JOURNAL HOME CME HOME THIS MONTH PAST ISSUES ETOC COLLECTIONS
AUTHORS REVIEWERS EDITORIAL BOARD FEEDBACK RSS HELP
A&A International Anesthesia Research Society
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a colleague
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Inoue, S.
Right arrow Articles by Patel, P. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Inoue, S.
Right arrow Articles by Patel, P. M.
Related Collections
Right arrow Mechanisms
Right arrow Neuroanesthesia
Right arrow Pharmacology

Anesth Analg 2006;102:1548-1555
© 2006 International Anesthesia Research Society
doi: 10.1213/01.ane.0000202381.40516.8d

NEUROSURGICAL ANESTHESIA

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, MD, John C. Drummond, MD, Daniel J. Cole, MD, and Piyush M. Patel, 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

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 davismd{at}cox.net.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
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 awake-vehicle, isoflurane-vehicle, awake-IETD, or isoflurane-IETD 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 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 vehicle-treated 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.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Animal 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 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 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 broad-spectrum 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
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
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, isoflurane-IETD, or isoflurane-vehicle.

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-IETD-fmk (0.5 µg/5 µL 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 µg 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-mm-diameter 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 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 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 µm 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 (x400), 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 ({propto} = 0.05, ß = 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
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
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).


View this table:
[in this window]
[in a new window]
  		Table 1. Physiologic Variables

 

Figure 142
View larger version (17K):
[in this window]
[in a new window]
  		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.

 

Of the original 100 animals, 15 were excluded for misplacement of a guide cannula, development of subarachnoid hemorrhage, or technical experimental 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 awake-vehicle 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 isoflurane-vehicle groups (P < 0.01 for both). The difference in cortical infarction volumes between the awake-IETD and awake-vehicle groups did not achieve statistical significance (P = 0.09). No statistically significant differences in subcortical infarct volume were observed.


Figure 242
View larger version (24K):
[in this window]
[in a new window]
  		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 342
View larger version (28K):
[in this window]
[in a new window]
  		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 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).


Figure 442
View larger version (27K):
[in this window]
[in a new window]
  		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).

 

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.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
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 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 {alpha}-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 {gamma}-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 {gamma}-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 {alpha}-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 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 z-IETD-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.

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 inflammation-induced 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 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.


   Footnotes
 
Accepted for publication December 6, 2005.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Warner DS, McFarlane C, Todd MM, et al. Sevoflurane and halothane reduce focal ischemic brain damage in the rat: possible influence on thermoregulation. Anesthesiology 1993;79:985–92.[Web of Science][Medline]
  2. Warner DS, Ludwig PS, Pearlstein R, Brinkhous AD. Halothane reduces focal ischemic injury in the rat when brain temperature is controlled. Anesthesiology 1995;82:1237–45.[Web of Science][Medline]
  3. Baughman VL, Hoffman WE, Miletich DJ, et al. Neurologic outcome in rats following incomplete cerebral ischemia during halothane, isoflurane or N2O. Anesthesiology 1988; 69:192–8.[Web of Science][Medline]
  4. Baughman VL, Hoffman WE, Thomas C, et al. Comparison of methohexital and isoflurane on neurologic outcome and histopathology following incomplete ischemia in rats. Anesthesiology 1990;72:85–94.[Web of Science][Medline]
  5. Miura Y, Grocott HP, Bart RD, et al. Differential effects of anesthetic agents on outcome from near-complete but not incomplete global ischemia in the rat. Anesthesiology 1998;89:289–91.[Web of Science][Medline]
  6. Soonthon-Brant V, Patel PM, Drummond JC, et al. Fentanyl does not increase brain injury after focal cerebral ischemia in rats. Anesth Analg 1999;88:49–55.[Abstract/Free Full Text]
  7. Saito R, Graf R, Hubel K, et al. Reduction of infarct volume by halothane: effect on cerebral blood flow on perifocal spreading depression-like depolarization. J Cereb Blood Flow Metab 1997;17:857–64[Web of Science][Medline]
  8. Du C, Hu R, Csernansky CA, et al. Very delayed infarction after mild focal cerebral ischemia: a role for apoptosis? J Cereb Blood Flow Metab 1996;16:195–201.[Web of Science][Medline]
  9. Li Y, Chopp M, Jiang N, et al. Temporal profile of in situ DNA fragmentation after transient middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab 1995; 15:389–97.[Web of Science][Medline]
  10. Kawaguchi M, Kimbro JR, Drummond JC, et al. Isoflurane delays but does not prevent cerebral infarction in rats subjected to focal ischemia. Anesthesiology 2000;92:1335–42.[Web of Science][Medline]
  11. Eilers H, Bickler PE. Hypothermia and isoflurane similarly inhibit glutamate release evoked by chemical anoxia in rat cortical brain slices. Anesthesiology 1996;85:600–7.[Web of Science][Medline]
  12. Patel PM, Drummond JC, Cole DJ, Goskowicz RL. Isoflurane reduces ischemia-induced glutamate release in rats subjected to forebrain ischemia. Anesthesiology 1995;82:996–1003.[Web of Science][Medline]
  13. Puil E, el-Beheiry E. Anaesthetic suppression of transmitter actions in neocortex. Br J Pharmacol 1990;101:61–6[Web of Science][Medline]
  14. Yang J, Zorumski CF. Effects of isoflurane on N-methyl-d-aspartate gated ion channels in cultured rat hippocampal neurons. Ann NY Acad Sci 1991;625:287–9.[Web of Science][Medline]
  15. Puil E, el-Beheiry H, Baimbridge KG. Anesthetic effects on glutamate-stimulated increase in interneuronal calcium. J Pharmacol Exp Ther 1990;255:955–61.[Abstract/Free Full Text]
  16. 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.[Web of Science][Medline]
  17. Harada H, Kelly PJ, Cole DJ. Isoflurane reduces N-methyl-d-aspartate toxicity in vivo in the rat cerebral cortex. Anesth Analg 1999;89:1442–7.[Abstract/Free Full Text]
  18. Kimbro JR, Kelly PJ, Drummond JC, et al. Isoflurane and pentobarbital reduce AMPA toxicity in vivo in the rat cerebral cortex. Anesthesiology 2000;92:806–12.[Web of Science][Medline]
  19. Patel PM, Drummond JC, Cole DJ, et al. Isoflurane and pentobarbital reduce the frequency of transient ischemia depolarizations during focal ischemia in rats. Anesth Analg 1998;86:773–80.[Abstract]
  20. Graham SH, Chen J. Programmed cell death in cerebral ischemia. J Cereb Blood Flow Metab 2001;21:99–109.[Web of Science][Medline]
  21. Hara H, Friedlander RM, Gagliardini V, et al. Inhibition of interleukin 1 beta converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage. Proc Natl Acad Sci U S A 1997;94:2007–12.[Abstract/Free Full Text]
  22. Endres M, Namura S, Shimizu-Sasamata M, et al. Attenuation of delayed neuronal death after mild focal ischemia in mice by inhibition of the caspase family. J Cereb Blood Flow Metab 1998; 18:238–47.[Web of Science][Medline]
  23. Li H, Colbourne F, Sun P, et al. Caspase inhibitors reduce neuronal injury after focal but not global cerebral ischemia in rats. Stroke 2000;31:176–82.[Abstract/Free Full Text]
  24. Himi T, Ishizaki Y, Murota S. A caspase inhibitor blocks ischaemia-induced delayed neuronal death in the gerbil. Eur J Neurosci 1998;10:777–81.[Web of Science][Medline]
  25. Ding C, He Q, Li PA. Activation of cell death pathway after a brief period of global ischemia in diabetic and non-diabetic animals. Exp Neurol 2004;188:421–9.[Web of Science][Medline]
  26. Le DA, Wu Y, Huang Z, et al. Caspase activation and neuroprotection in caspase-3-deficient mice after in vivo cerebral ischemia and in vitro oxygen glucose deprivation. Proc Natl Acad Sci U S A 2002;99:15188–93.[Abstract/Free Full Text]
  27. Yin XM, Luo Y, Cao G, et al. Bid-mediated mitochondrial pathway is critical to ischemic neuronal apoptosis and focal cerebral ischemia. J Biol Chem 2002;277:42074–81.[Abstract/Free Full Text]
  28. Cao G, Luo Y, Nagayama T, et al. Cloning and characterization of rat caspase-9: implications for a role in mediating caspase-3 activation and hippocampal cell death after transient cerebral ischemia. J Cereb Blood Flow Metab 2002;22:534–46.[Web of Science][Medline]
  29. Plesnila N, Zinkel S, Le DA, et al. BID mediates neuronal cell death after oxygen/glucose deprivation and focal cerebral ischemia. Proc Natl Acad Sci U S A 2001;98:15318–23.[Abstract/Free Full Text]
  30. Jin K, Graham SH, Mao X, et al. Fas (CD95) may mediate delayed cell death in hippocampal CA1 sector after global cerebral ischemia. J Cereb Blood Flow Metab 2001;21:1411–21.[Web of Science][Medline]
  31. Benchoua A, Guegan C, Couriaud C, et al. Specific caspase pathways are activated in the two stages of cerebral infarction. J Neurosci 2001;21:7127–34[Abstract/Free Full Text]
  32. Felderhoff-Mueser U, Taylor DL, Greenwood K, et al. Fas/CD95/APO-1 can function as a death receptor for neuronal cells in vitro and in vivo and is upregulated following cerebral hypoxic-ischemic injury to the developing rat brain. Brain Pathol 2000;10:17–29[Web of Science][Medline]
  33. Inoue S, Drummond JC, Davis DP, et al. Combination of isoflurane and caspase inhibition reduces cerebral injury in rats subjected to focal cerebral ischemia. Anesthesiology 2004;101:75–81[Web of Science][Medline]
  34. Garcia-Calvo M, Peterson EP, Leiting B, et al. Inhibition of human caspases by peptide-based and macromolecular inhibitors. J Biol Chem 1998;273:32608–32613.[Abstract/Free Full Text]
  35. Cao G, Luo Y, Nagayama T, et al. Cloning and characterization of rat caspase-9: implications for a role in mediating caspase-3 activation and hippocampal cell death after transient cerebral ischemia. J Cereb Blood Flow Metab 2002;22:534–46.[Web of Science][Medline]
  36. Henshall DC, Bonislawski DP, Skradski SL, et al. Cleavage of bid may amplify caspase-8-induced neuronal death following focally evoked limbic seizures. Neurobiol Dis 2001;8:568–80.[Web of Science][Medline]
  37. Zea-Longa E, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 1989;20:84–91.[Abstract/Free Full Text]
  38. Sarraf-Yazdi S, Sheng H, Miura Y, et al. Relative neuroprotective effects of dizocilpine and isoflurane during focal cerebral ischemia in the rat. Anesth Analg 1998;87:72–8.[Abstract/Free Full Text]
  39. Rogers DC, Campbell CA, Stretton JL, Mackay KB. Correlation between motor impairment and infarct volume after permanent and transient middle cerebral artery occlusion in the rat. Stroke 1997;28:2060–5.[Abstract/Free Full Text]
  40. Swanson R, Morton M, Tsao-Wu G, et al. A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab 1990;10:290–3.[Web of Science][Medline]
  41. Lei B, Popp S, Capuano-Waters C, et al. Effects of delayed administration of low-dose lidocaine on transient focal cerebral ischemia in rats. Anesthesiology 2002;97:1534–40.[Web of Science][Medline]
  42. Palkovits M, Brownstein MJ. Maps and guide to microdissection of the rat brain. New York: Elsevier, 1988.
  43. Nakanishi S, Nakajima Y, Masu M, et al. Glutamate receptors: brain function and signal transduction. Brain Res Brain Res Rev 1998;26:230–5.[Medline]
  44. Barker JL, Ransom BR. Pentobarbitone pharmacology of mammalian central neurones grown in tissue culture. J Physiol 1978;280:355–72.[Abstract/Free Full Text]
  45. Jones MV, Brooks PA, Harrison NL. Enhancement of gamma-aminobutyric acid-activated Cl- currents in cultured rat hippocampal neurones by three volatile anaesthetics. J Physiol 1992;449:279–93.[Abstract/Free Full Text]
  46. Valtysson J, Hillered L, Andine P, et al. Neuropathologic end points in experimental stroke pharmacotherapy: the importance of both early and late evaluation. Acta Neurochir (Wien) 1994;129:58–63.[Medline]
  47. Li Y, Chopp M, Jiang N, et al. Induction of DNA fragmentation after 10 to 120 minutes of focal cerebral ischemia in rats. Stroke 1995;26:1252–8[Abstract/Free Full Text]
  48. Kawaguchi M, Kimbro J, Drummond J, et al. Effect of isoflurane on neuronal apoptosis in rats subjected to focal ischemia. J Neurosurg Anesthesiol 2000;12:385.
  49. 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.[Medline]
  50. Dirnagl U, Iadecola C, Moskowitz MA. Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci 1999;22:391–7.[Web of Science][Medline]
  51. Loddick SA, Rothwell NJ. Neuroprotective effects of human recombinant interleukin-1 receptor antagonist in focal cerebral ischaemia in the rat. J Cereb Blood Flow Metab 1996;16:932–40.[Web of Science][Medline]
  52. Iadecola C, Salkowski CA, Zhang F, et al. The transcription factor interferon regulatory factor 1 is expressed after cerebral ischemia and contributes to ischemic brain injury. J Exp Med 1999;189:719–27.[Abstract/Free Full Text]
  53. Hara H, Fink K, Endres M, et al. Attenuation of transient focal cerebral ischemic injury in transgenic mice expressing a mutant ICE inhibitory protein. J Cereb Blood Flow Metab 1997;17:370–5.[Web of Science][Medline]
  54. Boldin MP, Goncharov TM, Goltsev YV, Wallach D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1-and TNF receptor-induced cell death. Cell 1996;14:803–15.
  55. Medema JP, Scaffidi C, Kischkel FC, et al. FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). EMBO J 1997;16:2794–804.[Web of Science][Medline]
  56. Velier JJ, Ellison JA, Kikly KK, et al. Caspase-8 and caspase-3 are expressed by different populations of cortical neurons undergoing delayed cell death after focal stroke in the rat. J Neurosci 1999;19:5932–41.[Abstract/Free Full Text]
  57. Benchoua A, Guegan C, Couriaud C, et al. Specific caspase pathways are activated in the two stages of cerebral infarction. J Neurosci 2001;21:7127–34.[Abstract/Free Full Text]
  58. Morita-Fujimura Y, Fujimura M, Yoshimoto T, Chan PH. Superoxide during reperfusion contributes to caspase-8 expression and apoptosis after transient focal stroke. Stroke 2001;32:2356–61.[Abstract/Free Full Text]
  59. Hossmann KA. Periinfarct depolarizations. Cerebrovasc Brain Metab Rev 1996;8:195–208.[Web of Science][Medline]
  60. Back T, Ginsberg MD, Dietrich D, Watson BD. Induction of spreading depression in the ischemic hemisphere following experimental middle cerebral artery occlusion: effect on infarct morphology. J Cereb Blood Flow Metab 1996;16:202–13.[Web of Science][Medline]
  61. Kawaguchi M, Drummond JC, Cole DJ, et al. Effect of isoflurane on neuronal apoptosis in rats subjected to focal cerebral ischemia. Anesth Analg 2004;98:798–805.[Abstract/Free Full Text]
  62. Sullivan BL, Leu D, Taylor DM, et al. Isoflurane prevents delayed cell death in an organotypic slice culture model of cerebral ischemia. Anesthesiology 2002;96:189–95.[Web of Science][Medline]
  63. Wise-Faberowski L, Aono M, Pearlstein RD, Warner DS. Apoptosis is not enhanced in primary mixed neuronal/glial cultures protected by isoflurane against N-methyl-d-aspartate excitotoxicity. Anesth Analg 2004;99:1708–14.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a colleague
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Inoue, S.
Right arrow Articles by Patel, P. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Inoue, S.
Right arrow Articles by Patel, P. M.
Related Collections
Right arrow Mechanisms
Right arrow Neuroanesthesia
Right arrow Pharmacology

Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press