypotension 7 Anesthetics and neuroprotection with anesthetics 10 PAPER 1: ISO FLURANE PLUS REPETITIVE CASPASE INHIBITOR ADMINISTRATION REDUCES LONG TERM HISTOLOGICAL DAMAGE AFTER MCAO IN RATS 23 Abstract 24 Introduction 27 Materials and M ethods 29 Results 39 Discussion 47 References 50 PAPER 2: HIPPOCAMPAL CELLULAR LOSS AFTER BRIEF HYPOTENSION 54 Abstract 55 Introduction 57 Materials and M ethods 59 Results 69 Discussion 79 References 83 PAPER 3: ISOFLURANE PROTECTS FROM LEARNING IMPAIRMENT CAUSED BY BRIEF HYPOXIA AND HYPOTENSION IN SPRAGUE DAWLEY RATS 88 Abstract 89

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! ii Introduction 92 Materials and M ethods 94 Results 103 Discussion 109 References 112 CONCLUSION S 118 ABOUT THE AUTHOR End Page

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! iii LIST OF TABLES PAPER 1 Table 1: Study design 29 Table 2: Time course 30 Table 3: Neurological score 32 Table 4 : Physiological variables 40 PAPER 2 Table 1: Study design 59 Table 2: Time course 60 Table 3 : Forty eight points neurological score 6 3 Table 4 : Physiological variables 70 PAPER 3 Table 1: Study design 94 Table 2: Time course 95 Table 3 : Forty eight points neurological score 98 Table 4 : Physiological variables 105

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! iv LIST OF FIGURES INTRODUCTION Figure 1: Macrosco pic pathology of focal Ischemia 3 Figure 2: Cel l death after cerebral ischemia 4 Figure 3: Apoptotic cascade 6 PAPER 1 Figure 1 : Neurological performance 41 Figure 2 : Relative infarct area to compensate for edema 42 Figure 3 : Microphotographs of the inf arcts at day 14 after de injury 43 Figure 4 : Estimated total number of TUN EL positive cells in the stroke hemisphere 45 Figure 5: Estimated total number of Cleaved Caspase 3 positive cells in the stroked hemisphere 46 PAPER 2 Figure 1 : Neurological Score 72 Figure 2 : Passive Avoidance 73 Figure 3 : Effect of 3 minutes o f hypotension in frontal cortex 74

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! v Figure 4 : Pictures from frontal cortex 74 Figure 5: Effect of 3 minutes of hypotension in the CA1 area 75 Figure 6: Pictures f rom CA1 area of the hippocampus 76 Figure 7: Estimated total Number of Ni ssl cells in frontal cortex 77 Figure 8: Estimated total number of Nissl cells two weeks after the i nsult in the CA1 area of the hippocampus 78 PAPER 3 Fi gure 1: Neurological score 106 F igure 2: Passive avoidance 107 Figure 3: NeuN positive cells in animals subject to 3 min of hypotension/hypoxia 108 Figure 4 : Nissl cells in CA1 area two weeks after the insult 10 9

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! vi Neuroprotection With Anesthetics i n T w o Models o f Cerebral Ischemia Rafael Eduardo Chaparro Buitrago, M.D. ABSTRACT Neuroprotection with anesthetics has been studied for many decades; important advances in this field have modified the way Anesthesiologist s treat patients in the operating room. Animal models have played an important role in the study of ischemia in the operating room Recent studies have demonstrated that the effect of anesthetics seems to be different in different animal models. We decided to evaluate anesthetics in a well known mod el of cerebral ischemia and also in hypotensive models designed by us. We used a model of cerebral ischemia (MCAO) to test anesthetics neuroprotective effect in a two week period. Then, we used a model of hypotens ion to characterize the damage caused by this type of insult. Finally we characterized a model of hypotension plus hypoxia that can mimic real situations in the OR. We found that anesthetics alone do not have a neuroprotective effect after two weeks in the MCAO model; but the combination of anesthetics with caspase

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! vii inhibitors can decreas e the damage caused by ischemia. The caspase inhibitor by it self did not show a significant neuroprotective effect. We also found that repetitive periods of profound hypotension can cause important damage in the hippocampus but no memory or neurological changes were s een. The induction of only one episode of hypotension plus hypoxia did not alter the morphology of the h ippocampus although induced memory changes that were reverted by the use of anesthetics.

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1 INTRODUCTION : Periopera t ive neuronal injury. Neuronal injury during anesthesia although infrequent, can result in subclinical neurocognitive deficit, long term disability or even death T he insult is caused by i schemia or hypoxia. Recent implementation of different neuroprotective maneuvers ha s reduc ed neurological morbidity and mortality. S everal surgical procedures are more likely to cause brain injury (1) F or this reason clini cians have been identifying these procedures and exploring possible neuroprotective therapies for more than six decades (2) Pathophysiology of Ischemic Cerebral I njury: Ischemia is defined as a local or global and temporary deficiency of blood supply, which may be accompanied by consequent cell alterations In humans a normal cerebral blood flow (CBF) is accepted as a blood flow between 45 to 50 ml/min/100g of tissue, which is usually generated b y a mean arterial pressure between 60 to 130 mmHg. I f the CBF drops below 20 to 30 ml/min/100g of tissue the cerebral cortex is considered ischemic. Cerebral ischemia can be localized (focal ischemia) or generalized (global ischemia). M acroscopically they have different characteristics but the pathophysiology at the cellular level is similar. In focal ischemia the sudden loss

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2 of cerebral blood flow causes immediate necrosis T his area is called "isc hemic core". A round the core lies another re gion called th e "ischemic penumbra"; in this region there is a p roliferation of apoptotic cells. I f uncorrected the ischemic damage will cause these cells to die in a few weeks increasing the size of the infarct (3,4) as shown in Figure1 Figure 1 Macroscopic pathology of focal Ischemia. Early in the course of the lesion, the necrotic component (core) is the most important factor. Over time, the penumbra area is responsible for the growth of the lesion (5) Neurotransmitters are implicated in the pathogenesis of ischemic cell injury, e specially glutamate and GABA (6,7) After ischemia there is depletion in

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3 energy stores. T his depletion can lead to acidosis failure of the Na + / K + pump which produces membrane depolarization and /or an excessive release of glutamate into the synaptic clef t Glutamate activates N methyl d aspartate (NMDA) and amino 3 hydr oxy 5 methylisoxazole 4 propionic acid ( AMPA) recepto rs in the postsynaptic neuron. T hese receptors activate the voltage gated calcium channels and in consequence there is an increase in the intracellular calcium. High intracellular Ca ++ levels produces ac tivation of nitric o xide synthetase (NOS), li pas es, proteases and endonucleases. F rom here two pathways can be activated. First, apoptosis can lead to an irreversible cell damage and cell death. The other pathway implicates NOS I ncrease d production of ni tri c oxide (NO) and free radicals can lead to lipid peroxidation also causing irreversible cell damage and cell death. In some cases the ischemic injury is not su stained and reperfusion occurs. W hen this happens besides the formation of free radicals, there is also inflammation with a subsequent cytokine release and irreversible cell damage and cell death (4,8 10) These pathways are depicted in Figure 2.

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4 Figure 2 The neurodeg enerative cascade (5) Ischemia leads to c ell death after cerebral ischemia through a series of intermediate steps. In the penumbra area cells are mostly apoptotic. Apoptosis is defined as programmed cell death in multicellular organisms. I t is widely recognized that there are three ap optotic pathways. T wo are caspase dependent and one is caspase independent. The first caspase dependent pathway is c all ed the intrinsic pathway, and is the mitochondrial mediated pathway. T he second one is a receptor mediated cascade and is call ed the extrinsic pathway (11) The exact mechanism of activation on both pathways is not exactly known, bu t the cascade after activation has been described In the intrinsic pathway cythoc h rome C is released from the mitochondria. Pro apoptotic Bax proteins induce cytochrome C release by opening the voltage dependent anion

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5 channels (VDAC) all owing cytochrom e C to pass through the channel. Bcl 2 and Bcl XL proteins have the exact opposite effect T hey close the VDAC channels and in this way prevent ap optosis. The cytochrome C activates pro caspase 9 and converts it to activated caspase 9. At this point the intrinsic and extrinsic pathway merge in to the common pathway and activate procaspase 3 conve rting it to activated caspase 3. T his caspase will cause cellular death (12,13) The extrinsic pathway is a receptor mediated pathway. It is activated when the Fas receptor (Fas R) receive s the Fas ligand (Fas L). The Fas R is a type two transmembrane protein that is part of the tumor necrosis factor (TNF ) family. The Fas associated protein with death d omain (FADD) is a molecule that serves as a bridge between the Fas R and th e procaspase 8, converting it to activated caspase 8. A fter this point the extrinsic pathway merge s with the common pathway and activated caspase 8 activates procaspase 3 resul ting in apoptosis (14,15) The caspase independ ent pathway is mediated by the apoptosis inducing f actor (AIF) released from the mitochondr ia. T his factor induce s apoptosis without using caspase proteins (8)

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6 Figure 3 Signaling pathway of ischemia induced apoptosis (8) Three pathways culminating in apoptosis are depicted. Hypotension. Hypotension has been defined by the World Health Organization as a systolic pressure below 110 mmHg in males and 100 mmHg in females D iastolic pressure is not taken into consideration (16) Chronic hypotension in humans is responsible for a variety of symptoms like fatigue, d izziness, palpitations and even cogniti ve alterations C hronic hypotension does appear as damaging (17) and there are autoregulatory mechanisms that protect specific organs against its effects. Recent data suggests that hypotension is responsible for alterations in cognitive performance (17,18) S ome researchers have demonstrated that even

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7 short periods of profound hypotension can cause cell damage in animal studies (19) During hemorrhagic hypote nsion, intravascular volume is lost and regulatory mechanisms rapidly star t compensatory mechanisms. The first event occurs in the heart L ack of volume cause s a decrease in cardiac output. T he body reacts by decreasing the blood supply to organs with less oxygen consumption like skin, intestines or muscle, while preferential flow is shifted to the brain and the heart. Im mediate vasoconstriction caus ed by l ocal mediators tends to stop bleeding and this mechanism is reinforced by an activation of the sympathetic system that causes more vasoconstriction and also an increase in cardiac output. When hypotension persist s for long periods or when profound hyp otension is present the s e mechanisms are not s ufficient to maintain cell viability and the cells die by necrosis or apoptosis (20) Cognitive dysfunction has been l inked with hypotension (17,21) especially in elderly patients (22 24) Several studies have studied the relation between hypotension during surgery and neurological performance after surgery but so far a clear link has not been found (25) The largest study that evaluated this point was "The International Study of Postoperativ e Cognitive Dysfunction" but an association between surgical blood pressure and postoperative cognitive dysfunction was not found (26) In contrast other researchers have found a clear overall link between surgery and blood pressure during surgery (18,26,27)

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8 Another important concept fo r the purpose of this presentation is hypoxia reduction in O2 availability to tissue. T his can be caused by a variety of respiratory or circulatory factors There are direct and indirect measures to evaluate oxygen in the blood. O ne of th e indirect indexes is the pulse oximeter, a device that measures the oxygen saturation. N ormal ranges are from 95% to 100%, while values below 90% are consider ed hypoxic. A direct measurement can be made wit h arterial blood gases analysi s: which measures pO 2 values The normal range is b etween 80 to 100 mm Hg During ischemia or hypotension, hypox ia is the main critical factor since low levels of oxygen are responsible for cell injury. All living carbon base d organisms use oxygen for their metabolism. When cell s do not receive enough oxygen they enter in an inefficient anaerobic state and if this situation is maintained for long periods they will die. After the hypoxic insult there is an increase in intracellular calcium that can lead either to necrosis or apoptosis (28) Anesthetics and neuroprotection with anesthetics. Anesthetics have been shown to decrease ischemic/ hypoxic cerebral injury, but their action only last for short periods of time. T his princi ple is correct in some types of inj ury but not in others, so the pu rpo se of this review is to study the typical anesthetics and their actions in different models of cerebral ischemia/hypoxia. There are two anesthetics that have been studied widely as neuroprot ective agents, p ropofol, an intravenous anesthetic and i soflurane, an inhalational anesthetic.

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9 Propofol (2,6 diisopropylphenol) is a nonbarbiturate intravenous general anesthetic; Propofol can be used alone with a continuous infusion or in combination with inhalational anesthetics. Propofol can induce anesthesia more rapidly than barbiturates and recovery is a lot faster and devoid of side effects Its mechanism of action is not well understood but the data suggest s that p ropofol is a GABA A receptor agonist and also has inhibitory action at the glutamate NMDA subtype receptor. Propofol is also used to treat other pathologies like sta tus epilepticus, delirium t remens and refractory migraines; these uses are considered a consequence of its glutamate antagonism or its GABA antagonism. Propofol decrease s intracranial pressure, decrease s cerebral oxygen consumption, increases cerebrovascular resistance and decreases cerebral blood flow. In addition, p ropofol also has antie metic properties by reducing serotonin concentration in the dorso lateral reticular formation Also p ropofol has been shown to attenuate neuronal injury (29) The dosage used depends on the type of procedure, age of the patient or the pathology that is be ing treated. For induction in adult neuro sur gical patients slow administrati on is recommended using bolus of 20 mg every 10 seconds Slower boluses or infusion of p ropofol for induction of anesthesia, titrated to clinical response, will generally result in reduced induction dosage requirements (1 to 2 mg/kg). Maintenance dosage is 100 to 200 m i c ro g r /kg/minute (6 to 12 mg/kg/hour) IV (30) Isoflurane is a general a nesthetic, classified as h alogenated anesthetic, a nd is a nonflammable colorless liquid that require s a vaporizer for its clinical

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10 use. I soflurane provides a rapid induction and rapid recovery I t is also a muscle relaxant a very useful characteristic in the operating room. The mechanism of action is unknown but some studies sugge st that i soflurane can a lter the membrane function by decreasing the number of molecules that alternate between gel and crystalline states (30) After inhalation, i soflurane is absorbed by the pulmo nary capillary system. I t und ergo es little biotransformation. A lmost 95% is exhaled unchanged and only 0.17% i s found in urine as metabolites The minimum alveolar concentration for an adult man is 1.15% but this decrease s with age. For induction in adults the recommended dose is 1.5 to 3%, which induces, anesthesia in 5 to 10 minutes. Anesthesia maintenance in adults is 1.5 to 3.5% when inhaled in o xygen. In children 0.5 to 1.5% is the recommended average dosage. For induc tion the concentration of i soflurane should not be higher than 3% and no more than 2.5%, in children (30) Neuroprotection with anesthetics is a practice that has been used for almost sixty years (1) The first anesthetics used for this purpose were the barbiturates, and their neuroprotective activity was attributed to a reduction in the metabolic rate Research has shown that the anesthetics go even fur ther and have specific actions o n specific receptors. The majority of anesthetics have an antagonistic effect on the glutam ate receptors (NMDA and AMPA). S ome researchers also believe that anesthetics can increase reuptake and decrease the release of glut amate in the presynaptic neuron. A dditionally anesthetics have an agonistic effect o n GABA A receptors increasing i their inhibitory activity. As a result, a decrease in the intracellular Ca ++ may block the deleteri ous effects of

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11 ischemia/hypoxia (1,31 33) Bickler et al ( 2006) ; have demonstrated that protection of hypoxic co rtical neurons with anesthetics more specifically i soflurane, involves signaling that inclu des changes in intracellular Ca + + regulation, several MAP kinase pathways and modulation of apoptosis regulators (34) There are also two pore domain potassium channels KC (K2P) that provide regulation of membrane potential. T hese TREK channels are present i n the central nervous system and are activated by acidosis, temperature, polyunsaturated fatty acids, membrane stretch and also by volatile anesthetics T h ese channels have neuroprotective activity and their activation explain s why some polyunsaturated fat ty acids have neuroprotective actions (35) Brain relaxation is another one of the anesthetics' properties useful in the operating room This is very important i n neurosurgery especially in patients with intracranial hypertension caused by a reduced blood volume ; relaxation is a neuroprotective measure in this type of patients. Also p ropofol is known as a drug that can reduce regional cerebral blood flow and metabolism, charact eristics that ar e also known as neuroprotective (36,37) Anesthetics have been demonstrated to be protective only for two days post ischemia in cases of severe focal ischemia, but this effect was not maintained at 14 days (38 40) T his is probably due to the apoptotic activity in the penumbra area (38) A nesthetics inhibit necrotic dam age but are incapable of reducing apoptotic injury (39) It is also im portant to mention that anesthetics prevent necrotic injury only if they are administered during ischemia; they have

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12 no action if administered after ischemia (41,42) Preconditioning with anesthetics is another pract ice that has shown to be effective in reducing ischemic injury (43) Anesthetics also have neuroprotective action if the ischemic injury is mild and although it does not produce necrosis, it can trigger apoptosis (44) If the ischemic insult is mild, anesthetics by themselves are protective enough and can decrease the neurologic morbidity (40) In the operating room, ischemic insult s vary from temporary vessel occlusion to profound systemic hypotens ion. In general, insult s tend to be temporal, mild and usual ly last for no more than 70 minutes. I T is generally accepted that ischemia lasting more than ten minutes causes brain damage and it is also accepted that in the presence of anesthetics this damage i s substantially reduced (40) Recent publications have shown that anesthetics can act as a necrosis inhibitors and this action could buy some time to initiate therapies that modify apoptosis Some researchers have been studying apoptosis and antiapoptotic therapies as a possible treatment for cerebral injury (45) Kawaguchi et al (2006) ; use d caspase inhibitors to prolong the neuroprotec tive effe ct of anesthetics in models of m i d dle cerebral artery o c clu sion with positive protective results (46) However; unfortunately there are no proven antiapoptot ic therapies available for humans Sakai et al (2007) ; repor ted long lasting protection by Isoflurane wi th no need for the use of caspase inhibitor s (48) and provided good evidence that the difference with Kawaguchi et al (2004) was caused to the later using an MCAO model that integrat ed permanent carotid occlusion and thus persistent

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13 hypoperfusion (38) On the other hand, Elsersy et al (2004) showed evidence of decli ne of the protective effect of Isoflurane over time; suggesting that the protection between local and global ische mia is different (47) ;

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14 References : 1. Fukuda S, Warner DS. Cerebral protection. Br J Anaesth 2007;99:10 7. 2. Bigelow WG, Lindsay WK, Greenwood WF. Hypothermia; its possible role in cardiac surgery: an investigation of factors governing survival in dogs at low body temperatures. Ann Surg 1950;132:849 66. 3. Muir KW, Buchan A, von Kummer R, Rother J, Baron JC. Imaging of acute stroke. Lancet Neurol 2006;5:755 68. 4. Dirnagl U, Iadecola C, Moskowitz MA. Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci 1999;22:391 7. 5. Clarkson AN. Anesthetic mediated protection/preconditioning during cerebral ischemia. Life Sci 2007;80:1157 75. 6. Benveniste H. Glutamate, microdialysis, and cerebral ischemia: lost in translation? Anesth esiology 2009;110:422 5. 7. Weigl M, Tenze G, Steinlechner B, Skhirtladze K, Reining G, Bernardo M, Pedicelli E, Dworschak M. A systematic review of currently available pharmacological neuroprotective agents as a sole intervention before anticipated or ind uced cardiac arrest. Resuscitation 2005;65:21 39.

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15 8. Kawaguchi M, Furuya H, Patel PM. Neuroprotective effects of anesthetic agents. J Anesth 2005;19:150 6. 9. Amir G, Ramamoorthy C, Riemer RK, Reddy VM, Hanley FL. Neonatal brain protection and deep hypothe rmic circulatory arrest: pathophysiology of ischemic neuronal injury and protective strategies. Ann Thorac Surg 2005;80:1955 64. 10. Suwanwela N, Koroshetz WJ. Acute ischemic stroke: overview of recent therapeutic developments. Annu Rev Med 2007;58:89 106. 11. Zheng Z, Zhao H, Steinberg GK, Yenari MA. Cellular and molecular events underlying ischemia induced neuronal apoptosis. Drug News Perspect 2003;16:497 503. 12. Fujimura M, Morita Fujimura Y, Kawase M, Copin JC, Calagui B, Epstein CJ, Chan PH. Manganese superoxide dismutase mediates the early release of mitochondrial cytochrome C and subsequent DNA fragmentation after permanent focal cerebral ischemia in mice. J Neurosci 1999;19:3414 22. 13. Fujimura M, Morita Fujimura Y, Murakami K, Kawase M, C han PH. Cytosolic redistribution of cytochrome c after transient focal cerebral ischemia in rats. J Cereb Blood Flow Metab 1998;18:1239 47.

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16 14. Martin Villalba A, Herr I, Jeremias I, Hahne M, Brandt R, Vogel J, Schenkel J, Herdegen T, Debatin KM. CD95 liga nd (Fas L/APO 1L) and tumor necrosis factor related apoptosis inducing ligand mediate ischemia induced apoptosis in neurons. J Neurosci 1999;19:3809 17. 15. Rosenbaum DM, Gupta G, D'Amore J, Singh M, Weidenheim K, Zhang H, Kessler JA. Fas (CD95/APO 1) play s a role in the pathophysiology of focal cerebral ischemia. J Neurosci Res 2000;61:686 92. 16. WHO. Arterial Hypertension. Technical Report Series World Health Organization 1978;628. 17. Duschek S, Schandry R. Reduced brain perfusion and cognitive performa nce due to constitutional hypotension. Clin Auton Res 2007;17:69 76. 18. Schutz C, Stover JF, Thompson HJ, Hoover RC, Morales DM, Schouten JW, McMillan A, Soltesz K, Motta M, Spangler Z, Neugebauer E, McIntosh TK. Acute, transient hemorrhagic hypotension d oes not aggravate structural damage or neurologic motor deficits but delays the long term cognitive recovery following mild to moderate traumatic brain injury. Crit Care Med 2006;34:492 501.

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17 19. Yamauchi Y, Kato H, Kogure K. Hippocampal damage following re peated brief hypotensive episodes in the rat. J Cereb Blood Flow Metab 1991;11:974 8. 20. Dutton RP. Current concepts in hemorrhagic shock. Anesthesiol Clin 2007;25:23 34, viii. 21. Wharton W, Hirshman E, Merritt P, Stangl B, Scanlin K, Krieger L. Lower bl ood pressure correlates with poorer performance on visuospatial attention tasks in younger individuals. Biol Psychol 2006;73:227 34. 22. Zuccala G, Onder G, Pedone C, Carosella L, Pahor M, Bernabei R, Cocchi A. Hypotension and cognitive impairment: Selecti ve association in patients with heart failure. Neurology 2001;57:1986 92. 23. Yap PL, Niti M, Yap KB, Ng TP. Orthostatic hypotension, hypotension and cognitive status: early comorbid markers of primary dementia? Dement Geriatr Cogn Disord 2008;26:239 46. 2 4. Qiu C, Winblad B, Fratiglioni L. The age dependent relation of blood pressure to cognitive function and dementia. Lancet Neurol 2005;4:487 99.

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18 25. Williams Russo P, Sharrock NE, Mattis S, Liguori GA, Mancuso C, Peterson MG, Hollenberg J, Ranawat C, Salv ati E, Sculco T. Randomized trial of hypotensive epidural anesthesia in older adults. Anesthesiology 1999;91:926 35. 26. Moller JT, Cluitmans P, Rasmussen LS, Houx P, Rasmussen H, Canet J, Rabbitt P, Jolles J, Larsen K, Hanning CD, Langeron O, Johnson T, L auven PM, Kristensen PA, Biedler A, van Beem H, Fraidakis O, Silverstein JH, Beneken JE, Gravenstein JS. Long term postoperative cognitive dysfunction in the elderly ISPOCD1 study. ISPOCD investigators. International Study of Post Operative Cognitive Dysfu nction. Lancet 1998;351:857 61. 27. Yocum GT, Gaudet JG, Teverbaugh LA, Quest DO, McCormick PC, Connolly ES, Jr., Heyer EJ. Neurocognitive performance in hypertensive patients after spine surgery. Anesthesiology 2009;110:254 61. 28. Sugawara T, Fujimura M, Noshita N, Kim GW, Saito A, Hayashi T, Narasimhan P, Maier CM, Chan PH. Neuronal death/survival signaling pathways in cerebral ischemia. NeuroRx 2004;1:17 25. 29. Adembri C, Venturi L, Tani A, Chiarugi A, Gramigni E, Cozzi A, Pancani T, De Gaudio RA, Pell egrini Giampietro DE. Neuroprotective effects of

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19 propofol in models of cerebral ischemia: inhibition of mitochondrial swelling as a possible mechanism. Anesthesiology 2006;104:80 9. 30. Inc GS. Drug Information: MD Consult, 2008. 31. Warner DS. Pharmacolog ic protection from ischemic neuronal injury. J Neurosurg Anesthesiol 2004;16:95 7. 32. Elsersy H, Mixco J, Sheng H, Pearlstein RD, Warner DS. Selective gamma aminobutyric acid type A receptor antagonism reverses isoflurane ischemic neuroprotection. Anesthe siology 2006;105:81 90. 33. Chen HS, Lipton SA. The chemical biology of clinically tolerated NMDA receptor antagonists. J Neurochem 2006;97:1611 26. 34. Bickler PE, Fahlman CS. The inhaled anesthetic, isoflurane enhances Ca2+ dependent survival signaling in cortical neurons and modulates MAP kinases, apoptosis proteins and transcription factors during hypoxia. Anesth Analg 2006;103:419 29, table of contents. 35. Franks NP, Honore E. The TREK K2P channels and their role in general anaesthesia and neuroprote ction. Trends Pharmacol Sci 2004;25:601 8.

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20 36. Hans P, Bonhomme V. Why we still use intravenous drugs as the basic regimen for neurosurgical anaesthesia. Curr Opin Anaesthesiol 2006;19:498 503. 37. Kaisti KK, Langsjo JW, Aalto S, Oikonen V, Sipila H, Teras M, Hinkka S, Metsahonkala L, Scheinin H. Effects of sevoflurane, propofol, and adjunct nitrous oxide on regional cerebral blood flow, oxygen consumption, and blood volume in humans. Anesthesiology 2003;99:603 13. 38. Kawaguchi M, Drummond JC, Cole DJ, Kelly PJ, Spurlock MP, Patel PM. Effect of isoflurane on neuronal apoptosis in rats subjected to focal cerebral ischemia. Anesth Analg 2004;98:798 805, table of contents. 39. Kawaguchi M, Kimbro JR, Drummond JC, Cole DJ, Kelly PJ, Patel PM. Isoflurane delays but does not prevent cerebral infarction in rats subjected to focal ischemia. Anesthesiology 2000;92:1335 42. 40. Warner DS. Perioperative neuroprotection: are we asking the right questions? Anesth Analg 2004;98:563 5. 41. Nussmeier NA. Neuropsychiatric complications of cardiac surgery. J Cardiothorac Vasc Anesth 1994;8:13 8.

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21 42. Sarraf Yazdi S, Sheng H, Brinkhous AD, Pearlstein RD, Warner DS. Effects of postischemic halothane administration on outcome from transient fo cal cerebral ischemia in the rat. J Neurosurg Anesthesiol 1999;11:31 6. 43. Steiger HJ, Hanggi D. Ischaemic preconditioning of the brain, mechanisms and applications. Acta Neurochir (Wien) 2007;149:1 10. 44. Du C, Hu R, Csernansky CA, Hsu CY, Choi DW. Very delayed infarction after mild focal cerebral ischemia: a role for apoptosis? J Cereb Blood Flow Metab 1996;16:195 201. 45. Dubois Dauphin M, Pfister Y, Vallet PG, Savioz A. Prevention of apoptotic neuronal death by controlling procaspases? A point of view Brain Res Brain Res Rev 2001;36:196 203. 46. Inoue S, Davis DP, Drummond JC, Cole DJ, Patel PM. The combination of isoflurane and caspase 8 inhibition results in sustained neuroprotection in rats subject to focal cerebral ischemia. Anesth Analg 2006;102: 1548 55. 47. Elsersy H, Sheng H, Lynch JR, Moldovan M, Pearlstein RD, Warner DS. Effects of isoflurane versus fentanyl nitrous oxide anesthesia on long term outcome from severe forebrain ischemia in the rat. Anesthesiology 2004;100:1160 6.

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22 48 Sakai H, Sheng H, Yates RB, Ishida K, Pearlstein RD, Warner DS. Isoflurane provides long term protection against focal cerebral ischemia in the rat. Anesthesiology 2007;106:92 9; discussion 8 10.

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23 PAPER 1: ISOFLURANE PLUS REPETITIVE INTRAPERITONEAL CASPASE INHIBITOR ADMINISTRATION REDUCES LONG TERM HISTOLOGICAL DAMAGE AFTER MCAO IN RATS. Rafael E. Chaparro M D .* ** Carolina Quiroga M.D.* **, Diana Erasso MS** Chrystal Price MS Aakash Hansoti M .D. Devanand Mangar M.D. ^ Enrico M. Camporesi M.D .* Department of Molecular Pharmacology and Physiology, University of South Florida 12901 Bruce B Downs Blvd, Tampa, Florida 33612, USA. **Department of Neurosciences, University of South Florida 12901 Bruce B Downs Blvd, Tampa, Florida 33612, USA. ^ Chief of Staff Tampa General Hospital 1 Tampa General Hospital Circle Tampa FL 33606 in Tampa

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24 Abstract : Introduction: Neuroprotection researchers have used caspase inhibitors to reduce neuronal damage caused by ischemia. Most of the trials have shown positive effects when these substan ces were administered intracrani al l y (1) There is very limited evidence that caspase blockers can be used systemically and still have a beneficial effect. We hypothesized that caspase inhibitors could cross the hematoencephalic barrier during ischemic events like str oke. Materials and methods: To test our hypothesis we used 250 to 350g male Sprague Dawl ey rats of an average age of three months examined after MCAO +CCAO The control group (group 1) received no treatment following the surgery (n=10). Gro up 2 received 90 minutes of 1% Isoflurane while inside an exposure chamber (n=7). Group 3 received 35 mg/kg/h of Propofol i.v. for 90 minutes (n=7). Group 4 received the 1% Isoflurane treatment for 90 minutes followed by 3 systemic injections of a caspase 3 inhibitor (1 00 g Z DEVD FMK, BD Pharmingen, San Jose, CA) that were given 3 times: immediately after, 24 hours after and one week after Isoflurane treatment (n=7). Group 5 received 35 mg/kg/h of Propofol for 90 minutes followed by the same caspase 3 inhibitor treatmen t regimen as group 4 (n=6) Group 6 received only Caspase 3 inhibitor as described before (n=6). Neuroscore evaluation was made on days 0, 4 and 14. Animals were euthanized two weeks after MCAO +CCAO Brains were harvested,

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25 sectioned, and a series of adjac ent sections were stained for H&E, TUNEL and cleaved caspase 3 Infarct size as a percentage of the normal hemisphere was reported. The numbers of positively stained neurons were counted within the infarct ed hemisphere using unbiased Stereology (Stereology Resource Center, Chester, MD). Statistical analysis was performed using Graphpad Prism 5 with Bonferroni test at a 95% confidence interval, after ANOVA (* p<0.05). Results: The combination of Isoflurane plus a caspase 3 inh i bitor significantly reduces the infarct size two weeks after the infarct; the rest of the groups did not show a statistically significant difference The number of neuron s that were positive for TUNEL were not significantly different in the groups that wer e treated with Propofol and Isoflurane alone compared to the control group. However, the number of positive TUNEL neurons were significantly lower in animals that received Z DEVD FMK in comparison with the animals that did not received the caspase inhibitor These results were confirmed with the significant reduction in cleaved caspase 3 positive neurons in the groups treated with the caspase inhibitors compared to the control group and the rats treated with anesthetics alone. Conclusion: Isoflurane plus s ystemic administration of caspase 3 inhibitor has a positive effect in the histological outcome of ischemic brains. With the significant reduction in apoptotic cells, we demonstrated that the caspase inhibitor can

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26 reach intracerebral targe ts after intra peritoneal injections and block the apoptotic cascade, decreasing the deleterious e ffects of programmed cell death

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27 Introduction : Neuronal injury during anesthesia although infrequent, can result in subclinical neurocognitive deficit, long te rm disability or even death, being the i nsult usually due to ischemia, hypoxia or both Innovative neuroprotective maneuvers have shown to decrease neurol ogical morbidity and mortality. Some surgical procedures are more likely to cause brain injury (2) for this reason clinicians have been identifying th ese procedures and working in possible neuroprotective therapies for more than six decades (3) Researches have demonstrated that anesthetics can decrease the ischemic damage, in some cases the neuroprotective effect was only evident for short periods of time (4) in contrast ; other researchers have found positive effect s up to 8 weeks after the insult (5) Sakai et al; evaluated the effect of anesthetics in different models of is chemia and found th at the effect of anesthetics can change depending on the model used to test the neuroprotective effect of the drugs (4,5) Independently of the model use d the histological damage increase s day after day for extended periods of time and these changes can be divided in two stages: an early stage and late stage E arly changes are related to acute neuronal death mediated by glu tamate activity and neuronal depolarization ; and anesthetics like isoflurane have shown to be effective in blocking glutamate activity and neuronal depolarization (6 9) Delayed damage is caused mainly by apoptosis; however, other factors like inflammation play an important role. Researchers have use d caspase inhibitor s to block the apoptotic cascade with remarkable success (1,10 12)

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28 Researchers do not agree in how to manage the ischemic injury but in general there is an agreement that no single therapy is sufficient (13) W e consider that the combination of an anesthetic plus caspase inhibitors can reduce brain damage compared w ith each therapy used alone Materials and Methods. Regulations : The present study was presented, evaluated and approved by the division of Comparative Medicine at the University of South Florida (USF). The experiments were done in following the guidelines of the IACUC of the University of South Florida's College of Med icine. Table 1. Study design Z DEVD FMK caspase 3 inhibitor from Bio sciences pharmaceutical. IV = intravenus. DMSO: Dimethyl Sulfoxide.

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29 The animal s were divided in 6 g roups as shown in table 1a. A nimal s were treated with anesthetics alone or in combination with caspase 3 inhibitors A group was treated only with the caspase inhibitor and al l groups were compared with the control s Table 2. Time course The project was designed to evaluate the neuroprotective effect of anesthetics alone or in combination with caspase inhibitors in a 2 week period. Table 2 shows t he specific order of occurrence of the events. Day 0 is the day of the surgery. Animals : Male Sprague Dawley rats from Harlan Laboratories ( Indianapolis, IN), with age s between 60 to 90 da ys and weights between 250 to 350 grams were used for this experiment. Upon arrival to the USF College of Medicine V ivarium the rats were housed in a climate controlled room in plastic cages in groups of two with free access to water and food and were left in quarantine for a we ek before the experiment took place. In general, all animals received the same !"#$ %&'() $$ *+)(&$ ,-#*.)/)0.+$ 1"(."2/&*$ 345674456$ 58&*9-&:+$$ ;,;$ <'9-"8.=&$ !" #" #" #" #" #" $" #" %" #" &" #" $%" #" "'()*+,-." #"

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30 anesthesia for the vascular occlusion surgery (see description below). In addition t he animals were divided in 6 groups: 1. C ontrol group, these animals received a Middle Cerebral Artery occlusion plus C ommon Carotid Artery Occlusion (MCAO + CCAO) with no treatment 2. Isoflurane group received 90 minutes of 1% Isoflurane and 100% Oxygen in a se aled chamber after the surgery. 3. Propofol group received 90 minutes of Propofol intravenously (tail vein ) at 35mg/kg/h after the surgery. 4. Isoflurane plus caspase 3 inhibitor (C3I ) group : received Isoflurane 1% and 100% Oxygen in a sealed chamber for 90 minutes after the surgery and C3I intraperitoneal100 g in Dimethyl sulfoxide ( DMSO ) 0.1ml The caspase 3 inhibitor used was Z DEVD FMK from DB Pharmingen (San Jose CA.) the animals were injected 3 times: immediately after the surgery, the next day after the surgery and a w eek after the surgery. 5. Propofol plus C3I ; rec eived 90 minutes of Propofol intravenously (tail vein catheter) at 35mg/kg/h after the surgery plus C3I intraperitoneal100 g in ( DMSO ) 0.1ml intraperitoneal 3 times: immediately after the surgery, the next day after the surge ry and a week after the surgery. 6. C3I only, this group received the caspase 3 inhibitor after surgery with the same dosage a nd schedule as the other groups.

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31 Neurological Assessment : We used a seven point neurologic al score to evaluate the animals ( Table 2) (1) The test was done at four different time points The first evaluation was the day before the surgery. This test was performed to assure that neurologically the rats were intact. No animals were withdrawn from the study for failing this evaluation Table 3 Neurological score. All animals were evaluated before the surgery, at day one after the surgery and two weeks after the surgery (1) The neurol ogical evaluatio n was done in all the groups: one day after the surgery, four days after the surgery and fourteen days after the surgery D ead animals were not included in the neurological score; mortality information was recorded and is presented in the results section. !"#$%& '%(")*& !" #$"%&'()*" +" ,-)./0&"*$"&1*&#%" ($#*0-.-*&0-. ",$0&2-3",/..4" 5" %&(0&-6&%"70)2"$," ($#*0-.-*&0-. ",$0&.)89"3:).&"*-)."2/..&%"" ;" 62$#*-#&$/6"8$<&8&#*")#"-.."%)0&(=$#6>" ($#*0-.-*&0-. "()0(.)#7"$#.4")," 2/..&%"94"*-)." ?" ()0(.)#7"$0"3-.@)#7"*$".&A"$0"0)7:*" B" 3-.@)#7"$#.4"),"6=8/.-*&%" C" /#0&62$#6)<&#&66"*$"6=8/.-=$#>"3)*:"-"%&20&66&%".&<&."$," ($#6()$/6#&66" D" %&-%""

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32 Anesthesia : After the weight was recorded, the animal was anesthetized in an induction chamber with 5% Isoflurane in 100% Oxygen After the rat was fully anesthetized we changed the animal from the induction chamber to a mask with 1 to 2% Isoflurane in 100% oxygen. Doppler Study : After shaving the head and neck the animal was placed in a stereotactic frame. A sag it t al incision was made to expose the skull; all the connective tissue was removed with a cotton applicator until the bregma was fully identified A Dremel 400 Series XPR rotary tool was attached to the frame to precisely microdrill a hole at 1 mm posterior and 4 mm lateral to the bregma in the same hemisphere were the middle cerebral artery occlusion plus common carotid artery occlusion occurred An i ntracerebral guide cannula from Harvard Biosciences Company was inserted in the hole and was seal ed in place with dental cem ent; a L aser D o ppler optic rigid prove ( 500 m ) attached to a Laser Doppler monitor from Moor Instrumets LTD, was inserted into the guide cannula. The intracerebral blood pressure was then recorded using Laser Doppler W indows compatible software from Moor Laboratories pr eviously installed in a Dell computer. The animal was then taken off the stereotactic frame and place d on a custom made stainless steal surgical board from Calven de Oriente C.A. During the surgery we were looking for a sharp decrease in blood flow, the a nimals that

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33 did not reach al least 50% decrease in blood flow were not included in the study. An average of 77% decrease in blood flow was recorded in the animals after the arterial occlusion. After the surgery, prob e and can nula were removed and the skin sutured with a skin stapler. After the surgery the animal w as pl ace d in a fresh cage for recover y Permanent Middle Cerebral Artery Occlusion plus Common Carotid Artery Occlusion : A medial incision on the middle line of the neck was done with a 15 blade scalpel, the common carotid, external carotid and internal carotid arteries were identified, and permanent ligation of the common carotid artery with a 4 0 silk was follow ed by an in cision in the arterial bifurcati on of the common carotid artery. A 4 cm Ethicon 4 0 Prolene monofilament was inserted trough the arterial incision into the internal carotid artery and advanced until the middle cerebral artery was reach ed and resistance wa s felt. The tread was secured in place with a 4 0 silk. The skin was closed with a skin stapler (14) Physiological parameters : We measured physiological parameters to assure that the results we obtained were due to the treatment and could not be confused with alterations in physiologic changes. The variables we measured were: Weight, t emperature taken with a rectal thermometer ( a heated pad was used to maintain body tem peratur e during the procedure ) ; a puls e o ximeter was used to measure hemoglobin saturati on (Hb Sat) ; heart rate (HR) and blood pressure (BP) were taken directly from an arterial catheter place d on the femoral artery (SurgiVet

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34 Advisor Monitor. Model number 92V303100 was used for Hb Sat, HR and BP) ; hematocrit was measured using Damon IEC MB Micro Hematocrit Desk Top Lab Centrifuge The measurements were taken before and after the ischemic event. Weight was measur ed right be fore the surgery and at day 14 Brain Collection and Sectioning The animals were euthanatized with an overdose of CO 2 ; immediately after the animal was perfuse d with normal saline solution 0. 9% followed by 4% paraformaldehyde. The brains were harvested, stored in plastic tubes with 4% paraformaldehyde for 24 hours, and then changed to 10%, 20% and 30% sucrose every 24 hours. After that b rains were sectioned with a cryostat at 50m ; s ections w ere collected as soon as the infarct area appeared and two of every 5 section s were collected for TUNEL and Cleaved Capase 3 stain ing until the infarct area was no longer visible (15) A total of 5 to12 sections per brain were collected depending on the infarct size S ections were stored in PBS plus Azide and then mounted for histopathology analysis. Hematoxylin and Eosin staining : H and E analysis was used to quantify the infarct size; the staining method used consisted in: 2 changes in a bsolute ethyl alcohol for 1 minute. Then 2 changes in 95% ethyl alcohol for 1 minute and wash in r unning tap water for 1 2 minutes. Sections were immersed in Mayer's hematoxylin, for 5 10 minutes and then washed in running tap water for 10 minutes. Later submerged in working Eosin Phloxine for 2 3 minutes. 2 more changes in 95% ethyl alcohol for 2 minutes each wi th gentle agitation at each change. After that a bso lute ethyl

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35 alcohol for 2 minutes. Clean in 3 changes of Xylene of 2 minutes each Finally, c overslip directly from xylene (16) Terminal deox ynucleotidyl transferase dUTP nick end labeling (TUNEL) staining : TUNEL was used to evaluate the effect of Z DEVD FMK, Caspase 3 Inhibitor in the infracted hemisphere. ApopTag In Situ Apoptosis Detection Kit from Chemic on International, Temecula, CA. CAT #: S7100 KIT was used following the instructions of the supplier. Cleaved Caspase 3 Staining: We used Cleaved caspase 3 positive cells to confirm that the Z DEVD FMK injected intraperitonely reach specific targets in the brain. Permeabilize in ethanol/acetic acid, 2:1. Wash in 2 changes of PBS. Mounted slides were hydrated in graded ETOH washes to deionized water (5 minutes each). Antigen retrieval was performed using the heating method; sections were steamed in 0.1M citrate buffer, pH 6.0, for 25 minutes, and cooled for 20 minutes. The tissues were washed in PBS for 10 minutes, followed by quenching of endogenous peroxidase activity with 3% H 2 O 2 wash for 10 minutes. The tissues were washed in PBS (3 x 5 minutes each) followed by a blockade o f nonspecific protein binding with 10% normal goat serum, 2% BSA, PBS for 30 minutes at room temperature. The tissues were incubated in avidin for 15 minutes, followed by biotin for 15 minutes, and then incubated at Negative controls were incubated without the primary antibody. Following a PBS wash (4 x 5 minutes), the tissues were incubated with the secondary antibody (biotinylated anti rabbit IgG) at

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36 room temperature for 30 minutes. Following a PBS wash (4 x 5 minutes each), the tissues were incubated wit h Vector ABC (elite Kit) for 20 minutes at room temperature. Following a final PBS wash (4 x 5 minutes each), the sections were incubated in a DAB substrate for visualization of the peroxidase reaction. The sections were washed in deionized water (5 minute s), counterstained, and dehydrated (graded ETOH, 5 minutes each step). S ections were then washed in xylene (3 x 5 minutes each) and coverslipped with permount (15) Statistical Analysis : The data are presented as Mean SE. To measure the infarct size and count the number of TUNEL and Cleaved Caspase 3 positive cells we used unbiased stereology (17) (Cavalieri estimator and optical dissector respectivel y ) measured with the Stereologer from Stereology Resource Center, Chester, Maryland We evaluated the volume of the infarct and also the volume of the opposite hemisphere; results are presented as a stroke area as percentage of the area of the opposite hemisphere to compensate for edema T he results were analyzed using GraphPad Prism 5.0 for Mac. Analysis of Variance was follow by Fisher LSD formula To quantify the number of TUNEL and Cleaved Caspase 3 positive cells in the infracted hemispher e we used unbiased stereology (Optical fractionator) the results are an estimate of the total number of cells. The results were analyzed using GraphPad Prism 5.0 for Mac. 1 way Analysis of Variance was follow by Bonferroni's Multiple Comparison Test. p va lue of <0.05 were considered statistically significant.

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37 Results We designed the experiment with 70 animals; 6 of them ( 1 animal per group ) were excluded because the Doppler never showed a sharp de crease in blood flow (at least 6 0% decrease) W e believe that either the intra arterial tread or the Doppler were misplaced. 9 more animals died during the surgery from subarachnoid hemorrhage, bleeding from the arterial incision and other technical issues; this include 3 animals in the control group, 1 animal in the Isoflurane group 2 animal s in the Isoflurane plus caspase 3 inhbitor, 2 animal s in the propofol plus caspase 3 inhibitor and 1 in the caspase 3 group. 12 ani mals died or were euthanized due to neurological dysfunction between the day of th e sur gery and day 14; this included 6 animals in the control group, 1 in the Isoflurane group, 2 rats in the propofol group 1 in the propofol plus caspase 3 inhibitor group and 2 in the caspase 3 inhibitor group. No replacement animals were added to the study. As p reviously described several physiological variables were considered. We evaluated rectal temperature, mean arterial pressure (MAP), heart rate (HR), hemoglobin saturation (Hg Sat), a nd hematocrit. The results are shown in the t able below We di d not find any statistically significant differences between the pre operative values and the post operative values. We also did not find statistically significant changes in the initial and final weight measured before the ischemic event and at day 14 Physiological variables are shown on Table 4.

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38 ! Table 4 Physiological Variables. Values are mean SD. MAP = Mean Arterial Blood Pressure, HR = Heart rate, T0 = Temperature, C3I = Caspase 3 inhibitor, MCAO = Middle Cere bral A rtery Occlusion, BPM = Bea ts Per Minute, Hb = Hemoglobin. Neurofunction index was calculated in the animals as shown in the next f igure (Neurological Scores ) (1) Behavior measurements on postoperative day 4 demonstrated a reduction of impairment in both groups receiving the caspase inhibitor ( p < 0.05) This trend is voided by day 14, when all animals approached baseline scores. The test used for this purpose wa s an eight point neurological score (from 0 to 7) the animals that score 0 were perfectly normal and the damage progress until a score of 7 when the animal is dead. This score only

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39 measures motor behavior; it does not include the sensory system, memory or any other abilities. Figure 1 Neurological Performance. Behavior measurements on postoperative day 4 demonstrated a reduction of impairment in both groups receiving the caspase inhibitor. This trend is voided by day 14, when all animals approached baseline scores 0 2 4 6 C o n t r o l I s o f l u r a n e P r o p o f o l I s o f l u r a n e + C a s p a s e i n h i b i t o r P r o p o f o l + c a s p a s e i n h i b i t o r C a s p a s e i n h i b i t o r + + + + + + + + + + + + + + + + + + * N e u r o l o g i c s c o r e D a y 1 D a y 4 D a y 1 4

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40 We used Stereology (Cavalieri Method) (17) to accurat ely measure infarct volume. To compensate for edema, we compared the ipsilateral infarcted area with the contra lateral hemisphere area. Significant differences in infarct volume were observed. T he groups treated with anesthetics alone ( Isoflurane and Propofol ) showed no significant differences in infarct size compare d to the control group However, the addition of caspase 3 inhibitors to Isoflurane resulted in up to 50% reductions in infarct size 14 days after permanent focal cerebral ischemia ( p < 0. 05 ) See Figure 2. Figure 2. Relative infarct area to compensate for edema. The graph shows the quantification of the average volume of infarct size 14 days after permanent focal cerebral ischemia as determined by stereological analysis of H&E histochemistry. C3I (Caspase 3 Inhibitor) Control Iso Pro Iso+C3I Pro+C3I C3I 0 10 20 30 40 50 G r o u p s % o f r e l a t i v e i n f a r c t a r e a

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41 Infarct volumes are shown in the figure photomicrographs of brain sections were taken from MCAO rats in all groups. Total infarct volume including cortical and subcortical structures w ere mea sured. T he only group that showed a statistical significant difference was the group treated with Isoflurane plus the caspase 3 inhibitor ( p < 0.05 ). Figure 3 Microphotographs of the infarcts at day 14 after the injury. Isoflurane plus caspase 3 inhibitor reduce the infarct size in Sprague Dawley rats 14 days after stroke. C3I = Caspase 3 Inhibitor.

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42 Neither p ropofol n or i soflurane alone show ed any statistically demonstrable benefit after 14 days. Interestingly the combination of p ropofol plus the caspase inhibitor and the caspase inhibitor by itself did not show a demonstrable benefit. Thus, elevations in caspases that follow focal ischemia appear to contribute to the ischemic injury and are critical targets to reduce infarct size only in combination with i soflurane for this particular ischemic model The number of neuro ns that were positive for TUNEL two weeks after permanent occlusion of the middle cerebral artery plus common carotid artery occlusion, were not significantly different in the groups that were treated with p ropofol and Isoflurane al one compared to the control group. However, the number of positive TUNEL neurons were significantly lower in animals that received the caspase 3 inhibitor in comparison to the animals that did not ( p < 0.05 for the three groups)

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43 ! Figure 4 Estimated total number of TUNEL positive cells in the stro ke hemisphere. The number of TUNEL positive cells is lower in the animals that received the C3I ( Caspase 3 Inhibitor ). These results were confirmed with the significant reduction in cleaved caspase 3 positive cells in the groups treated with the ca spase 3 inhibitor compared to the control group and with the rats treated with anesthetics alone (p < 0. 05 for the three groups ) Control Isoflurane Propofol Isoflurane+C3I Propofol+C3I C3I 0 10000 20000 30000 40000 * G r o u p s T U N E L p o s i t i v e c e l l s i n s t r o k e h e m i s p h e r e

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44 ! Figure 5 Estimated total number of cleaved Caspase 3 positive cells in the str oked hemisphere. The number of cleaved caspase 3 positive cells is significantly lower in the animals treated with C3I ( Caspase 3 Inhibitor ) Discussion : The results suggest that the in tra pe ritoneal injection of Z DEVD FMK, caspase 3 inhibitor in combination with i soflurane has a neuroprotective effect in a model of permanent cerebral ischemia two weeks after the initial injury. It is important to mention that the volume size of the Z DEVD FMK group is statistically significant when the statistic al analysis is done using Anova followed by Bonferroni test but it disappear ed when we used Fisher LSD as post test. W e Control Isoflurane Propofol Isoflurane+C3I Propofol+C3I C3I 0 5000 10000 15000 20000 25000 G r o u p s E s t i m a t e d t o t a l n u m b e r o f C l e a v e d C a s p a s e 3 p o s i t i v e c e l l s *

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45 decided to publish only the Fisher LSD result s because it is the most w idely used test in ischemia research At day four the n eurological score only showed significance in the groups that received the combination, this is another reason to consider that the caspase 3 inhibitor by it self does not have a neuroprotective effect in this model of permanent ischemia The neurological findings were correlated with the histological findings, showing that the combination of Z DEVD FMK, caspase 3 inhibitor and isoflurane is indeed neuroprotective. Isoflurane or p ropofol are not neuroprotective when administered alone in this model of perma nent ischemia two weeks after the insult. Al though a poptosis plays an important role after ischemia and blocking it seems to be the right answer ; we have demonstrated that apoptosis is not the only answer in neuroprotection, it is also important to block the necrosis and anesthetics have demonstrated to be useful in this area (5) In previous studies, researchers have used intracerebral injections of caspase inhibitors (11,18) in this study we used intraperitoneal injection s of a caspase 3 inhibitors, w e believe that since the hemato encephalic barrier is broken after a stroke that facilitates the entrance of the caspase inhibitor. We measure d the number of apoptotic cells in the infarcted hemisphere and we found a significant decrease in the TUNEL positive cells in all the groups that received the caspase inhibitor. We also counted the number of cleaved caspase 3 positive cells in the infarcte d hemisphere to confirm t he results found in the TUNEL analysis and the results were similar

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46 Since not all the groups that received the caspase inhibitor showed a significant decrease in infarct size we conclude that Isoflurane plays an important role as neuroprotective agent. We used a model of permanent cerebral ischemia, the presence of the surgical treat in the arterial side cause s a continuous local reaction leading to apoptosis, and this is one of the factors that may explain the positive effect of caspase inhibitors in this model. In this study we corroborate that in a model of MCAO plus CCAO the addition of caspase inhibitors is neces sary to achieve neuroprotection that anesthetics alone do not offer. The differences found with results reported by Sakai et al ; (5) could be justified by the fact that we created permanent global ischemia since we permanently occluded the common carotid artery Sakai reported persistent protection from Isoflurane with no need for the use of a caspase inhibitor and provided good evidence that the d ifference with Kawaguchi et al ; (12) was due to the later using an MCAO model that integrated permanent carotid occlusion and t hus persistent hypoperfusion. On the other hand, Elsersy et al ; (19) showed evidence of decline of the protective effect o f Isoflurane over time; suggesting that the protection between local and global ischemia is different. It seems clear now that volatile anesthetics have a neuroprotective action under specific conditions (mild to moderate severity) (20)

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47 References : 1. Inoue S, Davis DP, Drummond JC, Cole DJ, Patel PM. The combination of isoflurane and caspase 8 inhibition results in sustained neuroprotection in rats subject to focal cerebral ischemia. Anesth Analg 2006;102:1548 55. 2. Fukuda S, Warner DS. Cerebral protection. Br J Anaesth 2007;99:10 7. 3. Bigelow WG, Lindsay WK, Greenwood WF. Hypothermia; its possible role in cardiac surgery: an investigation of factors governing survival in dogs at low body temperatures. Ann Surg 1950;132:849 66. 4. Kawaguchi M, Kimbro JR, Drummond JC, Cole DJ, Kelly PJ, Patel PM. Isoflurane delays but does not prevent cerebra l infarction in rats subjected to focal ischemia. Anesthesiology 2000;92:1335 42. 5. Sakai H, Sheng H, Yates RB, Ishida K, Pearlstein RD, Warner DS. Isoflurane provides long term protection against focal cerebral ischemia in the rat. Anesthesiology 2007;10 6:92 9; discussion 8 10. 6. Bickler PE, Buck LT, Hansen BM. Effects of isoflurane and hypothermia on glutamate receptor mediated calcium influx in brain slices. Anesthesiology 1994;81:1461 9.

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48 7. Kimbro JR, Kelly PJ, Drummond JC, Cole DJ, Patel PM. Isoflura ne and pentobarbital reduce AMPA toxicity in vivo in the rat cerebral cortex. Anesthesiology 2000;92:806 12. 8. Patel PM, Drummond JC, Cole DJ, Kelly PJ, Watson M. Isoflurane and pentobarbital reduce the frequency of transient ischemic depolarizations duri ng focal ischemia in rats. Anesth Analg 1998;86:773 80. 9. Weigl M, Tenze G, Steinlechner B, Skhirtladze K, Reining G, Bernardo M, Pedicelli E, Dworschak M. A systematic review of currently available pharmacological neuroprotective agents as a sole interve ntion before anticipated or induced cardiac arrest. Resuscitation 2005;65:21 39. 10. Graham SH, Chen J. Programmed cell death in cerebral ischemia. J Cereb Blood Flow Metab 2001;21:99 109. 11. Inoue S, Drummond JC, Davis DP, Cole DJ, Patel PM. Combination of isoflurane and caspase inhibition reduces cerebral injury in rats subjected to focal cerebral ischemia. Anesthesiology 2004;101:75 81. 12. Kawaguchi M, Drummond JC, Cole DJ, Kelly PJ, Spurlock MP, Patel PM. Effect of isoflurane on neuronal apoptosis in rats subjected to focal cerebral ischemia. Anesth Analg 2004;98:798 805, table of contents.

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49 13. Kayiran O, Uysal A, Cuzdan SS, Kocer U. Struggling with ischemia reperfusion injury. J Craniofac Surg 2007;18:457 8. 14. Woitzik J, Schneider UC, Thome C, Schroeck H, Schilling L. Comparison of different intravascular thread occlusion models for experimental stroke in rats. J Neurosci Methods 2006;151:224 31. 15. Price CD, Yang Z, Karlnoski R, Kumar D, Chaparro R, Camporesi EM. Effect of continuous infusion of asialoerythropoietin (aEPO) on short term changes in infarct volume, penumbra apoptosis and behavior following MCAO in rats. Clin Exp Pharmacol Physiol 2009. 16. Gamble M WI. The Hematoxylins and Eosin. Theory and Practice of Histological Techniques Fif th Edition Edinburgh: Churchill Livingstone., 2002:125 38. 17. Mouton P. Principles and Practices of Unbiased Stereology: An Introduction for Bioscientists. 1 edition ed. Chester, Maryland: The Johns Hopkins University Press, 2002. 18. Li H, Colbourne F, Sun P, Zhao Z, Buchan AM, Iadecola C. Caspase inhibitors reduce neuronal injury after focal but not global cerebral ischemia in rats. Stroke 2000;31:176 82.

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50 19. Elsersy H, Sheng H, Lynch JR, Moldovan M, Pearlstein RD, Warner DS. Effects of isoflurane vers us fentanyl nitrous oxide anesthesia on long term outcome from severe forebrain ischemia in the rat. Anesthesiology 2004;100:1160 6. 20. Bickler P E M D Ph.D.; Patel, Piyush M. M.D., Ph.D. Anesthetic Neuroprotection: Some Things Do Last. Anesthesiology 2007;106:8 10.

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51 PAPER 2: HIPPOCAMPAL CELLULAR LOSS AFTER BRIEF HYPOTENSION. Rafael E. Chaparro M.D.* **, Diana Erasso MS**, Carolina Quiroga M.D.* **, Rick Varlotta *, Devanand Mangar M.D.^, Enrico M. Campor esi M.D*. *Department of Molecular Pharmacology and Physiology, University of South Florida 12901 Bruce B Downs Blvd, Tampa, Florida 33612, USA. **Department of Neurosciences, University of South Florida 12901 Br uce B Downs Blvd, Tampa, Florida 33612, USA. ^Chief of Staff Tampa General Hospital. 1, Tampa General Hospital Circle Tampa FL 33606 in Tampa.

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52 Abstract: Introduction: Maintenance of adequate blood flow to the brain is necessary in the course of general anesthesia in order to assure safe recovery and normal brain functio n after surgical intervention. Brief episodes of hypotension have been shown to cause acut e brain damage in animal models We used a rat hemorrhagic shock model to assess functional outcome and to measure the relative neuronal damage at 1, 4 and 14 days post injury. Methods: 250g to 350g Sprague Dawley male rats were subjected to severe hypotension induced by withdrawal of arterial blood from the right femoral artery, while under Isoflurane anesthesia. The mean arterial blood pressure was maintained between 20 30 mm Hg for one minute per hour, for a tot al of 3 times per rat in a two hour period. Shed blood was immediately returned to venous circulation, returning systemic pressure to normal. The rats were separat ed into 4 groups as follow: groups 1, 2 and 3 received 3 minutes of hyp otension 1 minute ev ery hour and were evaluated at 1, 4 and 14 days, respectively. An additional group of rats, group 4, received a sham operation. A neurological assessment including motor abilities, sensory system evaluation and retrograde memory was performed at 1, 4 and 14 days post hypotensive insult. Brains were harvested and stained for F luorojade C and Nissl at 1, 4 or 14 days. Stereology was used to analyze F luorojade C and Nissl stained brain sections to quantitatively detect

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53 neuronal damage after the hypotensive i nsult. Statistical analysis was performed using Graphpad Prism 5 with the Bonferroni test at a 95% c onfidence interval, after ANOVA and for passive avoidance evaluation, Mixed Effect Model was used. Results: We used a rat hemorrhagic shock model to assess behavior and functional outcome and to quantify the relative neuronal damage at 1, 4 and 14 days post hypotension. S ignificant differences in cell injury were seen between control rats and rats that received 3 minutes of hypotension solely at one day after the insult represented by numerous Fluorojade positive cell in the hippocampus quantified by using stereology (optical dissector) A lso a significant decrease in the number hippocampal cells was seen at da y 14. No changes i n frontal c ortical cells were evident at any time. Discussion: Our observation s suggest that in this hemorrhagic model in Sprague Dawley rats, even brief periods of hypotension result in cell damage. We did not find any significant cha nges in behavior in this study,

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54 Introduction: Maintenance of adequate blood flow to the brain is necessary in the course of general anesthesia for all patients in order to assure safe recovery and normal brain function after surgical intervention (1,2) Predictable models of neuronal loss after progressive low blood pressure insults have been developed. Yamauchi et al; have described selective progressive damage to regions of the brain after 2, or 3 minute epi sodes of profound hypotension (low blood pressure, 25 mmHg) after one week of recovery (3,4) These changes were carefully attributed to neuronal necrosis, or accidental cell death, measured one week after all rats survived the hypotensive challenge. However, this study did not measure functional behavior a fter recovery from hypotension. Cognitive dysfunction has been linked with hypotension (5,6) especially in elderly patie nts (7 9) Several studies have studied the relation between hypotension during surgery and neurological performance after surgery but so far a clear link has not been found (10) The largest study that evaluated this matter was The International Study of Postoperative Cognitive Dysfunction but no association between surgical blood pressure and postoperative cognitive function was found (11) In contrast other researchers have found a link bet ween postoperative cognitive function and blood pressure during surgery (12,13) With this in mind we designed an animal study that was able to show a clear link between blood p ressure during surgery and long term functional alterations. The investigation of short bouts of hypotension with lack of brain activity will provide evidence of the amount of brain damage caused at a n

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55 extended two week period. It is likely that the damage can be more effectively quantified w ith combined behavioral and tissue analysis at extended time points. A secondary objective of this study is to characterize an animal model of hypotension that would produce cerebral ischemia useful for testing of anesthetic protection. Materials and Metho ds. Regulations : The present study was presented, evaluated and approved by the division of Comparative Medicine at the University of South Florida (USF). The experiments were done in following the guidelines of the IACUC of the University of South Florida's College of Medicine. Table 1. Study Design. The rats were separa ted into four groups as follows: groups 1, 2 and 3 received 3 minutes of hy potension 1 minute every hour and were evaluated at 1, 4 and 14 days, respectively. An additional group of rats, group 4, received a sham operation.

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56 Table 2 Time course. N eurological assessment including motor abilities, sensory system and retrograde m emory w ere evaluated at 1, 4 and 14 days post hypotensive insult. Brains were harvested at 1, 4 or 14 days Animals: Male Sprague Dawley rats from Harlan Laboratories ( Indianapolis, IN), with age s between 60 to 90 days and weights between 250 to 350 gra ms were used for this experiment. Upon arrival to the USF College of Medicine Vivarium, the rats were housed in a climate controlled room in plastic cages in groups of two with free access to water and food and were left in quarantine for at least a week before the experiment took place.

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57 Groups: The animals were divided in 4 groups: 1. Control group; these animals received no hypotension 2. These animals received 3 minutes of hypotension and were euthanized 24 hours after the surgery 3. These animals received 3 minutes of hypotension and were euthanized 4 days after the surgery. 4. These animals received 3 minutes of hypotension and were euthanized 14 days after the surgery.

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58 N eurological Assessment : We used a forty eight point neurologic al score to evaluate the animals (14) The test was done at 2 different time points. The first evaluation was the day before the surgery this test was done to assure that neurologically the rats were intact; having an animal with any kind of neurol ogical deficit would prevent the animal to be part of the study. No animals were withdrawn from the study under those criteria The second evaluation was before tissue collection The neurological scale is shown in T able 3

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59 Table 3 Fourty eight points neurological scale. This scale has been used in previous papers and inclu des general status and motor and sensory abil ities (14) We wanted to evaluate memory function ; to achieve this we used the passive avoidance paradigm Habituation is the first step, t he animal was placed TEST 0 1 2 3 4 Spontaneous activity (5min) Normal Calm, quiet, explores slowly Somnolent; minimal exploration Stuporous; some movements in place No spontaneous movement Body symetry Normal Slight asymmetry Moderate asymmetry Prominent asymmetry Extreme asymmetry Gait (open bench top) Normal Stiff inflexible Limping Trembling, drifting, falling Does not walk Front limb symetry Normal Light asymmetry Marked asymmetry Prominent asymmetry No body/limp movement Circling/bench top Not present Predominantly one sided turns Circles to one side Circles constantly to one side Pivoting, swaying or no movement Circling/holding tail Not present Tendency to turn to one side Circles to one side Pivots to one side sluggishly Does not advance Hind limb placement Normal Slow placement No placement Vertical screen climbing Normal Climbs with strains; limb weakness present Holds onto slope; does not slip or climb Slide down slope Slides immediately; no effort to prevent fall Beam walking Walks to the end of the beam Walks to the middle of the beam No walking, stays more than 10 s Unsuccessful effort to prevent fall Falls immediately Fore limb touch (needle) Normal Withdraws slowly No withdrawal Hind limb touch (needle) Normal Withdraws slowly No withdrawal Trunk touch (needle) Symmetric Light asymmetry Prominent asymmetry Absent ipsilateral and diminished contralateral response Response absent bilaterally Vibrassae touch Symmetric Light asymmetry Prominent asymmetry Absent ipsilateral and diminished contralateral response Response absent bilaterally Face touch (needle) Normal Withdraws slowly No withdrawal Complex motor General status Simple motor Sensory POINTS

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60 on a platform in a pl exi glass box and we m easured the time they remained o n the platf orm before they step down of it. Ne xt day is training day: we place d the animal on the platform and once the animal ha d positioned the four limbs down the platform it received an electric shock (0.5mA) for three seconds. The animal learned that the safe place to be was the platform. In thi s case we were testing retrograde memories so the animals were trained before the surgery. On the day before tissue collection; the animal was placed in the platform and we measured the time it took to step down the platform up to a maximum of 5 minutes (300 seconds). Latencies greater than 300 seconds were assigned this value Fina lly on the eu thanasia day we measured the time the animal stayed on the platform up to 5 minutes. Anesthesia : After the weight was recorded, the animal was anesthetized in a n induction chamber with 5% Isoflurane in 100% Oxygen After the rat was fully anesthetized we changed the animal from the induction chamber to a mask with 1 to 2% Isoflurane in 100% oxygen The rats were continuously anesthetized during the three episodes of hypotension. Surgery: After shaving the neck, the area was cleaned with iodine and a medial linear incision was made. Plastic catheters were inserted in the jugular vein and both carotid arteries (bilateral) The arterial lines were used for blood pressure quantification and blood aspiration ; the venous line was used for blood reinfusion. The blood was rapidly aspirated until the mean arterial pressure

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61 (MAP) reach ed a point below 20 ; if the animal did not rea ch a MAP below 20 at any of the 3 episodes, it was excluded from the experiment. When MAP was below 20, the chronometer was set for 60 seconds. Between 8 cc to 15 cc of blood were taken to reach MAP of 20 or less. After the minute, the blood was reinfused using the venous catheter. During the procedure the animals were place d on a heated pad to prevent hypothermia. At the end of the third hypotensive episode the catheters were removed and skin was closed with a skin stapler. T he rats recovered in a fresh c age. Physiological parameters: The following variables we re measured: Weight, temperature measured with a rectal thermometer (a heated pad was used to maintain body temperature during the procedure); a puls e oxi meter was used to measure hemoglobin saturation (Hb Sat); heart rate (HR) and blood pressure (BP) were obtained directly from an arteri al catheter place d in the carotid artery (SurgiVet Advisor Monitor. Model number 92V303100 was used for Hb Sat, HR and BP) Readings were made be fore and after each ischemic event. Weight was measure d right before the surgery and at day 14. Brain Extraction and Sectioning The animals were euthanatized with an overdose of CO 2 ; immediately after the animal was perfuse d with normal saline solution 0. 9% followed by 4% paraformaldehyde. The brains were harvested, stored in plastic tubes with 4% paraformaldehyde for 24 hours, and then changed to 10%, 20% and 30% sucrose every 24 hours. Brains were the n sectioned at 40 m with cryostat HM

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62 550 from MICROM International, the chamber tempera ture was turned down to minus 22¡C. A series of 5 coronal sections spaced approximately 960 microns apart were stained. Fluorojade C and Nissl were used for histological analysis. The sections were mounted for histopathology analysis and remaining sections were stored in PBS plus Azide Fluoro J ade C : Fluoro Jade C stain is commonly used in ischemia research to label degenerating neurons regardless of the insult Fluoro Jade C analysis was used to quantify the number of degenerating cells in cortex and CA 1 area of the hippocampus ; the method used is described in detail (15) The sect ions were mounted on slides and air dry ed overnight. Then, dipped in absolute ethyl alcohol 3 times and 1 minute in 70% ethyl alcohol; washed in r unning tap water 1 minute Stain ed with potassium permanganate to oxidize tissue for 15 min while shaking gently 120 mg of potassium permanganate (KMnO4) 0.06% was diluted in 200 mL of PBS. At this point the slides were protected f rom light. Staining with Fluoro Jade C 0.001% for 30 minut es was follow by 3 changes of water for 1 minute and air dry overnight. The next day under the fume hood, cle ar in xylene for 2 minutes, 3 times. Coverslip with DPX directly from xylene. Fluoro Jade st ock was made with 0.01% Fluoro J ade C in water (dilute 2 mg/ 20 ml). The working solution was made with 0.001% Fluoro Jade C in 0.1% acetic acid. Dilute 20 ml stock with 180 mL of water plus 200 uL of acetic acid.

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63 Nissl: Nissl is a classic stain used for detection of Nissl bodies in the cytoplasm of cells, which will be stained purple blue. S ections were mounted and let a ir dry on a slide warmer overnight. Next day they were d ehydrate d through 100% and 95 % alcohol to distilled water. After that were stained in 0.1% cresyl violet solution for 5 minutes and then rinsed with distilled water. Following they were i mmerse d in 95 % ethyl alcohol for 30 minutes and posterior dehydrated in 100% alcohol 2 times for 5 minutes. Under the fume hood they were clear ed in xylene for 2 min utes, 3 times and then immediately c overslip with DPX Statistical Analysis : The data is presented as Mean SE. Counting of Fluoro Jade C positive cells and Nissl cells was done using unbiased stereology ( Optical fractionator ) as described in by M o uton (16) F or this purpose the Stere ologer from Stereology Resource Center, Chester, Maryland was used T he results are an estimate of the total number of cells. Neurological score data were also evaluated. One way Analysis of Variance was follow by Bonferroni's Multiple Comparison test. The results were analyzed using GraphPad Prism 5.0 for Mac. P assive avoidance was evaluated using Mixed Effect Model p value of <0.05 were considered statistically significant.

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64 Results W e designed the experiment with 40 animals, 7 of them died during surgery 3 from the group that undergo euthanize at day 1, 2 animals belong to the group that was euthanized at day 4 and 2 more from the group that was euthanized at day 14. Necropsies showed that pulmonary embolism and technical complications were the main culp rits. 5 more animals died after surgery; bleeding from the arterial incision and neurological deficits were the cause ; this include d 2 animals in the group that was euthanize d at day 4 and 3 animal s in the group that was euthanized at day 14. Dead animals were not replace d (See Table 4) P hysiological variables were considered. The resu lts are shown in the next figure The mean arterial pressure combining all the groups before the first insult was 100.3 + 42, 94.3 + 38 before the second insult and 102 + 40 before the last insult. After the insult the mean arterial pressure was 87.7 + 56.2, 94.5 + 57.2 and 92.7 + 43.2 respectively. During the hypotension period, the blood was withdrawn until the MAP reach 20 or less.

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65 Table 4 Physiological variables. Values are mean SD. MAP = Mean Arterial Pressure, HR = Heart rate, To = Temperature, BPM = Bits Per Minute, Hb = Hemoglobin, F¡= Fahrenheit. No significant differences were found. We did not find any statistically significant differences between the pre operative values and the post operative values. We also compared the weight before the ischemic event and at day 14, and did not find any s tatistically significant change Control ( n =10) Euthanize day 1 ( n =7) Euthanize day 4 ( n =6) Euthanize day 14 ( n =5) Initial Weight (g) 333 + 18 352 11 334 12 344 22 Final Weight (g) 370 + 20 349 13 361 29 380 39 Initial T¡ (F¡) 94.8 2.2 93.02 2.6 92.58 4.3 Final T¡ (F¡) 89.6 7.5 91.88 2.6 88.8 3.1 First MAP 19 2 20 2 19 2 Second MAP 19 1 19 1 20 1 Third MAP 20 2 18 1 19 1 Initial HR ( bpm ) 254 43 271 50 248 61 Final HR ( bpm ) 267 49 233 59 250 54 Initial Hb Saturation 97 2 94 6 95 4 Final Hb Saturation 95 5 94 4 98 7

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66 Neurological performance was evaluated in all animals as shown in the next f igure (Neurological Scores). Behavior measurements on postoperative day 1, 4 and 14 were compared with control; at day one there were positive findings (palpebral ptosis). These findings were not present at day 4 or 14. Although there was neurological impairment 24 hours after the injury, statistically, these changes were not significant (p > 0.05). It is important to mention that some animals also showe d some degree of paralysis right after the surgery but by the time of the ne urological evaluation it was no longer present. Figure 1 Neurological Score. The figure show no significant changes in the neurological sc ore. The only change found at 24 hours was palpebral ptosis in some animals, this finding was not present at day 4 or 14. Passive avoidance as described by Sapo rta (17) was used to tested the ability to recall old memories. During the habituation and training, animals

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67 learned to stay on the platform to avoid an electric shock. We confirmed learni ng by m easuring the time the rat stayed on the p latform before hypotension and later we retested the animals after hypotension. We did not find any memory impairment in the animals after the surgery in comparison with the control animals.

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68 Figure 2 Passive avoidance. Non e of the animals have any memory deficit after the procedure at any of the check points. Stereology was used to estimate the total number of positive Fluoro Jade C ce lls in the frontal cortex and CA 1 area of the hippocampus. No significant differences i n cell injury represented by numerous fluoro j ade positive cortical cells were seen between control rats and rats that received 3 minutes of hypotension (p > 0.05)

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69 Effect of 3 mi nutes of hypotension in frontal cortex Figure 3 Effect of 3 minutes of hypotension in frontal cortex. Esti mated total number of Fluoro Jad e C positive cells in animals subject to 3 min of hypotension.

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70 Figure 4 Pictures from frontal cortex. Fluoro jade C positive cells in cortical areas in animals subject to 3 minutes of hypotension. On the other hand significant differences in cell injury were seen between control rats and rats that received 3 minutes of hyp otension solely at one day after the insult represented by numerous fluoro jade C positive cells in the CA1 area of the hippocampus (p < 0.05). See Figure 5 and 6. Figure 5 Effect of 3 minutes of hypotension in the CA1 area. Group 1 = Stereology cell count in CA1 region, euthanize at day 1; Group 4 = Stereology cell count in CA1 region, euthanize at day 4; Group 14 = Stereology cell count in CA1 region, euthanize at day 14. There is a statistically significant difference in between control and group 1.

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71 Figure 6 Pictures from CA1 area of the hippocampus. One day after injury there are more fluoro jade positive cells than in the rest of the groups We also count ed Nissl cells in the frontal cortex and CA1 area of the hippocampus and we found that the cortex was not statistically affected by the 3 episodes of 1 minute of hypotension at any of the 3 time points. In contrast, the CA1 area of the hippocampus suffered a conti nuous decrease in the number of ce lls statistically significant only at day 14 after the 3 non consecutives minutes of profound hypotension. Results of the Nissl staining confirmed the findings of fluoro jade c; the number of cells did not change in the f rontal cortex but in the CA1 hippocampal area there was a significant detriment in the number of cells evidenced two weeks after the insult. The results from the Nissl cortical counting in the CA1

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72 area of the hippocampus are shown in figure 10 and 11 respe ctively See Figures 7 and 8. Figure 7 Estimated total number of Nissl positive cells in frontal cortex. There are no statistically significant differences between control and the animals euthanized at day 1, day 4 and day 14.

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73 Figure 8 Estimated total number of Nissl cells two weeks after the insult in the CA1 area of the hippocampus. At day 14 after the insult there is a statistically significant difference in comparison with control. The results in the CA1 area are congruent with the results found in the Fluoro Jade C staining; Fluoro Jade C shows an increase in the num ber of cells that suffered some degree of damage and in this case the day after the surgery there was a significant increase of damaged cells. At day 14 as shown with the Nissl stain, the cells that were injured at day 1 are gone and the number of normal cells in the CA1 area is significantly lower.

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74 Discussion: The present observations evidenced that repeated hypotensive episodes lead to hippocampal damage, which may have clinical implications. Patients with hemodynamic TIAs cerebral arteriosclerotic disease, or orthostatic hypotension may experience repeated nonfatal circulatory deficiencies. Our results suggest that in this rat hemorrhagic model, brief periods of hypotension result in neuronal damage or distress in the hip pocampal CA1 region one day after insult. By day 14, surviving cells are significantly reduced in the hippocampus. We did not find any significant changes in cortical cells. W e evaluate d the changes in weight in all the groups and the re was not a significant change; we believe that although the animals show ed some histological changes in the CA1 area of the hippocampus the damage is not enough to induce weight changes. Hypothermia has also shown to reduce ischemic damage (18) to prevent a hypothermic state in our animals a heated pad was place d underneath the rat preventing the presence of variables of confusion. There were not significant changes in the initial and fina l temperature. The mean initial temperature was 93.5 + 3 and the final was 90 + 4.4. Between 8 cc to 12 cc of blood were taken each time t o achieve a mean a rterial blood pressure below 20. The mean arterial blood pressure for all the groups at the beginning of the hypotensive period was 19.2 + 1.1. Yamauchi (3,4) found histopathological changes after one week ; in the hippocampus with a

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75 MAP of 25 with 2 or 3 minutes of hypotension. In our hands these changes continue to be present two week after the insult. The forty eight points neurological scale did not show any st atistically significant changes I nterestingly we found temporal paralysis in the upper body that las t ed only few hours ; by the time of the neurological evaluation the paralysis was gone and could not be recorded as a positive finding. A common finding after 24 hours of recovery was palpebral ptosis but statistically this finding was not significant; in a ll the cases w h ere palpebral ptosis was found, recovery was complete by day 4. In humans the dissection of the Internal carotid artery, typically manifests as an oculo sympathetic palsy (myosis and palpebral ptosis) (19) we placed the catheters in the common carotid artery but since t he surgical area is small, it is possible that we had manipulated the internal carotid artery leading to this finding. Passive avoidance paradigm has been used for memory evaluation; Bekker et al. have demonstrated using this paradigm that hypotension can cause disruption in consolidation of long term memory. In the present study we did not find any memory alterations (20) We hypothesize that this difference is due to e i ther the type of hypotension ; since they used nitroglycerine and we withdrawn the blood. The other important differ ence is the time of hypotension; Bekker caused the hypotension early after the learning ; in fact the latest injection was g iven 3 hours after the training. I n our study we trained the animals 24 hours before the surgery and tested 24 hours after the surgery giving the m enough time to consolidate their memories. This study demonstrated that although there is

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76 damage in the hippocampus after 3 separate period s of profound hypotension, memories that are already consolidated were not affected. Fluoro Jad e C stain is a fluorescein derived u sed to label degenerating neurons We used this staining to evaluate brains on 1, 4 and 14 days after hypotension. I n a pilo t study we evaluated the fluoro jade c stained brains ; t he samples were viewed with a fully automated, upright Zeiss Axio ImagerZ.1 microscope (Carl Zeiss Inc., Germ any) with a 20x /0.5NA objective and a FITC filter cubes. Images were produced using the AxioCam MRm CCD camera and Axiovision version 4.6 software suite (Carl Zeiss Inc., Germany). The resulting images were analyzed for number and intensity of degenerativ e neurons using Image Pro Plus 6.2 (Media Cybernetics Inc., Silver Springs, Maryland). We used the confocal microscope to have a better visualization and also to take pictures. Samples were viewed with a Leica DMI6000 inverted microscope, TCS SP5 confocal scanner, and a 40X/1.30NA Plan Apochromat oil immersion objective (Leica Microsystems, Germany). An argon laser line was applied to excite the samples and tunable filters were used to minimize backgrou nd fluorescence. Image were captured with photomultipli er detectors and prepared with the LAS AF software version 1.6.0 build 1016 (Leica Microsystems, Germany). At this point we found a significant difference in the in the number of fluoro jade c positive cells in the frontal cortex at day 1 (21) however this significant difference was not evident when stereology was used. Results found in Fluoro jade c cells at day on e in the CA1 hippocampal area were significant with the two methods.

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77 We concluded that one day after repetitive period s of hypotension there is brain damage that can persist after 2 weeks.

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78 References : 1. Rubio A, Hakami L, Munch F, Tandler R, Harig F, Weyand M. Noninvasive control of adequate cerebral oxygenation during low flow antegrade selective cerebral perfusion on adults and infants in the aortic arch surgery. J Card Surg 2008;23:474 9. 2. Moritz A, Koci G, Steinlechner B, Holzenbein T, Nasel C, Grubhofer G, Dworschak M. Contralateral stroke during carotid endarterectomy due to abnormalities in the circle of Willis. Wien Klin Wochenschr 2007;119:669 73. 3. Yamauchi Y, Kato H, Kogure K. Brai n damage in a new hemorrhagic shock model in the rat using long term recovery. J Cereb Blood Flow Metab 1990;10:207 12. 4. Yamauchi Y, Kato H, Kogure K. Hippocampal damage following repeated brief hypotensive episodes in the rat. J Cereb Blood Flow Metab 1991;11:974 8. 5. Duschek S, Schandry R. Reduced brain perfusion and cognitive performance due to constitutional hypotension. Clin Auton Res 2007;17:69 76.

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79 6. Wharton W, Hirshman E, Merritt P, Stangl B, Scanlin K, Krieger L. Lower blood pressure correlate s with poorer performance on visuospatial attention tasks in younger individuals. Biol Psychol 2006;73:227 34. 7. Zuccala G, Onder G, Pedone C, Carosella L, Pahor M, Bernabei R, Cocchi A. Hypotension and cognitive impairment: Selective association in pati ents with heart failure. Neurology 2001;57:1986 92. 8. Yap PL, Niti M, Yap KB, Ng TP. Orthostatic hypotension, hypotension and cognitive status: early comorbid markers of primary dementia? Dement Geriatr Cogn Disord 2008;26:239 46. 9. Qiu C, Winblad B, F ratiglioni L. The age dependent relation of blood pressure to cognitive function and dementia. Lancet Neurol 2005;4:487 99. 10. Williams Russo P, Sharrock NE, Mattis S, Liguori GA, Mancuso C, Peterson MG, Hollenberg J, Ranawat C, Salvati E, Sculco T. Rand omized trial of hypotensive epidural anesthesia in older adults. Anesthesiology 1999;91:926 35. 11. Moller JT, Cluitmans P, Rasmussen LS, Houx P, Rasmussen H, Canet J, Rabbitt P, Jolles J, Larsen K, Hanning CD, Langeron O, Johnson T,

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80 Lauven PM, Kristensen PA, Biedler A, van Beem H, Fraidakis O, Silverstein JH, Beneken JE, Gravenstein JS. Long term postoperative cognitive dysfunction in the elderly ISPOCD1 study. ISPOCD investigators. International Study of Post Operative Cognitive Dysfunction. Lancet 1998; 351:857 61. 12. Schutz C, Stover JF, Thompson HJ, Hoover RC, Morales DM, Schouten JW, McMillan A, Soltesz K, Motta M, Spangler Z, Neugebauer E, McIntosh TK. Acute, transient hemorrhagic hypotension does not aggravate structural damage or neurologic motor deficits but delays the long term cognitive recovery following mild to moderate traumatic brain injury. Crit Care Med 2006;34:492 501. 13. Yocum GT, Gaudet JG, Teverbaugh LA, Quest DO, McCormick PC, Connolly ES, Jr., Heyer EJ. Neurocognitive performance i n hypertensive patients after spine surgery. Anesthesiology 2009;110:254 61. 14. Yokoo N, Sheng H, Mixco J, Homi HM, Pearlstein RD, Warner DS. Intraischemic nitrous oxide alters neither neurologic nor histologic outcome: a comparison with dizocilpine. Ane sth Analg 2004;99:896 903, table of contents.

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81 15. Ajmo CT, Jr., Vernon DO, Collier L, Hall AA, Garbuzova Davis S, Willing A, Pennypacker KR. The spleen contributes to stroke induced neurodegeneration. J Neurosci Res 2008;86:2227 34. 16. Mouton P. Principles and Practices of Unbiased Stereology: An Introduction for Bioscientists. 1 edition ed. Chester, Maryland: The Johns Hopkins University Press, 2002. 17. Saporta S, Borlongan CV, Sanberg PR. Neural transplantation of human neuroteratocarcinoma (hNT) neurons into ischemic rats. A quantitative dose response analysis of cell survival and behavioral recovery. Neuroscience 1999;91:519 25. 18. Fukuda S, Warner DS. Cerebral protection. Br J Anaesth 2007;99:10 7. 19. Eschmann L, Favrat B, Botez S, Wue rzner K. [Partial Horner's syndrome and facial pain: a diagnosis one should not miss]. Rev Med Suisse 2006;2:544 6. 20. Bekker A, Haile M, Li YS, Galoyan S, Garcia E, Quartermain D, Kamer A, Blanck T. Nimodipine prevents memory impairment caused by nitrog lycerin induced hypotension in adult mice. Anesth Analg 2009;109:1943 8.

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82 21. Rafael E. Chaparro MD, Carolina E. Quiroga, M.D., M.B.A., Rachel Karlnoski, Ph.D., Devanand Mangar, M.D., Enrico Camporesi, M.D. Brief systemic hypotension results in cortical ne uronal loss in Sprague Dawley rats. American Society of Anesthesiology. Orlando, 2008.

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88 PAPER 3 : ISOFLURANE PROTECTS FROM LEARNING IMPAIRMENT CAUSED BY BRIEF HYPOXIA AND HYPOTENSION IN SPRAGUE DAWLEY RATS Rafael E. Chaparro M.D.* **, Diana Erasso MS**, Carolina Quiroga M.D.* **, Melis s a Olsson *, Rachel Karlnoski PhD. Devanand Mangar M.D.^, Enrico M. Camporesi M.D* *Department of Molecular Pharmacology and Physiology, University of South Florida 12901 Bruce B Downs Blvd, Tampa, Florida 33612, USA. **Department of Neurosciences, University of South Florida 12901 Bruce B Downs Blvd, Tampa, Florida 33612, USA. ^Chief of Staff Tampa General Hospital. 1, Tampa General Hospital Circle Tampa FL 33606 in Tampa

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89 Abstract: Introduction: Previous studies in our lab have demonstrated that short periods of hypotension can cause histological changes in the hippocampal CA1 area, while behav ior remains unchanged (1) We believe that a n even stronger insult may also cause behavioral changes. We used a rat hemorrhagic shock model plus temporary hypoxia to assess functional outcome at different time points post injury. Methods: Spragu e Dawley rats were subjected to brief, severe hypotension induced by withdrawal of arterial blood from the right femoral artery. Mean arterial blood pressure was reduced to 20 to 30 mm Hg for three consecutive minutes, while the an imal was breathing 12% O2 in N2. M ean Hb saturation was maintai ned at 74.2 %. After the 3 minutes s hed blood was immediately returned to venous circulation, returning systemic pressure to normal. At the end of the hypotensive period the animals were returned to breath ing air. Anima ls were evaluated at di fferent time points before their brains were harvested for quantitative his tology. Group 1: C ontrol normal anima l s with no injury. N eurological evaluation at day 0, 1, 4 and 14, passive avoidance at day 3, 4, 7 and 14, euthanize d a t day 14. Group 2 received hypotension plus hypoxia : N eurological evaluation before the surgery and at day 1, euthanize d at day 1; Group 3 re ceived hypotension plus hypoxia: N eurological evaluation before the surgery and at day 4, euthanized at

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90 day 4 ; and Group 4 received hypotension plus hypoxia : neurological evaluation at day 0, 1, 4 and 14, passive avoidance at day 3, 4, 7 and 14, euthanize d at day 14 Group 5 received hypotension plus hypoxia and was treated with isoflurane per 90 minutes after the ins ult: neurological evaluation at day 0, 1, 4 and 14, passive avoidance at day 3, 4, 7 and 14, euthanized at day 14 We used a 48 point neurological evaluation scale, in which the higher the number the worse the deficit (2) Histopatology cal analysis with NeuN and Nissl was performed Statistical evaluation with ANOVA followed by Bonferroni test for NeuN and Nissl results ; for neurological evaluation we used Dunnett's multiple comparison test after AN OVA and for passive avoidance a M ix ed Effect M o del was used Significance was defined as p < 0.05. Results: Neurological assessment showed that hypotension plus hypoxia resulted only in little abnormalities, but no statistically significant changes were found In the pas sive avoidance test we found that new memories were still created after the injuries but animals with no hypotension/hypoxia had a better performance in comparison with the hypotension/h ypoxia. T he use of i soflurane 90 minutes after the insult showed to protect the animals from memory alterations. No histopatological changes were found in any of the groups.

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91 Discussion: This observation suggests that in this model of hypotension plus hypoxia there is mild cerebral damage that is reflected by memory changes and this deficit is maintain ed two weeks after the insult. Exposure to i soflurane after the insult can prevent the onset of memory deficits.

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92 Introduction: Recent publications have shown that hypotension during surgery can produce a range of effects from post operative memory deficits (3) to a n increase in mortality during the next year after surgery ; specially in elderly patients (4) Permanent, stable and a dequate blood flow to the brain is necessary dur in g general an esthesia in order to assure safe recovery and normal brain function after a surgical intervention (5,6) Some researche r s have demonstrated that hypotension can have deleterious effects in brain histology Yamauchi et al ; described progressive damage to several regions of the brain after three separated 1 minute episodes of hypotension (Mean Arter ial Pressure 25 mmHg) after seven days of recovery in rats (7,8) Although Pos t Operative Cognitive D ysfunction (POCD) does not have a clear etiology, some physicians believe Intra Operative Hypotension (IOH) may be the culprit (9,10) Schutz et al; found that a 30 minute period of hypotension does not have any effect in struc tural and motor deficits but has some effects in recovering cognition (11) Hypertensive patients seem to be more sensitive to IOH, Yocum et al; has found a clear relation between IOH in hypertensive patients and dec line in cognitive function (12) Patients with advance d age seem to be more vulnerable to POCD after IOH (13 15) but not all the studies have shown a clear relati on between IOH and POCD (16,17) In our laboratory, using a rat model of hypotension, we have found a clear relation between IOH and histological changes in the hippocampus but not the cortex and memory was not affected (1)

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93 Animal models of hypotension and hypotension plus hypoxia are necessary to study the effect of these variables and their effects in the central nervous system. In this study we evaluated the effect of a short period of profound hypotension plus hypoxia. Histology and behavioral testing was used in order to clearly underst and the relation between these variables. Materials and Methods. Regulations : The present study was presented, evaluated and approved by the division of Comparative Medicine at the University of South Florida (USF). The experiments were done in following the guidelines of the IACUC of the University of South Florida's College of Med icine. Table 1 Study design. T he rats were separated into five groups as follows; group 1 was the cont rol (no injury), the other 4 groups received 3 consecutives minutes of hypo tension plus hypoxia and were euthanized at day 1, 4 or 14; the last group received treatment with i soflurane per 90 minutes Five groups were used in this study; the first 4 groups shown were design to characterize the damage caused by hypotension plus hypoxia and the !"#$%& '(%#)*+,-#+&%.$,&/(%#0-12& 3$)/1+-4*& + & !"#$%"&' ("' )*+',-' ,.' /0$1*#234'5*+',' 647' )*+',' ,.' /0$1*#234'5*+'-' 647' )*+'-' ,.' /0$1*#234'5*+',-' 647' )*+',-' ,.' 87"90%*#4' 647' )*+',-' ,.'

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94 i s oflurane group was added to determine if the expected damage could be reversed with this anesthetic. Table 2 Time course Neurological evaluation and memory test were used to evaluate the damage caused by hypotension plus hypoxia. The creation of new memories after the insult were evaluated using the passive avoidance paradigm as described by Saporta (18) A fourty eight point scale was used for neurological assessment at different time points in a two week period (2) Animals: Male Sprague Dawley rats from Harlan Laboratories ( Indianapolis, IN), with age s between 60 to 90 days and weights between 250 to 350 grams were used for this experiment. Upon arrival to the USF College of Medicine Vivarium, the rats were housed in a climate controlled room in plastic cages in groups of two with free access to water and food and were left in quarantine for at least a week before the experiment took place. !"#$ %&'()'#$ *)&'+ $$ ,-+')$ .",,/0)$ "0+/1"2-)$ 3"4/5&"6+2$ .",,/0)$ "0+/1"2-)$ 7'"/2/2($ .",,/0)$ "0+/1"2-)$ 7),62($ 8&59"2/:)$ !" #" #" $" #" #" #" %" #" &" #" '" #" #" #" (" #" #" $'" #" #" #"

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95 Groups: The animals were divided in 5 groups: 1. Control group, these animals received no hypotension 2. These animals received 3 minutes of hypotension plus hypoxia and were euthanized 24 hours after the surgery 3. These animals received 3 minutes of hypotension plus hypoxia and were euthanized 4 days after the surgery. 4. These animals received 3 minutes of hypotension plus hypoxia and were euthanized 14 days after the surgery. 5. These animals recei ved 3 minutes of hypotension plus hypoxia, they also received treatment with isoflurane per 90 minutes after the insult and were euthanized 14 days after the surgery. Neurological Assessment : Neurological evaluation was perform ed twice in every animal The first evaluation was the day before the surgery this test was done to assure that neurologically the rats were intact; having an animal with any kind of neurological deficit would prevent it to be part of the stud y. No animals were wit hdrawn from the study for this reason The second one was done before tissue collection We used a forty eight point neurological score to evaluate the animals This scale was taken Yokoo et al, it is the result of combining elements of several systems; 0 points indicate normal function, a total of 48 deficit point is possible (2) See Table 3.

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96 Table 3 Fourty eight points neurological score. This scale inclu des general conditions, motor and sensory abilities giving a complete asses sment of the neurological status of the animal. TEST 0 1 2 3 4 Spontaneous activity (5min) Normal Calm, quiet, explores slowly Somnolent; minimal exploration Stuporous; some movements in place No spontaneous movement Body symetry Normal Slight asymmetry Moderate asymmetry Prominent asymmetry Extreme asymmetry Gait (open bench top) Normal Stiff inflexible Limping Trembling, drifting, falling Does not walk Front limb symetry Normal Light asymmetry Marked asymmetry Prominent asymmetry No body/limp movement Circling/bench top Not present Predominantly one sided turns Circles to one side Circles constantly to one side Pivoting, swaying or no movement Circling/holding tail Not present Tendency to turn to one side Circles to one side Pivots to one side sluggishly Does not advance Hind limb placement Normal Slow placement No placement Vertical screen climbing Normal Climbs with strains; limb weakness present Holds onto slope; does not slip or climb Slide down slope Slides immediately; no effort to prevent fall Beam walking Walks to the end of the beam Walks to the middle of the beam No walking, stays more than 10 s Unsuccessful effort to prevent fall Falls immediately Fore limb touch (needle) Normal Withdraws slowly No withdrawal Hind limb touch (needle) Normal Withdraws slowly No withdrawal Trunk touch (needle) Symmetric Light asymmetry Prominent asymmetry Absent ipsilateral and diminished contralateral response Response absent bilaterally Vibrassae touch Symmetric Light asymmetry Prominent asymmetry Absent ipsilateral and diminished contralateral response Response absent bilaterally Face touch (needle) Normal Withdraws slowly No withdrawal Complex motor General status Simple motor Sensory POINTS

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97 Passive Avoidance: Memory was assessed using the passive avoidance paradigm (PA) ; habituation, training and testing were done after the hypotensive/hypoxic in sult. Habituation occur red 24 hours after surgery: during habituation the animal was placed i n a pl exi glass box, the floor of the cage is a n exposed metallic grid that can be manually electrified; on habituation day the animal is place d on a platform that prevent the animal from touching the grid and animals tend to spontaneously step down the platform and the latency is recorded. On the next day the grid is electrified with 0.5mA and again we measured the time they remained in the platf orm before they step down of it; once the animal have placed the four limbs down the platform, the animal received an electric shock (0.5mA) for three seconds. If memory process is intact t he animal learns that the safe place to be is the platform. A day 3 4, 7 and 14 we placed the animals on the platform and me asured the time the animal took to step down of it, with a maximum of 5 minutes (300 seconds). Latencies greater than 300 seconds were assigned this value Fina lly on tissue collection day we took t he time the animal remained on the platform up to 5 minutes. P.A. was only measure d in the control group and the group euthanized at day 14 (18)

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98 Anesthesia : After the weight was recorded, the animal was anesthetized in an induction chamber with 5% isoflurane in 100% o xygen After the rat was fully anesthetized we changed the animal from the induction chamber to a mask with 1 to 2% isoflurane in 100% oxygen A nimals remained anesthetized at all times. A nimals in the isoflurane group re ceived 1% isoflurane in 100% o xy gen by mask for 90 minutes after the insult. Surgery: After shaving the neck, the area was cleaned with iodine and a medial linear incision was made. Plastic catheters were inserted in the jugular vein and both carotid arteries (bilateral). A rterial lines were used for blood pressure and blood suction; the venous line was used for blood reinfusion. B lood was aspirated until the mean arterial pressure (MAP) reach ed a point below 3 0 Hypoxia was induc e d with a mix ture of 12% o xygen and 8 8% n itrogen the animal breathed the mix ture for 5 minutes before the hypotensive period and during the hypotensive period. As soon as MAP was below 30, the chronometer was set for 18 0 seconds. Between 8 cc to 15 cc of blood were taken to reach the MAP of 3 0 or less. After the hypotensive episode t he blood was reinfused using the venous catheter and the hypoxic mixture was stopped and 100% Oxygen was given to the animal until the end of the surgery The catheters were r emoved and the wound closed with a skin stapler. During the procedure the animal s were place d on a heated pad to prevent hypothermia. Recovery from procedure was in a fresh cage.

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99 Physiological parameters: P hysiological parameters were measured to assure that the results we obtained due to the insult and could not related to alterations in physiologic changes. The variables we measured were: Weight, temperature taken with a rectal thermometer (a heated pad was used to maintain body te mperature during the procedure), hemoglobin saturation with a puls e o ximeter (Hb Sat), finall y heart rate (HR) and blood pressure (BP) were taken directly (SurgiVet Advisor Monitor. Model number 92V303100 was used for Hb Sat, HR and BP) Readings were taken before, during and after the ischemic event. Weight was measure d before the surgery and at day 14. Brain Extraction and Sectioning The animals were euthanatized with an overdose of CO 2 immediately after the animal was perfuse d with normal saline solution 0. 9% followed by 4% paraformaldehyde. The brains were harvested, stored in plastic tubes with 4% paraformaldehyde for 24 hours, and then changed to 10%, 20% and 30% sucrose every 24 hours. Brains were the n sectioned at 40 m with cryostat HM 550 from MICROM International, the chamber temperat ure was turned down to minus 22¡C. A series of 5 sect ions spaced approximately 960 microns apart were stained. NeuN and Nissl stains were used for histological analysis. S ections were mounted for histopathology analysis and the remaining sections were s tored in PBS plus Azide

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100 NeuN : NeuN (Neuronal Nuclei) is used to label alive neurons. Immunoreacti vity is observed after mitosis. NeuN is primarily localized in the neuronal nuclei with lighter staining in the cytoplasm. The staining was done following the instructions from the manufacturer (Chemicon) Nissl: Nissl is used for detection of Nissl bodies in the cytoplasm of Cells. The Nissl body stain purple blue. We used this staining to count the number of cells in the Hippocampus. Statistical Analysis : Data is presented as Mean SE. To count the number of NeuN positive cells and Nissl cells in we used unbiased stereology ( Optical fractionator ) as described in other publications (19 21) F or this purpose the Stereologer from Stereology Resource Center, Chester, Maryland was used T he results are an estimate of the total number of cells. Neurological score and passive avoidance data were also evaluated. The results were analyzed using GraphPad Prism 5.0 for Mac. One way Analysis of Variance was used follow ed by Bonferroni's Multiple Comparison Test for NeuN and Nissl F or neurological evaluation we used Dunnett's multiple com parison test after ANOVA. P assive avoidance w a s analyzed using the Mixed Effect Model to study the general differences between groups during the two week period; for specific time points we used One way Analysis of Variance follow ed by Bonferroni's Multiple Comparison Test p value of <0.05 were conside red statistically significant.

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101 Results W e designed the experiment with 5 0 animals, 10 of them died during or after surgery ( 2 per group ) Necropsies showed that pulmonary embolism, bleeding from the arterial incision and neurological deficits were the cause, these animals were not replaced and the experiment was completed with the remaining animals. P hysiological variables are presented on T able 4 The mean arterial pressure combining all the injured groups before the insult was 110, d uring the insult t he mean arterial pressure was 27 and a fter the bl ood was reinfused the MAP was 87 We did not find any statistically significant differences between the pre operative values and the post operative value s Normal animals with no injury, gained a n average of 24 grams in two weeks, the animals that received the insult gained an average of 25 grams in the same period; the animals that received the insult plus were treated with isoflurane 90 minutes after the insult, gained 8 grams in average. Statis tically we did not find any significant difference. The animals that received the insult have lower weight at day 1 and 4 after the injury but by day 14 the weight was comparable with the control animals. The mean hemoglobin saturation during the episode m easured immediately before the hypotensive insult was 74.2; the values bef ore and after the insult were 94.8 and 95.5 respectively. No significant changes in temp erature and heart rate were found

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102 Table 4 Values are mean SD. MAP = Mean Arterial Pressure, HR = Heart rate, To = Temperature, bpm = Bits Per Minute, Hb = Hemoglobin, F¡= Fahrenheit Neurological performance was evaluated in all animals and assigned a value in a neurological score, see Figure 1 Behavior measurements on postoperative day 1, 4 and 14 were evaluated ; the groups evaluated were: in group 1 are all the animals with the sham surgery that received no insult; in group 2 we have the animals that received the insult and were euthanized at day 14 and group 3 is the group that received the insult and also were treated with i soflurane 90 minutes. N o statistically significant changes were found (p > 0.05). It is important to

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103 mention that some animals also showed some degree of paralysis aft er the surgery but by the time of the ne urological evaluation it was no longer present. Figure 1 Neurological score. The figure shows small and no n significant changes in the neurological sc ore. Only the groups euthanized at day 14 were subject of neurological evaluation. Passive avoidance as described by Sapo rta (18) was used to tested the abili ty to recall new memories. The r e wa s a statistically significant difference in memory during the two week period this difference was more evident at day 7 and persist ed at day 14 demonstrating that profound hypotension plus hypoxia for short periods of time can impair the creation of new me mories Animals treated with isoflurane after the insult did no show any memory impairment. 1 2 3 1 2 3 1 2 3 1 2 3 0.0 0.5 1.0 1.5 2.0 N e u r o l o g i c a l s c o r e D a y 0 D a y 1 D a y 4 D a y 1 4 G R O U P S 1 S h a m : N o H y p o t e n s i o n / H y p o x i a 2 C o n t r o l : H y p o t e n s i o n / H y p o x i a 3 H y p o t e n s i o n / H y p o x i a T r e a t m e n t w i t h I s o f l u r a n e

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104 Figure 2 Passive avoidance. There is a statistically significant d ifference between groups 1 and 2 and 2 and 3. There is no statistically significant difference between groups 1 and 3. Stereology was used to estimate the total number of positive NeuN cells in the CA 1 area of the hippocampus. No difference in cell number s were seen between control rats and rats that received 3 minutes of profound hypotension plus hypoxia at any time point (p > 0.05) day 1 day 2 day 3 day 4 day 7 day 14 0 100 200 300 400 G R O U P S 1 S h a m : N o H y p o t e n s i o n / H y p o x i a 2 C o n t r o l : H y p o t e n s i o n / H y p o x i a 3 H y p o t e n s i o n / H y p o x i a T r e a t m e n t w i t h I s o f l u r a n e G r o u p 1 G r o u p 2 G r o u p 3 D a y s a f t e r h y p o t e n s i o n + h y p o x i a T i m e i n s e c o n d s *

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105 Figure 3 NeuN positive cells in animals subject to 3 min of hypoten sion/hypoxia. No statistically significant changes were found. Similar number of cells were found at all the time points analyzed. Nissl stained cells in the CA1 area of the hippocampus were counted and although we found a patter n of decrease in the number of cells (see day 14) there were no statistically significant changes at any time point after the hypotension plus hypoxia insult. Control 1 day 4 days 14 days Isoflurane 0 50000 100000 150000 G r o u p s E s t i m a t e d t o t a l n u m b e r o f N e u N + c e l l s

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106 Figu re 4 Nissl cells in CA1 area two weeks after the insult. No significant changes in the number of cell s in the CA1 area of the hippocampus were found. Discussion: Although this model of insult is a temporary model (3 minutes of hypotension p lus 8 minutes of hypoxia), we occluded the two common carotid arteries making it a model of constant hypoperfusion or global ischemia. However, even with this permanent ischemia, we did not find changes in the CA1 area. Weight is always a good indicative of general conditions normal animals increase in weight day after day ; animals th at received the insult lost a Control 1 day 4 days 14 days Isoflurane 0 200000 400000 600000 800000 G r o u p s E s t i m a t e d t o t a l n u m b e r o f N i s s l c e l l s

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107 statistically significant amount of weight after the insult but by day 14 this difference vanish among the gr o ups Oxygen saturation was recorded with a pulse o ximeter and maintained at an average of 74.2 + 6.6 assuring that a lthough we use d a facial mask for administration the mix ture of 12% oxygen plus 88% n itrogen was effective in diminishing the saturation levels Between 8 to 1 5 milliliters of blood were taken from the animal to decrease the blood pressure to a mean of 27.1 + 5.4 In previous experiments we have used lower MAP (below 20) (22) but in a preliminary study using that hypotension plus hypoxia the mortality was to o high, for this reason we maintained a higher MAP (MAP < 30). Temperature is a we ll known factor in neuroprotection, the results showed that the heated pad is a good device to control this variable. The temperature at the beginning of the surgery was 95.5 + 2.9 and 93.5 + 1.6 at the end of the surgery. Neurological scale evaluation showed some negative effect s related to the procedure but statistically results were not significant. Palpebral ptosis was the main motor alteration we found. In humans the dissection of the Internal carotid artery, typically manifests as an oculo sympathe tic palsy (myosis and palpebral ptosis) (23) Passive avoidance was used to test anterograde memories and a negative effect was seen one week after the insult, this memory deficiency persisted at two weeks after the insult. In a recent paper Bek ker et al; found

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108 impair ment after hypotension in long term associative memory (3) Althoug h we did not use the same model of hypotension and we also added hypoxia, the present study may highlight a similar mechanism This memory deficit was reverse by the use of i soflurane per 90 minutes after the insult. Shutz et al; have demonstrated that hem orrhagic hypotension does not cause structural damages but can cause cognitive alterations (11) Our study show ed a similar result, we found long term (2 weeks) memory impairment w ith out neurological changes or histological changes in the hippocampus.

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109 References : 1. Rafael E. Chaparro M D Carolina E. Quiroga, M.D., M.B.A., Rachel Karlnoski, Ph.D., Devanand Mangar, M.D., Enrico Camporesi, M.D.Ra. Delayed Cell Demise in Rat Hippocampus after Brief Hypotension. ASA Program Manual 2009; # A 1131. American Society of Anesthesiology. New Orleans, 2009. 2. Yokoo N, Sheng H, Mixco J, Homi HM, Pearlstein RD, Warner DS. Intraischemic nitrous oxide alters neither neurologic nor histologic outcome: a comparison with dizocilpine. Anesth Analg 2004;99:896 903, table of contents. 3. Bekker A, Haile M, Li YS, Galoyan S, Garcia E, Quarterma in D, Kamer A, Blanck T. Nimodipine prevents memory impairment caused by nitroglycerin induced hypotension in adult mice. Anesth Analg 2009;109:1943 8. 4. Bijker JBvK, Wilton A.; Vergouwe, Yvonne; Eleveld, Douglas J.; van Wolfswinkel, Leo; Moons, Karel G. M.; Kalkman, Cor J. Intraoperative Hypotension and 1 year mortality after non cardiac surgery. Anesthesiology 2009;111:1217 26.

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110 5. Rubio A, Hakami L, Munch F, Tandler R, Harig F, Weyand M. Noninvasive control of adequate cerebral oxygenation during low f low antegrade selective cerebral perfusion on adults and infants in the aortic arch surgery. J Card Surg 2008;23:474 9. 6. Moritz A, Koci G, Steinlechner B, Holzenbein T, Nasel C, Grubhofer G, Dworschak M. Contralateral stroke during carotid endarterectom y due to abnormalities in the circle of Willis. Wien Klin Wochenschr 2007;119:669 73. 7. Yamauchi Y, Kato H, Kogure K. Brain damage in a new hemorrhagic shock model in the rat using long term recovery. J Cereb Blood Flow Metab 1990;10:207 12. 8. Yamauchi Y, Kato H, Kogure K. Hippocampal damage following repeated brief hypotensive episodes in the rat. J Cereb Blood Flow Metab 1991;11:974 8. 9. Duschek S, Schandry R. Reduced brain perfusion and cognitive performance due to constitutional hypotension. Clin Auton Res 2007;17:69 76.

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111 10. Wharton W, Hirshman E, Merritt P, Stangl B, Scanlin K, Krieger L. Lower blood pressure correlates with poorer performance on visuospatial attention tasks in younger individuals. Biol Psychol 2006;73:227 34. 11. Schutz C, Stov er JF, Thompson HJ, Hoover RC, Morales DM, Schouten JW, McMillan A, Soltesz K, Motta M, Spangler Z, Neugebauer E, McIntosh TK. Acute, transient hemorrhagic hypotension does not aggravate structural damage or neurologic motor deficits but delays the long te rm cognitive recovery following mild to moderate traumatic brain injury. Crit Care Med 2006;34:492 501. 12. Yocum GT, Gaudet JG, Teverbaugh LA, Quest DO, McCormick PC, Connolly ES, Jr., Heyer EJ. Neurocognitive performance in hypertensive patients after s pine surgery. Anesthesiology 2009;110:254 61. 13. Zuccala G, Onder G, Pedone C, Carosella L, Pahor M, Bernabei R, Cocchi A. Hypotension and cognitive impairment: Selective association in patients with heart failure. Neurology 2001;57:1986 92. 14. Yap PL, Niti M, Yap KB, Ng TP. Orthostatic hypotension, hypotension and cognitive status: early comorbid markers of primary dementia? Dement Geriatr Cogn Disord 2008;26:239 46.

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112 15. Qiu C, Winblad B, Fratiglioni L. The age dependent relation of blood pressure to cognitive function and dementia. Lancet Neurol 2005;4:487 99. 16. Williams Russo P, Sharrock NE, Mattis S, Liguori GA, Mancuso C, Peterson MG, Hollenberg J, Ranawat C, Salvati E, Sculco T. Randomized trial of hypotensive epidural anesthesia in older adult s. Anesthesiology 1999;91:926 35. 17. 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, Benek en JE, Gravenstein JS. Long term postoperative cognitive dysfunction in the elderly ISPOCD1 study. ISPOCD investigators. International Study of Post Operative Cognitive Dysfunction. Lancet 1998;351:857 61. 18. Saporta S, Borlongan CV, Sanberg PR. Neural t ransplantation of human neuroteratocarcinoma (hNT) neurons into ischemic rats. A quantitative dose response analysis of cell survival and behavioral recovery. Neuroscience 1999;91:519 25.

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113 19. Shi L, Molina DP, Robbins ME, Wheeler KT, Brunso Bechtold JK. H ippocampal neuron number is unchanged 1 year after fractionated whole brain irradiation at middle age. Int J Radiat Oncol Biol Phys 2008;71:526 32. 20. Fitting S, Booze RM, Hasselrot U, Mactutus CF. Dose dependent long term effects of Tat in the rat hippo campal formation: A design based stereological study. Hippocampus 2009. 21. Skoglund TS, Pascher R, Berthold CH. Aspects of the quantitative analysis of neurons in the cerebral cortex. J Neurosci Methods 1996;70:201 10. 22. Rafael E. Chaparro MD, Carolin a E. Quiroga, M.D., M.B.A., Rachel Karlnoski, Ph.D., Devanand Mangar, M.D., Enrico Camporesi, M.D. Brief systemic hypotension results in cortical neuronal loss in Sprague Dawley rats. American Society of Anesthesiology. Orlando, 2008. 23. Eschmann L, Favr at B, Botez S, Wuerzner K. [Partial Horner's syndrome and facial pain: a diagnosis one should not miss]. Rev Med Suisse 2006;2:544 6.

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! 114 CONCLUSIONS: I schemia seems to be the cause of several disabilities and even death after surgical procedures In the quest for options to manage it s everal variables have been studied with mixed results A mong them are hypothermia, hyp erthermia, hyperoxemia, hypergly cemia, hyperventilation, etc. P harmacologica l agents have been used for these purpose s in animals and in humans with better results in the animals Under specific conditions anesthetics have shown to have a neuroprotective effect but t hey do not work equally well in different models of ischemia (1) The main objective of this research project was to test the neuroprotective effect of anesthetics in different models of cerebral ischemia. The results of our first study indicate that the combination of Isoflurane plus repeated administration of a caspase 3 inhibitor ( Z DEVD FMK ) r educes the infar ct size. We also observed a clear pattern of improvement with the use of Propofol in combination with Z DEVD FMK but it was not statistically significant. A similar but weaker protective pattern was found with the use of the caspase inhibitor by it self but the difference was not significant either It is possible that by using a bigger sample size these patterns c ould reach significance. It s eems clear that in a model of focal ischemia combined with mild global ischemia (middle cerebral artery occlusio n plus common carotid artery occlusion)

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! 115 the combination of volatile anesthetics with other ph armacological agents like caspase inhibitors is necessary to achieve a sustained protection (two weeks) (2) The positive result s found with Isoflurane plus Z DEVD FMK confirm ed that the apoptotic cascade is activated during the ischemic event and interventions directed to block apoptosis are possible targets of pharmacologic agents The synergi stic effect of anesthetics plus caspase inhibitors has been demonstrated by other researchers (3,4) and our results confirm ed the effectiveness of th e combination. Although the administration of Z DEVD FMK was done intraperitoneal ly the caspase inhibitor reach ed intracerebral targets. W e believe that the caspase inhibitor got access to the apoptotic ne urons thanks to the disruption of the blood brain barrier due to the ischemic insult We found a significant decrease in the number of Tunel positive cells in the brains of the animals that received th e caspase inhibitor and in order to confirm that this effect was related to the intraperitoneal administration of the casp a se inhibitor, we count ed the cells that w ere posi tive for cleaved caspase 3. These results w ere consistent with the Tunel results and showed that animals that received the intraperitoneal injection had an important decrease in the number of apoptotic cells. We demonstrated that systemic administration of the caspase inhibitors is effective and can be used for the treatment of ischemic events in ani mal models. Histological improvement was consistent with neurological performance at four days after the insult ; animals treated with anesthetics plus Z DEVD FMK

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! 116 showed a significant improvement in comparison with the animals that d id not receive the combi nation. B y day 14 all the animals showed an important improvement. We believe that the scale used was not sensitive enough to pick some of the alterations that were present at two weeks after the insult. A more comprehensive scale is necessary to have a be tter understand ing of the neurological outcome : for this reason, recently a 48 point scale was develop ed by connecting together several neurological scales making a more specific and reliable scale to measure neurological performance (5) I t i s clear now that anesthetics alone can also be protective and that this protection is sustained for long periods of time in models of focal ischemia (6) P rotection is evident in models of global ischemia only for short periods of time (7) Since we occluded the middle cerebral artery and also the common carotid artery, we create d a mix ed mo d el of focal and global ischemia; furthermore, t he fact that anesthetics alone did not work could be explained for the global ischemic effect created by the blockage of the common carotid artery In addition, i t is also known that Isoflurane activates caspase 3 (8) and this is probably at least part of the reason for the lack of effectiveness of Isoflurane in models of global ischemia. In our work w e used a caspase 3 inhibitor because it is part of the common pat hway in the apoptotic cascade, and we believe tha t blocking only the intrinsic or extrinsic apoptotic pathway is less effective than blocking the common pathway In addition the caspase 3 inhibitor can also block the caspase 3 activating effect of the Iso flurane granting a more potent neuroprotection.

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! 117 In the bibliography hypothermia has been reported to be an important neuroprotective influence (1) I n our study we contro l l ed t emperature to prevent bias. T o prevent a hypothermic state in our animals a heated pad was place d un derneath the rat There were not significant changes in the initial and final temperature. The mean initial body temperature was 93.5 + 3 and the final was 90 + 4.4. Although the temperature was lower than the normal, there are no statically significant differences in between the groups. We are confident that the results we obtained were not influenced by hypothermia since all the groups showed similar values Monitoring other parameters like arterial blood gases and glycemia was also important. W e did not control these variables because of technical difficulties but it is highly recommended to consider these parameters to be able to attribute the results to the actual treatment been evaluated and not to external factors that were not consider ed in the study. In our experiments a fter c ollecting the brains we review ed the placement of the filamen t in the middle cerebral artery. W e found that in a few cases the filament moved T herefore, we consider that the filament approach to occlusion of the middle cerebral artery is better for temporary focal ischemi a rather than permanent ischemia For permanent blockage of the middle cerebral artery the use of craniotomy and direct cauterization of the artery is recommended. T his al l o ws the surgeon to in sure that the artery will remain close d and permanent focal ischemia will follow. After finding the neuroprotective effect of anesthetics in combination with

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! 118 caspase inhibitors in the previous animal model we decided to look fo r another model of ischemia to verify the neuroprotective effect of anesthetics. We found that anesthesiologists were divid ed regarding the issue of continuous monitoring of arterial blood pressure during surgery. T he group in favor of direct a rterial pressure monitoring believe that even a short period of hypotension not detected by traditional monitor ing methods, can be the origin of conditions like post operative cognitive dysfunction With that idea in mind we decided to study a model of profound hypotension for short periods of time Initially the use of nitroprusside seemed to be the logical as the hypotensive agent. How ever, after a pilot s tudy with mixed results and uncertainty whether the results were related to hypotension or the cyanide released as a principal metabolite of nitroprusside ; we opted for a bleeding induced hypotension model In a pilot study we tested one, two and three minutes of profound hypotension (M ean A rterial Blood P ressure below 20) and w e did not find any changes either in the cortex no r in the hippocampus with only one or two minutes of hypotension In the three minutes of hypotension group w e found fluoro jade C positives cells in cortex and hippocampus. B ecause of the se findings we decided to conduct a study to detect if th ose changes were maintained Our observations evidenced that repeated hypotensive epis odes lead to hippocampal damage Our results suggest that in this rat hemorrhagic model, brief periods of hypotension result in neuronal damage in the hippocampal CA1 region one day after the insult. By day 14, surviving cells are significantly

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! 119 reduced in number in the hippocampus. We did not find any signifi cant changes in cortical cell numbers W e believe that more studies are necessary to elucidate subtler effect s of apparently innocuous periods of hypotension may not be accurately detected by indirect blood pressure monitoring. W e eval uate d the changes in weight in all the groups and the re was not a s ignificant change. W e believe that although the animals show ed some histological changes in the CA1 area of the hippocampus the damage was not sufficient to induce weight changes two weeks after the injury Although short periods of profou nd hypotension may not alter general conditions, they can be implicated in damage to very sensitive cells. M ore research is necessary to show whether th ose changes are limited to the brain or if other organs are affected. In our experimental model 8 cc to 12 cc of blood were removed each time to achieve a mean arterial blood pressure below 20. The mean arterial blood pressure for all the groups at the beginning of the hypotensive period was 19.2 + 1.1. Yamauchi et al (9,10) found histopathological changes after one week in the hippocampus with a MAP of 25 with 2 or 3 minutes of hypotension. In our experience these changes continue to be present two week s after the insult. Our findings support previous data that showed deleterious effects of short periods of hypotension. We found damages in the hippocampus and since this structure is part of the machinery involve d in creating memories, there is a possibili ty that short periods of hypotension c ould explain cognitive impairment seen after surgery in some patients.

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! 120 The forty eight point neurological scale did not show any st atistically significant changes I nterestingly we found temporal motor paralysis in the upper body that last ed only few hours B y the time of the neurological evaluation the paralysis was gone and could not be recorded as a positive finding. A common finding after 24 hours of recovery was palpebral ptosis but statistically t his finding was not significant. I n all the cases w h ere palpebral ptosis was found, recovery was complete by day 4. In humans the dissection of the Internal carotid artery, typically manifests as an oculo sympathetic palsy (myosis and palpebral ptosis) (11) we placed the catheters in the common carotid artery but since the surgical area is small, it is possible that we had manipulated the internal carotid artery leading to this finding. The p assive avoidance paradigm is used for memory evaluation (12) ; Bekker et al. (2009) have demonstrated using this paradigm that hypotension can cause disruption in consolidation of long term me mory (13) In the present study we did not find any memory alte rations We hypothesize that this difference is due to e i ther the type of hypotension ( they used nitroglycer ine and we withdrew the blood) or t he other important difference is the timeline of the hypotensive period. Bekker caused the hypotension early after the learning ; in fact the latest injection was g iven 3 hours after the training. I n our study we trained the animals 24 hours before the surgery and tested 24 hours after the surgery giving the m enough time to con solidate their memories. Our study demonstrated that although there is damage in the hippocampus after 3 separate

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! 121 period s of profound hypotension, memories that are already consolidated were not affected. We used fluoro jade C to evaluate brains on 1, 4 a nd 14 days after hypotension. W e found a significant difference in the in the number of fluoro jade c positive cell s in the frontal cortex at day 1. C ells were count ed using an automatic met h od as previously described (14) H owever this significant di fference was not evident when stereology was used. Results found in Fluoro jade c cells at day on e in the CA1 hippocampal area were significant with the two methods. We concluded that one day after repetitive period s of hypotension there is brain damage th at can persist after 2 weeks. Although the number of affected cells in cortex did not reach significance, we believe that the presence of these fluoro jade positive cells is important and should be consider ed; since the numbers were close to significant an d is possible that by using more animals numbers can reach significance. After finishing with this experiment we believed that although there w as some degree of damage, it was possible that the model was not severe enough to test the efficacy of anesthetic s as neuroprotective agents. For this reason we decided to conduct another study with a stronger insult in order to create more damage and in this way be able to tes t the effect of anesthetics in a new model. We developed a model of profound hypotension si milar to the one previously used; but instead of using one minute every hour during two hours for a total of three episodes of hypotension, we used three consecutive minutes of hypotension combined with eight minutes of hypoxia. After the insult w e exposed

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! 122 the animals to Isoflurane for ninety minutes to evaluate its effect We believed that because the insult was longer, the damage should be greater we were surprised we did not find any histological damage at any of the time points analy zed (1 day, 4 days and 14 days). The histology was done using NeuN staining NeuN was used because it is specific for neurons and should improve the counting methods W hen we realized that no changes were found an d taking into consideration that in the previous study we ha d found changes with the Nissl stain ing we decided to confirm our NeuN results with Nissl staining A gain no changes were seen. It is important to mention that we also stained the brains with fluoro j a de C only in the animals that were sacrifice d at day one and compared tho se results with control animals to see if any damage was evident but again no changes were seen. We did not show the fluoro jade c results in here because not all groups were represented Our theory is that to be able to cause some dam age in the brain it is necessary to have repetitive insults; one insult was not capable of causing histological changes. A closer look at the data show s that in this experiment we did achieve a mean arterial pressure (MAP) below 20 as used in our previous study and as used by other researche r s (14) I n a preliminary study using hypotension with a mean arterial pressure below 20 plus hypoxia the mortality was too high (80%) for this reason we maintained a higher MAP (MAP < 30). It is pos sible that this is a determining factor that can be responsible for the unexpected results. The n eurological scale showed some impairments related to the procedure but statistically the results were not significant. This is not surprising

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! 123 since histological evaluati on of one of the most ischemia sensible areas of the brain was not affected. Palpebral ptosis was the main motor alteration we found; again possibly related to the dissection of the Internal carotid artery, which manifests as an oculo sympathetic palsy in humans (myosis and palpebral ptosis) (11) Passive avoidance was used to test anterograde memories an d a negative effect was seen In a recent paper B ekker et al (2009) ; found impairment after hypotension in long term associative memory (13) Although we did not use the same model of hypotension and we also added hypoxia, the present study may highlight a similar mechanism. Other investigations have demonstrated that hemorrhagic hypotension does not cause structural damages but can cause cognitive alterations (15) Our study showed a similar result, we found long term (2 weeks) memory impairment without neurological changes or histological changes in the hippocampus. What we find interesting is the fact that using Isoflurane for 90 mi nutes after the insult could reverse the memory changes seen with a single episode of hypotension plus hypoxia We concluded that a single episode of hypotension and hypoxia is not enough to caus e histological changes in the CA 1 hippocampal area but it is enough to cause m emory changes that can be reversed with the use of i soflurane. As a general conclusion we demonstrated that anesthetics have a neuroprotective effect. T hey act in different ways according ly to th e insult that caused the injury and in some cases they need coadjuv ants to exert i t s beneficial

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! 124 effect I n the first experiment neither i soflurane and p ropofol show ed any long lasting protective effect by themselves, nor the coadjuvant drug alone, but the combination was neuroprotective. I n the second case although there were not histological changes there were memory changes that were prevented by the use of only anesthetics.

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! 125 References : 1. Fukuda S, Warner DS. Cerebral protec tion. Br J Anaesth 2007;99:10 7. 2. Inoue S, Drummond JC, Davis DP, Cole DJ, Patel PM. Combination of isoflurane and caspase inhibition reduces cerebral injury in rats subjected to focal cerebral ischemia. Anesthesiology 2004;101:75 81. 3. Inoue S, Davis DP, Drummond JC, Cole DJ, Patel PM. The comb ination of isoflurane and caspase 8 inhibition results in sustained neuroprotection in rats subject to focal cerebral ischemia. Anesth Analg 2006;102:1548 55. 4. Jin K GS, Mao X, et al. 2001;21:1411 21. Fas (CD95) may mediate delayed cell death in hippo campal CA1 sector after global ce rebral ischemia. J Cereb Blood Flow Metab 2001;21:1411 21. 5. Yokoo N, Sheng H, Mixco J, Homi HM, Pearlstein RD, Warner DS. Intraischemic nitrous oxide alters neither neurologic nor histologic outcome: a comparison with dizocilpine. Anesth Analg 2004;99:896 903, table of contents. 6. Sakai H, Sheng H, Yates RB, Ishida K, Pearlstein RD, Warner DS. Isoflurane provides long term protection against focal cerebral ischemia in the rat. Anesthesiology 2007;106:92 9; discussion 8 10.

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! 126 7. Elsersy H, Sheng H, Lynch JR, Moldovan M, Pearlstein RD, Warner DS. Effects of isoflurane versus fentanyl nitrous oxide anesthesia on long term outcome from severe forebrain ischemia in the rat. Anesthesiology 2004;100:1160 6. 8. Zhang G, Dong Y, Zhang B, Ichinose F, Wu X, Culley DJ, Crosby G, Tanzi RE, Xie Z. Isoflurane induced caspase 3 activation is dependent on cytosolic calcium and can be attenuated by memantine. J Neurosci 2008;28:4551 60. 9. Yamauchi Y, Kato H, Kogure K. Brain damage in a new hemorrhagic shock model in the rat using long term recovery. J Cereb Blood Flow Metab 1990;10:207 12. 10. Yamauchi Y, Kato H, Kogure K. Hippocampal damage following repeated brief hypotensive episodes in the rat. J Cereb Blood Flow Metab 1991;11:974 8 11. Eschmann L, Favrat B, Botez S, Wuerzner K. [Partial Horner's syndrome and facial pain: a diagnosis one should not miss]. Rev Med Suisse 2006;2:544 6. 12. Saporta S, Borlongan CV, Sanberg PR. Neural transplantation of human neuroteratocarcinoma (hNT) neurons into ischemic rats. A quantitative

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! 127 dose response analysis of cell survival and behavioral recovery. Neuroscience 1999;91:519 25. 13. Bekker A, Haile M, Li YS, Galoyan S, Garcia E, Quartermain D, Kamer A, Blanck T. Nimodipine prevents memory impai rment caused by nitroglycerin induced hypotension in adult mice. Anesth Analg 2009;109:1943 8. 14. Rafael E. Chaparro MD, Carolina E. Quiroga, M.D., M.B.A., Rachel Karlnoski, Ph.D., Devanand Mangar, M.D., Enrico Camporesi, M.D. Brief systemic hypotension results in cortical neuronal loss in Sprague Dawley rats. American Society of Anesthesiology. Orlando, 2008. 15. Schutz C, Stover JF, Thompson HJ, Hoover RC, Morales DM, Schouten JW, McMillan A, Soltesz K, Motta M, Spangler Z, Neugebauer E, McIntosh TK. A cute, transient hemorrhagic hypotension does not aggravate structural damage or neurologic motor deficits but delays the long term cognitive recovery following mild to moderate traumatic brain injury. Crit Care Med 2006;34:492 501.

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ABOUT THE AUTHOR Rafael Eduardo Chaparro received his Medical Doctor degree from the Po ntificia Universidad Javeriana in Bogota Colombia in 1996; he then work ed in Colombian rural areas serving communities and for the Colombian Army In 2001 he received his first Master's degree in Occupational Health Management fr om the Universidad del Rosario in Bogota and started a successful career in the Pharmaceutical Industry. He got married and moved to Tampa Right before entering the Ph.D. program at USF, Ra fael worked at the Alzheimer's disease Research Laboratory under the tutelage of Dr. Dave Morgan and Dr. Marcia Gordon. He got his second M aster 's degree in Medical Sciences from the University of South Florida in 2008. His Ph.D. research focused on neurona l injury during anesthesia and n europrotection with anesthetic agents ; receiving three awards f rom national and international Anesthesia S ocieties He successfully defended his doctoral dissertation in April 2010 at the University of South Florida.


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Neuroprotection with anesthetics in two models of cerebral ischemia
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ABSTRACT: Neuroprotection with anesthetics has been studied for many decades; important advances in this field have modified the way Anesthesiologists treat patients in the operating room. Animal models have played an important role in the study of ischemia in the operating room. Recent studies have demonstrated that the effect of anesthetics seems to be different in different animal models. We decided to evaluate anesthetics in a well known model of cerebral ischemia and also in hypotensive models designed by us. We used a model of cerebral ischemia (MCAO) to test anesthetics neuroprotective effect in a two-week period. Then, we used a model of hypotension to characterize the damage caused by this type of insult. Finally we characterized a model of hypotension plus hypoxia that can mimic real situations in the OR. We found that anesthetics alone do not have a neuroprotective effect after two weeks in the MCAO model; but the combination of anesthetics with caspase vii inhibitors can decrease the damage caused by ischemia. The caspase inhibitor by itself did not show a significant neuroprotective effect. We also found that repetitive periods of profound hypotension can cause important damage in the hippocampus but no memory or neurological changes were seen. The induction of only one episode of hypotension plus hypoxia did not alter the morphology of the hippocampus although induced memory changes that were reverted by the use of anesthetics.
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