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*Department of Anesthesiology, VA Medical Center and University of California, San Diego, San Diego, California, the
Department of Anesthesiology, University of California, San Diego, San Diego, California, and the
Department of Anesthesiology, Mayo Clinic, Scottsdale, Arizona
Address correspondence to Piyush M. Patel, MD, Anesthesia Service 125, VA Medical Center, 3350 La Jolla Village Drive, San Diego, CA 92161. Address email to ppatel{at}ucsd.edu
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IMPLICATIONS: The effect of isoflurane on neuronal apoptosis was investigated in rats subjected to focal cerebral ischemia. In isoflurane-anesthetized animals, ischemia-induced apoptosis occurred during the later stages of postischemic recovery. Isoflurane did not inhibit postischemic neuronal apoptosis.
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It has been proposed that at least two pathophysiologic processes, excitotoxicity and apoptosis, contribute to the development of ischemic brain injury (11,12). The effect of anesthetics on excitotoxicity has been well studied. Anesthetics can suppress excitotoxicity both in vitro and in vivo (13–20). The demonstration of the early neuroprotective efficacy of anesthetics has been attributed to this suppression of excitotoxicity. By contrast, apoptosis, which is initiated soon after ischemia but continues for several days into the postischemic recovery period, results in a gradual increase in the size of cerebral infarction (21). The lack of sustained neuroprotection by isoflurane suggests that anesthetics might not inhibit apoptosis. The effect of isoflurane on ischemia-induced apoptosis, however, has not been evaluated.
The present study was therefore conducted to investigate the effects of isoflurane on apoptosis in rats subjected to focal ischemia. The time course of the development of cerebral infarction and neuronal apoptosis in isoflurane-anesthetized and awake rats was determined. Apoptotic neurons were identified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin in situ nick labeling (TUNEL) staining and by immunohistochemical detection of active caspase-9 and caspase-3.
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Wistar-Kyoto rats (Simonson Laboratories, San Diego, CA) weighing 275–325 g were fasted overnight. Access to water was provided. The rats were anesthetized with an inspired concentration of 5% isoflurane (Ohmeda, Liberty Corner, NJ). The animals’ tracheas were intubated, and their lungs were mechanically ventilated with a mixture of 30% oxygen/70% nitrogen. 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, and the pericranial temperature was servocontrolled to 37.0°C ± 0.2°C. The tail artery was cannulated with PE-50 tubing. The mean arterial blood pressure (MAP) was monitored continuously. The right external jugular vein was cannulated with PE-50 tubing.
The animals were surgically prepared for occlusion of the middle cerebral artery (MCAO) according to the technique of Zea-Longa et al. (22). The right common carotid artery was exposed via a midline pretracheal incision. 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. The internal carotid artery was dissected distally to expose the origin of the pterygopalatine artery. The common carotid artery was then permanently ligated. Baseline values for arterial partial pressure of carbon dioxide (PaCO2), arterial partial pressure of oxygen (PaO2), arterial pH, plasma glucose, hematocrit, MAP, and heart rate were measured. Via a small arteriotomy, a 0.25-mm diameter nylon monofilament previously coated with silicone was inserted into the proximal common carotid artery and was advanced into the internal carotid artery to distance of 18–20 mm from the carotid artery bifurcation until slight resistance was felt.
The animals were then allocated randomly to one of two experimental groups. In the awake group (n = 40), isoflurane administration was discontinued. On resumption of spontaneous ventilatory effort, mechanical ventilation was discontinued and the endotracheal tube was removed. Thereafter, the animals were transferred to a heated and humidified incubator through which oxygen was continuously flushed. The animals were briefly anesthetized with isoflurane 6 min before the end of the 70-min ischemic interval. The pretracheal incision was reopened and the monofilament was removed from the common carotid artery at the end of the 70-min ischemic interval. The tail artery and jugular vein catheters were removed and the wound was sutured. Then the animals were allowed to awaken. In the isoflurane group (n = 40), the concentration of isoflurane was reduced to 1.8% end-tidal (approximately 1.5 minimum alveolar anesthetic concentration) (23) after MCAO. At the end of 70-min ischemic interval, the monofilament was removed. The tail artery and jugular vein catheters were removed and the wound was sutured. All wounds were infiltrated with 0.25% bupivacaine (total dose 0.5 mg). Isoflurane was then discontinued. On resumption of spontaneous ventilatory effort, mechanical ventilation was discontinued, and the endotracheal tube was removed. Thereafter, the animals were transferred to the incubator as described above.
The animals were anesthetized with chloral hydrate 7 h, 1 day, 4 days, or 7 days after the reperfusion. They were killed by transcardiac perfusion with 200 mL of heparinized saline followed by 200 mL of phosphate buffered paraformaldehyde. The animals were decapitated. The brains were removed carefully, immersed in fixative, and refrigerated at a temperature of approximately 4°C for 24–48 h. The brains were then prepared for histologic analysis. After dehydration in graded concentrations of ethanol and butanol, the brains were embedded in paraffin. Several 6-µm thick coronal brain sections were prepared from each brain. Of these, 4 consecutive sections, approximately at a level 1.3 mm rostral to the bregma, were selected for further analysis. This level was chosen to ensure the presence of a substantial amount of peri-infarct cortical tissue.
The area of cerebral infarction in each animal was measured in a coronal section stained with hematoxylin and eosin (H and E). Infarction was defined as the area in which more than 90% of neurons were injured (necrosis or apoptosis). Neuronal injury ranged from necrosis (pyknosis, karyorrhexis, and karyolysis, as well as cytoplasmic eosinophilia or loss of affinity for hematoxylin) to apoptosis (cell shrinkage, nuclear condensation, and presence of apoptotic bodies). Infarct area measurement was restricted to the cortex because the objective of the study was to analyze neuronal apoptosis at the boundary of infarcted and normal brain and because of the limited area of subcortex that was present in the sections chosen for analysis. The area of infarction was measured by image analysis of the H and E stained section with National Institutes of Health Image 1.60 software and an Apple Macintosh computer (Apple Computer, Cupertino, CA).
For the detection of DNA fragmentation, TUNEL staining was performed with the Apoptag Plus Kit (Intergen Company, Purchase, NY) (24–26). Briefly, after deparaffinizing with xylene and graded concentrations of alcohol, brain sections were exposed to Proteinase K for 15 min at room temperature. Endogenous peroxidase activity was quenched with 2% hydrogen peroxide. Sections were then incubated with TdT enzyme in a humidified chamber at 37°C for 1 h. After incubation with anti-digoxigenin-peroxidase for 30 min at room temperature, peroxidase was detected with diaminobenzidine (DAB). The specimens were then washed with distilled water and were counterstained with methyl green. The TUNEL method is based on the specific binding of TdT to 3'-OH ends of DNA and the ensuing synthesis of a polydeoxynucleotide polymer. TUNEL-positive neurons that contained apoptotic bodies were identified as being neurons undergoing apoptosis. Most apoptotic neurons contained multiple apoptotic bodies. In the present study, only cells containing more than 2 apoptotic bodies were referred to as apoptotic cells (11). TUNEL positive neurons that did not have apoptotic bodies were referred to as necrotic neurons. TUNEL staining in these cells was diffuse and faint. The number of apoptotic cells per 0.25 mm2 of tissue at the margin of the area of infarction was counted under high-power microscopic magnification (x400). Three contiguous fields that spanned the dorsal-ventral boundaries of the cortex were evaluated and the number of apoptotic cells was averaged.
For immunohistochemical analysis, the DAKO LSAB-2 Peroxidase kit (DAKO Corporation, Carpinteria, CA) was used. After deparaffinization, brain sections were placed in Target Retrieval Solution (S1700, DAKO Corporation). Endogenous peroxidase activity was quenched with 2% hydrogen peroxide for 5 min at room temperature. Blockade of nonspecific protein binding was performed with 5% goat serum. Sections were incubated with primary antibody for 50 min at room temperature. For caspase-3 analysis, a purified rabbit anti-caspase-3 polyclonal antibody (DAKO Pharmingen Laboratories, San Diego, CA) was used at a dilution of 1:100. This antibody has a 50-fold greater affinity for the active caspase-3 compared with the procaspase-3. For caspase-9 analysis, a polyclonal rabbit anti-caspase-9 antibody (Pharmingen Laboratories) was used at a dilution of 1:500. This antibody recognizes procaspase-9 as well as the active caspase-9. Negative control sections were incubated without the primary antibody. Sections were rinsed with PBS and were incubated with biotinylated rabbit anti-mouse immunoglobulin G (E0464, DAKO Corporation, Carpinteria, CA) for 30 min at room temperature. The sections were then incubated with streptavidin-horseradish peroxidase for 45 min at room temperature. A DAB substrate was used for visualization of the catalyzed peroxidase-reaction product. The sections were then rinsed with distilled water and were counterstained with hematoxylin. The number of caspase-3 and caspase-9 positive neurons per 0.25 mm2 of tissue at the margin of the infarction were counted under high-power microscopic magnification (x400). Three fields were evaluated and the number of caspase positive neurons was averaged. In the present study, (active) caspase-3 and caspase-9 positive neurons were defined as ones in which positive immunostaining was observed in both the nucleus and the cytoplasm (27,28).
Statistical analysis was performed with StatView 4.5 (Abacus Concepts, Berkeley, CA). The physiologic values between the awake and isoflurane groups were evaluated by repeated-measures analysis of variance (ANOVA). Percentage area of cortical infarction and numbers of apoptotic cells and caspase-positive neurons were analyzed by factorial ANOVA. Bonferroni/Dunnett test was used for post hoc tests. A P value < 0.05 was considered to be statistically significant. All data are presented as mean ± SD.
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Discussion |
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Isoflurane reduces neuronal injury in animal models of hemispheric severe cerebral ischemia, near-complete global ischemia, and focal ischemia after a relatively short recovery period (1–7). Although exact mechanisms by which isoflurane reduces neuronal injury after a short recovery period are not clear, the available data suggest that inhibition of excitotoxicity probably plays a significant role. Isoflurane has been shown to reduce glutamate release from anoxic brain slices in vitro (13) and from the cortex in rats subjected to incomplete forebrain ischemia (14). In addition, isoflurane can also inhibit postsynaptic glutamate receptor-mediated responses in neocortical and hippocampal cells (15–17) and rat brain slices (18). Data from our laboratory have also shown that isoflurane can reduce both N-methyl-D-aspartate (NMDA)- and 
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-mediated cortical injury in vivo (19,20). Collectively, these data indicate that isoflurane-mediated reduction of cortical infarction after a short recovery period may be attributable in part to the attenuation of excitotoxicity.
Apoptosis is characterized by degradation of nuclear chromatin, condensation of the cytoplasm and nucleus, and ultimately the fragmentation of the cell into "apoptotic bodies." A number of investigators have shown that apoptosis, demonstrated by DNA fragmentation on agarose gels and by TUNEL staining, contributes to ischemic neuronal injury (11,24–29). Li et al. have shown that the number of cells undergoing apoptosis peaks at 24–48 h postischemia (26). Our findings in the awake group, in which apoptosis peaked at 24 h postischemia, are consistent with their results. Apoptotic cells are localized primarily within the inner boundary zones of the evolving infarct (26). This suggests that apoptosis may contribute to the expansion of the ischemic lesion. By delaying the onset of apoptosis, isoflurane also delayed the enlargement of the infarct. Of interest is the finding of Li et al. (26) that apoptosis can be observed as late as 4 weeks after ischemia. This indicates that postischemic neuronal death is a dynamic process in which neurons continue to die for a long time after the initiating ischemic insult.
Integral to the process of apoptosis are the cysteine proteases called caspases (12,28–30). Caspase-3 is a principal effector of apoptosis. The mitochondria-dependent pathway has been proposed to be one of the major pathways that leads to the activation of caspase-3 (31). In vitro studies have shown that cytochrome-c released from the intermembrane space of mitochondria combines with apoptosis activating factor (apaf-1) and procaspase-9 to form an apoptosome. This subsequently leads to proteolytic cleavage and activation of caspase-9 (32,33). Activated caspase-9 then activates caspase-3. Postischemic activation of caspase-3 and caspase-9 has been shown in the experimental model of cerebral ischemia (27–29). Namura et al. (28) examined the activation of active-caspase-3 (p20) using immunohistochemistry in rats subjected to 2 hours of MCAO. Active caspase-3 (p20) immunoreactivity became prominent in neurons within the MCA territory at the time of reperfusion and was visualized in TUNEL-positive cells at 12–24 hours. Velier et al. (27) also demonstrated that active caspase-3 immunostaining was localized in the nucleus of cortical neurons 24 hours after permanent MCAO in the rat. Krajewski et al. (29) have reported that caspase-9 translocates from cytoplasm to the nucleus in neurons that are TUNEL-positive after global cerebral ischemia. Furthermore, intracerebroventricular administration of caspase-3 specific inhibitor (z-DEVF.fmk) and nonspecific caspase inhibitor (z-VAD.fmk) reduced neuronal injury after focal ischemia (34–37). Collectively, these data suggest that caspase activation plays an important role in the development of apoptosis after cerebral ischemia.
In the present study, the delayed activation of caspase-3 and caspase-9 probably contributed to the delayed development of apoptosis and cerebral infarction. Although the exact mechanism by which caspase activation was delayed is not known, the antiexcitotoxic effects of isoflurane might have contributed. In the awake group, lack of suppression of excitotoxicity might have increased the severity of the ischemic insult. As the number of apoptotic cells increases as a function of the severity of ischemic injury early after ischemia (26,38), more apoptotic cells might be observed 1 day after ischemia in the awake group. More neurons would therefore undergo early death. Neurons undergoing delayed death would not be prominent. By contrast, in the isoflurane group, isoflurane might have reduced the severity of the ischemic insult by reducing excitotoxicity. As a result, neurons would survive the initial insult and few neurons would undergo early apoptotic death. However, neurons that have been spared early death from excitotoxicity might undergo apoptosis during the later stages of postischemic recovery. There are experimental data to support this rather speculative proposition. For example, although antiexcitotoxic interventions such as glutamate antagonists attenuate excitotoxic death, they greatly increase the number of neurons that succumb to apoptosis (12,39–42). Gwag et al. (39) investigated the effect of glutamate receptor antagonists on excitotoxic necrosis and apoptosis in mouse cortical cultures exposed to transient oxygen-glucose deprivation. Both NMDA and AMPA/kainate receptor antagonists attenuated rapidly triggered excitotoxicity after 24 h. However, when cultures were examined 1 day later, many neurons that survived the initial excitotoxic insult were seen to go on to develop apoptosis. These data suggest that antiexcitotoxic interventions "unmask" apoptosis (43). The antiexcitotoxic properties of isoflurane might have had a similar "unmasking" effect on apoptosis during the later stages of postischemic recovery.
With regard to neuroprotection, our studies indicate that the administration of isoflurane during the ischemic insult may not be sufficient to achieve permanent neuroprotection. Nonetheless, our data, and those from other investigations, clearly demonstrate that isoflurane, by suppressing excitotoxicity, transiently reduces neuronal injury early after ischemia. This transient neuroprotection may increase the therapeutic window for drugs that have neuroprotective efficacy. Given that apoptosis is one of the major mechanisms by which neurons die in the postischemic period, drugs directed against apoptosis hold the greatest promise. The administration of anti-apoptosis drugs, in combination with anesthetics, might result in robust and sustained neuroprotection. Experimental support for this speculative premise is clearly needed.
In summary, in comparison with the awake state, isoflurane provided transient neuroprotection against ischemic injury. However, isoflurane only delayed the development of cerebral infarction caused by focal ischemia but did not prevent it. This was, in part, mediated by a delay in the activation of caspases and the development of apoptosis. Our data suggest that isoflurane is unlikely to provide sustained neuroprotection. However, by transiently protecting neurons, isoflurane might increase the therapeutic window for other drugs that can reduce neuronal injury. In this regard, the combination of anesthetics and drugs directed against apoptosis might provide permanent neuroprotection against ischemic injury.
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