The Combination of Isoflurane and Caspase 8 ... - Semantic Scholar

The Combination of Isoflurane and Caspase 8 Inhibition Results in Sustained Neuroprotection in Rats Subject to Focal Cerebral Ischemia Satoki Inoue, MD, Daniel P. Davis, and Piyush M. Patel, MD

MD,

John C. Drummond,

MD,

Daniel J. Cole,

MD,

Department of Anesthesiology, VA Medical Center and UC San Diego, Department of Emergency Medicine, UC San Diego, San Diego, California; Department of Anesthesiology, Loma Linda University, Loma Linda, California

Although isoflurane can reduce ischemic neuronal injury after short postischemic recovery intervals, data from our laboratory have demonstrated that this neuroprotection is not sustained and that delayed apoptotic neuronal death, mediated in part by activation of caspases, contributes to the gradual increase in the size of the infarction. We tested the hypothesis that the neuroprotective efficacy of isoflurane can be prolonged with the administration of z-IETD-fmk, a specific inhibitor of caspase 8. Fasted Wister rats were anesthetized with isoflurane and randomly allocated to awakevehicle, isoflurane-vehicle, awake-IETD, or isofluraneIETD groups (n ⫽ 25 per group). Animals were subjected to 60 min focal ischemia by filament occlusion of the middle cerebral artery (MCAO). Daily intracerebroventricular injections of z-IETD-fmk or vehicle were administered via an implanted cannula starting before ischemia and continuing until 14 days post-MCAO. Neurological assessment was performed 14 days after ischemia after which the volume of cerebral infarction and number of intact neurons in the peri-infarct cortex

A

nimal models of ischemic brain injury have demonstrated that volatile anesthetics can reduce neuronal injury in the setting of focal and global cerebral ischemia (1–7). In most of these studies, however, the recovery period was relatively short (1 to 7 days). Some data suggest that postischemic neuronal death is a dynamic process in which loss of neurons continues over weeks to months after the ischemic insult (8,9). Although isoflurane has demonstrated neuroprotection within the first 48 h, data from our laboratory have demonstrated that this protective effect is not sustained at 14 days (10). These data Accepted for publication December 6, 2005. Address correspondence and reprint requests to Daniel P. Davis, MD, UCSD Emergency Medicine, 200 W. Arbor Drive #8676, San Diego, CA 92103-8676. Address e-mail to [email protected]. DOI: 10.1213/01.ane.0000202381.40516.8d

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were determined. Total infarction volume was less in the isoflurane-IETD group than in awake-vehicle, isoflurane-vehicle, and awake-IETD groups. Infarction volume was also less in the awake-IETD group versus the awake-vehicle group. The number of intact neurons within the peri-infarct cortex was significantly less in the awake-vehicle group in comparison with the other three experimental groups. The isoflurane-IETD group had better neurologic outcomes than both vehicletreated groups at 14 days post-MCAO. These results suggest that a combination of isoflurane and a caspase 8 inhibitor can produce neuroprotection that is evident even after a recovery period of 14 days. This combination demonstrated greater efficacy than the administration of either isoflurane or z-IETD-fmk alone. These results are consistent with the premise that continuing apoptosis contributes to the enlargement of cerebral infarction during the recovery period and that its inhibition can provide sustained neuroprotection. (Anesth Analg 2006;102:1548 –55)

suggest that isoflurane delays, but does not prevent, the development of cerebral infarction. A number of mechanisms contribute to postischemic neuronal injury. During the ischemic and early reperfusion periods, rapid neuronal death occurs via glutamate-mediated excitotoxicity and ischemic neuronal depolarization, both of which are suppressed by isoflurane (11–19). In the later stages of postischemic recovery, the development of inflammation and emergence of neuronal apoptosis lead to delayed neuronal death and gradual expansion of the cerebral infarct. An important mechanism by which neuronal apoptosis occurs is via activation of caspases. Caspase activation results in the proteolytic cleavage of a number of vital cellular components, ultimately leading to neuronal apoptosis. Caspase activity appears to be an important mediator of neuronal cell death after a brain ©2006 by the International Anesthesia Research Society 0003-2999/06

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NEUROSURGICAL ANESTHESIA INOUE ET AL. ISOFLURANE AND CASPASE 8 INHIBITION IN FOCAL CEREBRAL ISCHEMIA

insult. The administration of caspase inhibitors has been reported to reduce the volume of infarction after focal cerebral ischemia (20 –24). Two distinct pathways for caspase activation have been identified. The intrinsic pathway appears to be initiated via the release of mitochondrial cytochrome c in response to ischemia, ultimately leading to caspase 3 and 7 activation through activation of caspase 9; an extrinsic caspase pathway has also been identified, with stimulation of the tumor necrosis factor (TNF) family of receptors leading to caspase 3 and 7 activation via activation of caspase 8 (25–32). The effects of isoflurane on postischemic neuronal apoptosis are unclear. However, available data suggest that isoflurane does not prevent neuronal apoptosis in vivo, potentially explaining the failure of isoflurane to prevent infarct expansion after focal ischemia. Previous work from our laboratory has documented that the combination of isoflurane and the broadspectrum caspase inhibitor zVAD-fmk provides sustained neuroprotection versus either drug alone (33). In the present study, we administered z-IETD-fmk, a drug that is 100-200 times more specific for caspase 8 than for other caspases, to rats undergoing focal cerebral ischemia (34). The following specific goals were addressed: 1) to confirm the additive effect of combining isoflurane with caspase 8 inhibition in producing neuroprotection after ischemia, and 2) to further define a role for the extrinsic apoptotic pathway in mediating ischemic neuronal death.

Methods The study was approved by the local institutional animal care and use committee. All experimental procedures were performed in accordance with the guidelines established in the Public Health Service Guide for the Care and Use of Laboratory Animals. Male Wistar rats (Simonson Laboratories, San Diego, CA) weighing 270 –330 g were fasted overnight. Access to water was provided. Rats were anesthetized with an inspired concentration of 5% isoflurane (Ohmeda, Liberty Corner, NJ). After endotracheal intubation, the lungs were mechanically ventilated with a mixture of 30% oxygen and 70% nitrogen, and the end-tidal concentration of isoflurane was reduced to 2.5%. A needle thermistor (Mon-a-Therm; Mallinckrodt, St. Louis, MO) was inserted between the temporalis muscle and the skull. Pericranial temperature was servocontrolled to 37.0°C ⫾ 0.2°C by surface heating or cooling. A cannula was inserted in the tail artery using PE-50 tubing, and the mean arterial blood pressure (MAP) was monitored continuously. Animals were randomly assigned to 1 of 4 groups (n ⫽ 25 per group): awake-IETD, awake-vehicle, isofluraneIETD, or isoflurane-vehicle.

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The anesthetized animal’s head was secured in a steretotactic frame (Kopf Instruments, Tujunga, CA). A midline scalp incision was made and a 1.5-mm burr hole was drilled 0.8 mm posterior and 1.5 mm right lateral from bregma. A 23-gauge guide cannula was inserted by micromanipulator to a depth 4.0 mm from the surface of the cranium and dental cement was used to secure it to the cranium. A 30-gauge stylet was placed in the cannula to maintain patency. The animal was then removed from the frame and transferred to the surgical table for right middle cerebral artery occlusion (MCAO). Intracerebroventricular administration of z-IETDfmk (0.5 ␮g/5 ␮L over 5 min) dissolved in vehicle or vehicle alone was performed at the following time points: 30 min before MCAO, 2 h after MCAO, and every 24 h for 14 days. The z-IETD-fmk dose was based on previous reports using an intraventricular infusion of 0.1–1.5 ␮g before and after ischemia (35,36). Drug vehicle was 0.4% dimethyl sulfoxide in artificial cerebrospinal fluid. The animals were prepared surgically for MCAO according to the technique of Zea-Longa et al. (37). In brief, the right common carotid artery was exposed via a midline pretracheal incision, and the vagus and sympathetic nerves were carefully separated from the artery. The external carotid artery was ligated 2 mm distal to the bifurcation of the common carotid artery, and dissection along the internal carotid artery was performed to expose the origin of the pterygopalatine artery. The common carotid artery was then ligated 5–10 mm proximal to its bifurcation. Baseline values for arterial oxygen (Pao2), carbon dioxide (Paco2), pH, plasma glucose concentration, hematocrit, MAP, and heart rate were measured and recorded. A 0.25-mmdiameter nylon monofilament previously coated with silicone was inserted into the proximal common carotid artery and advanced into the internal carotid artery to a distance of 18 –20 mm from the carotid artery bifurcation until slight resistance was felt. The monofilament was secured in place and the wound was sutured. After induction of focal ischemia, isoflurane was discontinued for awake-group animals. In the isoflurane groups, the end-tidal concentration of isoflurane was reduced to 1.8% (approximately 1.5 times the minimum alveolar concentration) after insertion of the monofilament (38). At the end of a 60-min ischemic interval, the monofilament was removed, the tail artery catheter was removed and the wound was closed with sutures. In the awake groups, animals were anesthetized 6 min before the end of the 60-min ischemic interval. The monofilament was removed 60 min postischemia. Isoflurane administration was discontinued and on resumption of spontaneous ventilation, mechanical ventilation was discontinued and the endotracheal tube

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was removed. Animals were then transferred to a heated and humidified incubator through which oxygen was flushed continuously. All wounds were infiltrated with 0.25% bupivacaine (total dose 0.5 mg). Isoflurane was then discontinued and animals were allowed to awaken. During the recovery period, the pericranial temperature was recorded at 1-h intervals for 3 h, after which time the temperature probe was removed. Rectal temperatures were monitored every 24 h for 14 days. Sham operations were performed in additional animals using the same procedure as for the awake and isoflurane groups except that MCAO was not performed; these brains were used as nonischemic controls to evaluate the effects of z-IETD-fmk administration on brain tissue (n ⫽ 3– 4 per each group). All animals subjected to MCAO underwent neurologic evaluation 2 h after the procedure. Animals that did not manifest clinical evidence of neurologic injury were then excluded from the study. Neurologic evaluations were also performed 14 days after ischemia. Each rat was assigned a score according to an 8-point behavioral rating scale: 0 ⫽ no neurologic deficit; 1 ⫽ failure to extend left forepaw fully; 2 ⫽ decreased grip of the left forelimb; 3 ⫽ spontaneous movement in all direction, contralateral circling only if pulled by the tail; 4 ⫽ circling or walking to the left (or right); 5 ⫽ walking only if stimulated; 6 ⫽ unresponsiveness to stimulation, with a depressed level of consciousness; and 7 ⫽ dead (39). Neurologic testing was performed by a single observer who was blinded as to group assignment. Animals were allowed to survive for 14 days after ischemia. The animal’s body weight was measured before the experiment and 14 days after ischemia. The animals were anesthetized with chloral hydrate after the final neurological examination at 14 days after ischemia. Animals were killed via transcardiac perfusion of 200 mL of heparinized saline followed by 200 mL 4% phosphate-buffered formaldehyde. Animals were then decapitated, and their brains were carefully removed, immersed in fixative, and refrigerated at approximately 4°C for 24 – 48 h. Brains then were prepared for histologic analysis. After dehydration in graded concentrations of ethanol and butanol, brains were embedded in paraffin. Six-micron-thick coronal sections were obtained at 0.75-mm intervals and stained with hematoxylin and eosin. During tissue processing, the implanted guide cannula placement was evaluated; animals in which the guide cannula was not determined to be in the lateral ventricle were excluded from the study. Animals with subarachnoid hemorrhage were also excluded. Infarction was assessed using light microscopy, with the area of infarction traced for each section. Infarction area was defined as pan-necrosis defined by

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the loss of affinity for hematoxylin. The area of infarction was determined by image analysis using National Institutes of Health Image 1.62 software and an Apple Power Macintosh G4 computer (Apple Computer, Cupertino, CA). The total volume of injury was determined by integration of the area of injury in each section (9 to 12 sections of the brain, spanning the entire region of ischemic injury, were analyzed) according to the technique of Swanson et al. (40). Assessment of infarct volume was performed by a single observer blinded as to experimental group assignment. For quantification of neuronal damage, a modification of the method of Lei et al. was used (41). Coronal sections at the level of the anterior commisure and 750 ␮m rostral and caudal to that level were used for counting neurons. Both regions at each level were delineated on each slice by reference to a rat brain atlas (42). Under high-power microscopic magnification (⫻400), the number of intact neurons per 0.25 mm2 of tissue were counted and averaged in the 3 peri-infarct cortical fields. The counting was performed by a single observer blinded as to experimental group assignment. The group sample size was determined based on our previous study data (10). A power analysis was performed, with a sample size of 17 animals per group determined to be sufficient (⬀ ⫽ 0.05, ␤ ⫽ 0.80). Given the expected animal mortality and potential failure of MCAO in a few animals, each group size was increased to 25 animals. For statistical analysis of physiologic values, infarction volumes, and neuronal cell counts, analysis of variance and repeated measures analysis of variance was used (StatView 4.5; Abacus Concepts, Berkeley, CA). If significant differences were detected, an unpaired Student’s t-test with Bonferroni correction was used for intergroup comparisons. Neurologic scores were analyzed by Friedman analysis of variance followed by Mann-Whitney U-test with Bonferroni correction. Statistical significance was assumed for P ⬍ 0.05. All data except for neurologic score are presented as mean ⫾ sd. Neurologic scores are reported as 10th, 25th, median, 75th, and 90th percentile ranges.

Results Physiologic variables are presented in Table 1. There were no statistically significant differences with regard to preischemic weight, MAP, heart rate, pH, Paco2, Pao2, glucose concentration, and hematocrit among the four experimental groups. There were also no statistically significant differences in pericranial and rectal temperatures (Fig. 1). Of the original 100 animals, 15 were excluded for misplacement of a guide cannula, development of subarachnoid hemorrhage, or technical experimental

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Table 1. Physiologic Variables

Pre-weight (g) MAP (mm Hg) Before MCAO At reperfusion HR (bpm) Before MCAO At reperfusion pH Paco2 (mm Hg) Pao2 (mm Hg) Hematocrit (%) Glucose (mg/dL)

Awake-IETD (n ⫽ 23)

Awake-vehicle (n ⫽ 21)

Isoflurane-IETD (n ⫽ 22)

Isoflurane-vehicle (n ⫽ 19)

296 ⫾ 18

303 ⫾ 25

295 ⫾ 25

299 ⫾ 23

93 ⫾ 12 89 ⫾ 10

92 ⫾ 11 88 ⫾ 7

90 ⫾ 10 85 ⫾ 5

91 ⫾ 8 87 ⫾ 7

382 ⫾ 28 395 ⫾ 14 7.43 ⫾ 0.02 37 ⫾ 2 132 ⫾ 11 44 ⫾ 3 121 ⫾ 15

367 ⫾ 32 394 ⫾ 14 7.43 ⫾ 0.02 38 ⫾ 2 127 ⫾ 11 44 ⫾ 2 118 ⫾ 16

385 ⫾ 27 396 ⫾ 14 7.44 ⫾ 0.02 38 ⫾ 3 130 ⫾ 10 44 ⫾ 2 113 ⫾ 13

390 ⫾ 34 390 ⫾ 29 7.43 ⫾ 0.02 38 ⫾ 3 129 ⫾ 14 45 ⫾ 2 117 ⫾ 12

Values are mean ⫾ sd. MAP ⫽ mean arterial blood pressure; HR ⫽ heart rate; MCAO ⫽ middle cerebral artery occlusion; Paco2 ⫽ arterial carbon dioxide partial pressure; Pao2 ⫽ arterial oxygen partial pressure.

Figure 1. Changes in pericranial and rectal temperature in the four experimental groups. Data are expressed as mean ⫾ sd. I-30, R-30, R-60, R-120, R-180 ⫽ pericranial temperatures 30 min after middle cerebral artery occlusion and at 30, 60, 120, and 180 min after reperfusion; R-1D through R-14D ⫽ rectal temperatures 1–14 days after reperfusion.

problems; this included 2 in the awake-IETD group, 4 in the awake-vehicle group, 3 in the isoflurane-IETD group, and 6 in the isoflurane-vehicle group. Of the remaining 85 animals, 12 animals died before histologic analysis; this included 3 in the awake-IETD group, 4 in the awakevehicle group, 2 in the isoflurane-IETD group, and 3 in the isoflurane-vehicle group. All of these animals were considered to have undergone neurologic deaths. Weight loss in the isoflurane-IETD group was less than in the awake-vehicle and isoflurane-vehicle groups (P ⬍ 0.01 and P ⬍ 0.05, respectively). The results of neurologic assessments are displayed in Figure 2. At 14 days after ischemia, neurologic outcomes were better in the isoflurane-IETD group than in both the awake-vehicle and the isoflurane-vehicle groups (P ⬍ 0.05 for both). Infarction volumes are presented in Figure 3. Total infarction volume (cortical and subcortical) was less in the isoflurane-IETD group (27 ⫾ 21 mm3, mean ⫾ sd)

than in awake-vehicle, isoflurane-vehicle, and awake-IETD groups (81 ⫾ 32 mm3, 71 ⫾ 35 mm3, 53 ⫾ 31 mm3; P ⬍ 0.01, P ⬍ 0.01, and P ⬍ 0.05, respectively). Total infarct volumes were also smaller in the awake-IETD group as compared with the awake-vehicle group (P ⬍ 0.05). Cortical infarction volumes were less in the isoflurane-IETD group as compared with the awake-vehicle and isofluranevehicle groups (P ⬍ 0.01 for both). The difference in cortical infarction volumes between the awakeIETD and awake-vehicle groups did not achieve statistical significance (P ⫽ 0.09). No statistically significant differences in subcortical infarct volume were observed. The number of histologically preserved neurons within the peri-infarct cortex in the four groups is presented in Figure 4. The awake-vehicle group had significantly fewer intact neurons in comparison with the other three experimental groups (P ⬍ 0.01).

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Figure 2. The isoflurane-IETD group demonstrated better neurologic outcomes at 14 days after middle cerebral artery occlusion versus both the awake-vehicle and isoflurane-vehicle groups (*P ⬍ 0.05 for both). Neurologic scores are reported as 25th, median, and 75th percentiles. Eight-point behavioral rating scale (29): 0 ⫽ no neurologic deficit; 1 ⫽ failure to extend left forepaw fully; 2 ⫽ decreased grip of the left forelimb; 3 ⫽ spontaneous movement in all directions, with contralateral circling only if pulled by the tail; 4 ⫽ circling or walking to the left; 5 ⫽ walking only if stimulated; 6 ⫽ unresponsiveness to stimulation, with a depressed level of consciousness; and 7 ⫽ dead. Three in the awake-IETD group, 4 in the awake-vehicle group, 2 in the isoflurane-IETD group, and 3 in the isoflurane-vehicle group underwent neurologic death.

Figure 3. Total, cortical, and subcortical infarction volumes at 14 days after middle cerebral artery occlusion. Data are presented as mean ⫾ sd. Total infarction volume was less in the isoflurane-IETD group versus awake-vehicle, isoflurane-vehicle, and awake-IETD groups (**P ⬍ 0.01, P ⬍ 0.01, and P ⬍ 0.05, respectively). Total infarct volume was smaller in the awake-IETD group as compared to the awake-vehicle group (*P ⬍ 0.05). Cortical infarction volume was with in the isoflurane-IETD group as compared with the awake-vehicle and isoflurane-vehicle groups (#P ⬍ 0.01 for both). Data from 12 animals who died before histologic analysis are not presented here. This included 3 in the awake-IETD group, 4 in the awake-vehicle group, 2 in the isoflurane-IETD group, and 3 in the isoflurane-vehicle group.

The administration of either vehicle or z-IETD-fmk in sham-operated rats not undergoing MCAO did not result in any histologic or clinical neurologic injury.

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Figure 4. Number of intact neurons in the peri-infarct cortex 14 days after middle cerebral artery occlusion in the four experimental groups. Data are expressed as mean ⫾ sd. The awake-vehicle group had significantly fewer intact neurons in comparison to the other three experimental groups (*P ⬍ 0.01).

Discussion The results of the present study indicate that a combination of isoflurane and caspase-8 inhibition can reduce cerebral injury produced by focal ischemia. The neuroprotective efficacy of this combination is apparent even after a 14-day recovery period. Although the administration of z-IETD-fmk alone appeared to be neuroprotective, the reduction in infarct volume was less than the combination of isoflurane and z-IETD-fmk. The histologic reduction in cerebral injury was consistent with an improvement in neurologic outcome in animals that received isoflurane and z-IETD-fmk. Isoflurane, when administered alone, did not reduce cerebral infarction 2 weeks postischemia, although the number of intact neurons was increased over the awake-vehicle group; this confirms the results from our previous investigation (10). These results are consistent with the premise that continuing apoptosis contributes to postischemic neuronal injury in rodents subjected to focal ischemia and that the reduction of apoptosis can provide sustained neuroprotection. The data reported here also support the findings of our previous study, which documented synergistic neuroprotection between isoflurane and the nonspecific caspase inhibitor z-VAD-fmk (33). A similar magnitude of neuroprotection was observed with z-VAD-fmk and z-IETD-fmk. Our results support a role for the extrinsic caspase pathway (which includes caspase 8) in mediating neuronal injury after focal ischemia. Isoflurane has been shown to reduce neuronal injury in animal models of hemispheric severe cerebral ischemia, near-complete global ischemia, and focal ischemia after a relatively short recovery period (3– 6). Although the precise mechanisms by which isoflurane reduces neuronal injury after a short recovery period remain unclear, the available data suggest that inhibition of excitotoxicity likely plays an important role. Isoflurane has been shown to reduce glutamate release

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from anoxic brain slices in vitro (11) and from the cortex in rats subjected to incomplete forebrain ischemia (12). In addition, isoflurane can also inhibit postsynaptic glutamate receptor-mediated responses in neocortical and hippocampal cells (13–15) and rat brain slices (16). Data from our laboratory have also demonstrated that isoflurane can reduce in vivo cortical injury mediated by both N-methyl-d-aspartate and ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (17,18). Collectively, these data indicate that isoflurane-mediated reduction of cortical infarction after a short recovery period may be a result of the attenuation of excitotoxicity. In addition, isoflurane may reduce neuronal injury through enhancement of inhibitory responses mediated by ␥-aminobutyric acid. Stimulation of glutamate receptors results in Na⫹ influx and rapid depolarization of postsynaptic neurons (43). Hyperpolarization of neurons could conceivably retard this depolarization; in this regard, isoflurane may not only increase but also prolong the hyperpolarizing action of ␥-aminobutyric acid, probably through enhanced influx of Cl⫺ (44,45). However, the relative contribution of attenuation of excitotoxicity and enhancement of inhibitory responses to the reduction in neuronal injury after a relatively short recovery period by isoflurane is not known. Although the short-term neuroprotective efficacy of isoflurane has been clearly established, our results indicate that isoflurane neuroprotection does not persist. The findings reported here suggest that isoflurane delays but does not prevent the development of infarction, confirming our previous work that demonstrated a decay in the neuroprotective efficacy of isoflurane after a survival period of 14 days (10). This phenomenon has also been reported for other neuroprotective drugs, including both ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and N-methyl-d-aspartate acid antagonists (46). Du et al. (8) demonstrated that cerebral infarction after focal cerebral ischemia undergoes a gradual expansion; the authors suggested that this expansion is attributable in part to delayed neuronal death via apoptosis. Li et al. (47) showed that apoptosis can be observed as late as 4 weeks after ischemia. Moreover, apoptotic cells are localized primarily within the inner boundary zones of the evolving infarct. These data suggest that apoptosis may contribute to the expansion of the ischemic lesion. We demonstrated that isoflurane delays but does not prevent the development of apoptosis, with changes characteristic of apoptosis present up to 7 days after focal ischemia (48). Collectively, these data indicate that isoflurane does not inhibit apoptotic neuronal death. Consequently, isoflurane does not prevent infarct expansion, and its neuroprotective efficacy is not sustained. A number of mechanisms contribute to the development of postischemic neuronal apoptosis. Mitochondrial injury, the release of cytochrome c and the

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subsequent activation of caspases 9 and 3 have received the most attention (49). More recent data also indicate that an inflammatory response, triggered by ischemia and neuronal injury, might also play an important role in the genesis of apoptosis and contribute to infarct expansion (50 –53). TNF is a major inflammatory cytokine, and signaling via the TNF receptor leads to direct activation of caspase 8. Activated caspase 8 then cleaves downstream caspases, ultimately resulting in caspase 3 activation and apoptosis (54,55). In the setting of focal ischemia, caspase 8 activation has been implicated as a contributor to the development of apoptosis (56 –58). The administration of z-IETD-fmk appears to prevent apoptosis by inhibiting caspase 8-mediated activation of caspase 3, as it does not directly inhibit caspase 3 (57). In the present study, intracerebroventricular administration of zIETD-fmk reduced cerebral injury. These results are consistent with the premise that caspase 8 activation occurs during and after focal ischemia, resulting in neuronal apoptosis. Thus, inhibition of caspase 8 would be expected to reduce apoptosis and provide sustained neuroprotection. Of interest is our observation that there were more neurons in the peri-infarct cortex in the animals that received either isoflurane or z-IETD-fmk. The mechanism by which isoflurane would preserve neurons in the peri-infarct cortex after ischemia but fail to decrease infarct size is not clear. One possible mechanism is the inhibition of ischemic depolarizations that occurs during focal ischemia. These have been shown to augment neuronal calcium influx during ischemia and increase brain injury, probably mediated via apoptosis (57,59). Data from Back et al. (60) suggest that ischemic depolarizations during focal ischemia do not increase infarct volume but contribute significantly to the development of scattered neuronal injury within the cortex adjacent to the infarct. Previous work in our laboratory demonstrated that isoflurane can reduce both the frequency of ischemic depolarizations during focal ischemia as well as infarct volume (19). Together, these studies suggest that the increase in the number of intact neurons in the penumbra might be mediated in part by a reduction in the frequency of ischemic depolarizations during isoflurane anesthesia, although the total number of intact neurons did not differ among the isoflurane-vehicle, isoflurane-IETD, and awake-IETD groups. Our previous work documents that neurons initially protected by isoflurane undergo delayed cell death via apoptosis, with gradual extension of the infarct area (61). Although this supports the hypothesis that the same neurons are protected from both an immediate excitotoxic death by isoflurane and a delayed apoptotic death by caspase inhibition, it is conceivable isoflurane and z-IETD-fmk protect a different subset of neurons and with a different time course of neuroprotection.

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It should be noted that others have failed to demonstrate the neuroprotective potential of caspase 8 inhibition. Cao et al. (28) and Li et al. (23) reported that preadministration of a caspase 8 inhibitor had no significant effect on neuronal survival in the hippocampal CA1 sector in a model of transient global ischemia. Moreover, Morita-Fujimura et al. (58) demonstrated that caspase 8 induction was not a substantial contributor to neuronal death in a model of permanent focal ischemia. In contrast, Velier et al. (56) have demonstrated procaspase 8 cleavage to active caspase 8 in a rodent model of focal ischemia, with activated caspase 8 expression observed in a large number of pyramidal cells in the ipsilateral cortex after MCAO. Their speculation that caspase 8 inhibition might decrease postischemic neuronal injury was experimentally confirmed in our study. In aggregate, the available data indicate that caspase 8 inhibition is likely to be most beneficial in models of transient focal ischemia and that it may be ineffective in models of global ischemia or permanent focal ischemia. In contrast to our work, Sullivan et al. (62) and Wise-Faberowski et al. (63) have shown that with in vitro models, isoflurane-mediated protection is not transient and it does not lead to delayed apoptosis. A fundamental difference between these studies and the present one is that although postischemic inflammation is a consistent feature of in vivo ischemia, such inflammation is not present in in vitro models. Collectively, these studies suggest that postischemic inflammation is a major contributor to delayed postischemic neuronal death and that some of this inflammationinduced injury may be mediated by caspase-8 activation. Several limitations should be considered when interpreting these data. Although IETD-fmk has previously been demonstrated to produce highly specific caspase 8 inhibition in vivo at the doses used here, we did not assess the effect on caspase 8 activity in our model (34 –36). In addition, the optimal postischemic time period to observe the entire pattern of injury is not clear, and although 14 days is longer than most studies on ischemia, other investigators have documented apoptosis occurring even after 1 month (47). Although we have considerable experience with our model of focal ischemia, animals do receive isoflurane before ischemia, which produces some degree of ischemic preconditioning. In summary, a combination of isoflurane and a caspase 8 inhibitor decreased cerebral infarction in a rat model of focal ischemia after a recovery period of 14 days. This combination demonstrated greater efficacy that the administration of either isoflurane or z-IETD-fmk alone. These results demonstrate that inhibition of continuing apoptosis is necessary to observe long-term neuroprotection from isoflurane anesthesia. Our findings are also consistent with the hypothesis that combination therapy with drugs that

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target different, although not mutually exclusive, aspects of the pathophysiology of cerebral ischemia are more effective in reducing ischemic cerebral injury than the administration of either drug alone.

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