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Departments of *Anesthesiology and Neurobiology and Anatomy, Wake Forest University School of Medicine, Winston-Salem, North Carolina
Address correspondence to David Zvara, MD, Department of Anesthesiology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27127-1009. Address e-mail to dzvara{at}wfubmc.edu.
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Abstract |
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Introduction |
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Anesthetic preconditioning (APC) is a cellular protective mechanism whereby exposure to a volatile anesthetic renders a tissue more resistant to a subsequent ischemic insult. This benefit is well established in models of myocardial protection (12,13). APC's benefit has been found in neurologic tissue, as well. Kapinya et al. (14) demonstrated that pretreatment for 3 h with one minimal alveolar anesthetic concentration (MAC) of isoflurane or halothane provided neurologic protection after permanent middle cerebral artery occlusion in a rat model. Xiong et al. (15) demonstrated a similar neurologic benefit in the brain using rats exposed to 0.75%, 1.5%, and 2.25% isoflurane in oxygen 1 h per day for 5 days. The repeated isoflurane exposure induced ischemic tolerance in rats in a dose-response manner, and the effect was obliterated by the administration of glibenclamide, a nonspecific KATP channel blocker.
In both of these studies, the reperfusion interval between the APC and the subsequent ischemic event was several hours or days. Such a wide time window of interval reperfusion suggests that more than one mechanism is present in APC protection of neurologic tissue. There are no published studies examining a brief reperfusion interval in a model of spinal cord ischemia. We hypothesized that spinal cord protection can be provided by both acute and chronic exposure to the anesthetic.
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Methods |
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Male Sprague-Dawley rats (body weight, 325 ± 2 g) were used in this experiment. The rats were allowed access to standard rat chow and water ad libitum. All rats were neurologically intact before anesthesia and instrumentation.
The rats were anesthetized with 3.5% sevoflurane in a 45% oxygen and air mixture by breathing spontaneously through a nonsealing facemask device. Arterial blood gas sampling was performed before any aortic occlusion. A thermistor probe was inserted 8.0 cm into the rectum to monitor core body temperature that was maintained at normothermia between 37.0°C and 38.0°C with a circulating warm water (37.5°C) underbody heating pad. The tail artery was cannulated with a polyethylene (PE-50) catheter to obtain arterial blood samples and to monitor mean distal aortic blood pressure (MDAP). The left femoral artery was isolated, and a Fogarty 2F balloon-tipped catheter (Baxter, Santa Ana, CA) was introduced for later advancement into the descending thoracic aorta. The left internal carotid artery was cannulated with a 20-gauge catheter for measurement of mean proximal aortic pressure (MPAP). The carotid artery cannula was connected to a heated blood-collection circuit (37.5°C) that included an 88-cm column of heparinized normal saline solution (1 U/mL). When the aorta was occluded, proximal aortic blood was allowed to bleed into the heparinized column, maintaining MPAP at 65 ± 3 mm Hg. MPAP, MDAP, and temperature were recorded at 1-min intervals by a PC-based data acquisition system (Micro-Med, Louisville, KY).
To induce spinal cord ischemia, the Fogarty 2F catheter in the left femoral artery was inserted retrograde into the descending thoracic aorta 10.0 cm from the femoral arteriotomy so that the tip of the catheter balloon lay just caudal to the left subclavian artery. After instrumentation, all rats were given 200 U of heparin sodium. To induce ischemia, the catheter was inflated with 0.05 mL of saline solution, and aortic occlusion was confirmed by reduction in MDAP. At the end of the IPC occlusion period, the vented blood from the carotid artery cannula was reinfused for 60 s, and the Fogarty catheter was retracted into the femoral artery. At the end of the 12-min ischemic injury period, blood from the heat exchanger was again reinfused for 60 s, 4 mg of protamine sulfate was administered, catheters were removed, surgical wounds were closed, and the rats returned to their cages for recovery.
The rats were randomized into 5 groups of 14 each (except in the sham group, which had only 3 rats): (a) IPC, (b) chronic APC (cSEVO), (c) acute APC (aSEVO), (d) control, and (d) sham surgery. Rats in the IPC group had 3 min of preconditioning ischemia, 30 min of reperfusion, and then 12 min of ischemia. The 30-min reperfusion interval and the 12-min ischemic insult are based on previous study results demonstrating protection with this protocol (11). The control group had a similar time for anesthesia (50 min) as the IPC group, but there was no preconditioning maneuver before the 12-min period of ischemia. The sham group had the same anesthesia time and surgical preparation but no ischemia. The 33-min ischemic-reperfusion period before the 12-min ischemic period was omitted from the cSEVO- and aSEVO-treated rats. Rats in the cSEVO group had 1 h of 3.5% sevoflurane exposure on each of 2 days before the 12-min period of ischemia on the third day. Rats in the aSEVO group had 1 h of 3.5% sevoflurane exposure before surgery, followed by a 1-h period of no anesthesia before being reanesthetized and then instrumented for the 12-min period of ischemia. At 24 h, and then daily for 7 days postischemia, all rats were tested to assess neurologic function. Tests were performed using the Tarlov Motor Scale (0 = normal, 1 = mild deficit, 2 = weight bearing support and one or two steps, 3 = no weight bearing with frequent movement, 4 = no weight bearing with barely perceptible movement, and 5 = no movement). Sensory function was assessed by a hindlimb withdrawal from a stimulus (1 = [+] = oral or tactile response to noxious stimulus applied to hindlimbs and 0 = [–] = no response to noxious stimulus). One member of the research team who was blinded to the treatment groups conducted all neurologic testing. After neurologic testing, the rats were killed in accordance with guidelines of the Institutional Animal Care and Use Committee. Rats with complete hindlimb paralysis for 72 h, hematuria, or 25% reduction in body weight were killed and perfusion-fixed before the 7-day evaluation for humanitarian reasons.
Rats selected for histologic analysis were deeply anesthetized via a saturated halothane chamber and were perfusion-fixed. After administration of a direct left ventricular bolus of 0.5 mL of heparin, rats were perfused with 100 mL of 0.9% normal saline solution (37°C) followed by methacarn-fixative (16) per 0.6 mL/g of body weight. Afterward, whole spinal cords were dissected free, blocked into rostral and caudal sections, and postfixed by immersion in methacarn for another 24 h. The spinal cords were further cut into two blocks corresponding with the third to fourth cervical segment (normal control) and the fourth to sixth lumbar segment (experimental region of ischemia). After postfixation, the methacarn was replaced with 70% ethanol using three washes with that solution, shaking for at least 1 h per wash. The tissue was refrigerated in 70% ethanol until processed for paraffin embedding. Serial transverse sections (12 µm) were prepared from both cervical and lumbar blocks. The slides were stained with hematoxylin and eosin method and evaluated for evidence of cellular degeneration and necrosis. Color digital images of typical cervical/thoracic and lumbar sections from each rat were captured by one of the authors blinded to treatment groups. These were graded using a 5-point qualitative scale. One point was awarded for each of the following characteristics: leukocyte infiltration, <3 motor neurons visualized, shrunken neurons, necrotic neurons, and dying motor neurons. An entirely normal spinal cord segment would score a 0, whereas a completely damaged segment would score a 5.
Fifty-nine rats were studied (IPC, n = 14; cSEVO, n = 14; aSEVO, n = 14; control, n = 14; and sham, n = 3). Fifty-four rats were perfusion-fixed for histologic analysis.
Statistical analysis of motor and sensory neurologic data was performed using two-way analysis of variance (ANOVA) and Student-Newman-Keuls method tests. Heart rate, MPAP, MDAP, and temperature were analyzed using one-way ANOVA and Holm-Sidak method for all pair-wise multiple comparisons. Survival rate was examined by 
2 test. Total histology score was examined by Kruskal-Wallis one-way ANOVA and Dunn method of all pair-wise multiple comparisons. For all statistical analysis, a P value of <0.05 was considered significant. Data are expressed as the mean ± se of the mean unless otherwise indicated.
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Results |
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The sham group and IPC group had significantly superior neurologic scores compared with the control, aSEVO, and cSEVO groups (P
0.03 at Day 1; P 0.002 at Days 2–7) (Fig. 2).
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There was a significant difference in withdrawal testing between the IPC and aSEVO groups at Days 1–7 (P < 0.001) (Fig. 3). IPC was significantly different than control Days 2 and 3 (P = 0.004), as well as Days 4–7 (P = 0.001). IPC compared with cSEVO was significantly different on Days 1–7 (P = 0.001).
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Histologic evaluation was conducted at Day 7 on 39 rats and on 15 rats killed early, as required by the humanitarian protocol. There was no significant difference in the cervical spinal cord (P = 0.108 among all groups). There was a significant difference in the lumbar cord sections (Fig. 4) between the IPC and sham groups and all other experimental groups (P < 0.050).
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Discussion |
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The absence of any beneficial effects of APC in spinal cord ischemic injury were unexpected in light of the numerous reports that this treatment did protect the brain and heart from ischemia, using several volatile anesthetics administered according to several different protocols (12–14,17–20). The reports of Xiong et al. (15), Payne et al. (21), and Kehl et al. (22) on protection of the brain from ischemic injury after isoflurane or sevoflurane preconditioning used protocols for APC similar to that used in our study and found neurophysiological and histological preservation of function after ischemia. Xiong et al. (15) demonstrated the dose-related benefit of APC with isoflurane in a model of neurologic injury via activation of adenosine triphosphate-regulated potassium channels after focal cerebral ischemia in rats. There are several potential reasons why this benefit was not observed in our spinal cord model as compared with cerebral models of APC. The most parsimonious is that there were minor technical differences in the experimental protocol that are important in its effectiveness in the spinal cord but less so in the brain. For example, the threshold dosing required to observe this benefit in the spinal cord may be very different than it is for the brain. There may be an anesthetic-specific benefit seen with some inhaled anesthetics and not others (i.e., isoflurane). Our protocol was designed using a volatile anesthetic with known preconditioning benefit in a common, clinically relevant, and previously established dose. Further experimentation with other anesthetics and varying doses and administration regimens are required to fully answer these questions.
There are other potential explanations for spinal cord-brain difference in APC-induced protection. It could be related to the fact that the spinal cord has much less collateral vasculature and volume of cerebrospinal fluid than the brain (23). Therefore, a given period of ischemia is likely to result in greater irreversible damage in the spinal cord than in the brain. However, the comparable effectiveness of IPC to protect both spinal cord (11) and brain (15,24) from ischemia suggests that such anatomical differences between the two are not responsible for the inability of APC to provide comparable protection.
Another reason for the apparent difference may come from the differences in the methods in this study used to assess protection compared with those in the brain. We evaluated functional recovery of the whole rat by behavioral tests of motor and sensory function, as well as survival postischemia, for up to a week after ischemia. The brain APC studies reported similar behavioral indices of function only at 24 hours after ischemia or relied on electrophysiological measures of small numbers of neurons in brain slices of the animals and histopathological changes (15,21,22). Because the effects of reperfusion injury in the brain and spinal cord cause progressive neuronal degeneration over a period of days after the injury (24,25), short-term protection may not accurately reflect the long-term outcome.
Our data demonstrate a strong benefit of acute mechanical IPC of the spinal cord. There were both a survival benefit and improved neurologic outcome in the IPC group. Potential mechanisms for this protection are suggested by a number of reports that tested the effects of inhibitors of specific cellular functions or examined differences in gene expression between conditioned and unconditioned animals subjected to acute ischemia (15,26,27). One likely mechanism common to both the central nervous system and heart involves the activation of adenosine triphosphate-regulated potassium channels (15,18,26,27). There are also suggestions that the heat shock protein system (Hsp) may come into play (25,28,29), but the short period between IPC and ischemic injury makes it unlikely that the protection is a result of accumulation of Hsp during the 30 minutes between the 2 events. However, it is possible that IPC may mobilize existing Hsps to sequester proinflammatory signaling proteins into inactive complexes called stress granules (30,31). Alternatively, acute preconditioning treatments may initiate the phosphorylation or nuclear import of Hsf-1 so that the Hsp expression system is primed and Hsp accumulation would occur faster than in unconditioned animals. A faster response enhances stress tolerance based on work with some nonsteroidal antiinflammatory drugs and plant compounds known to enhance cell stress tolerance in other models of metabolic stress (32,33).
The histology of the rat spinal cords at the end of the seven-day protocol is consistent with the behavioral observations. All cervical sections examined from both groups were normal. However, rats in the groups subjected to ischemia exhibited clear pathologic changes within the lumbar regions, including eosinophilic neurons, inflammatory cell infiltration, and gross necrosis of the neuropil. Further analyses of the tissues from these animals are currently in progress to begin to assess possible cellular mechanisms for the preservation of motor function in the IPC group, which include changes in distribution or accumulation of several Hsps and metallothioneins I and II, which are another set of proteins that have been linked to ischemic tolerance (33).
There are several limitations to the present study. Only one volatile anesthetic was studied and only one dose used. In our review of the literature, all volatile anesthetics exhibit APC effects, at least in the heart (27), and sevoflurane has preconditioning benefits in the brain (15,27). Although the evaluation of different inhaled anesthetics could yield different results, we consider this unlikely because their primary effects on neural tissue are similar (14,15,34). The dose of sevoflurane represents 1.5 MAC values in the rat, which is consistent with models in which benefit has been previously demonstrated. There may be a dose-related effect in either the MAC or the administration sequencing that could lead to different outcomes. That point remains to be tested. Another limitation in this study arises from the use of a noxious stimulus to provoke hindlimb withdrawal for sensory motor testing in rats with potential motor deficit. Indeed, if the rat is unable to withdraw its paw secondary to complete motor paralysis, then such a test may give a false negative. However, to guard against that possibility, we also watched for vocalization and other body withdrawal movements. By those criteria, we concluded that there was no evidence of pain with the paw pinch in rats with complete motor neurologic deficit.
Mechanical IPC has been shown to be effective in prevention of spinal cord ischemic injury (11). Unfortunately, the procedure is unfeasible in a clinical setting because of the potential for increased risks to the patient. Hence, the importance of identifying a drug that mimics the mechanical IPC protection is critical. Several groups have shown a potential clinical application in the use of volatile APC with isoflurane, a relatively low-risk procedure, to elicit a protected state in cerebral and cardiac tissues subjected to mechanical clamping (20). Our study suggests that the spinal cord does not respond in the same way as the brain. Nonetheless, this work supports the strong benefit of IPC in neurologic protection and encourages further investigation to find a pharmacologic substitute to mimic these effects. Such drugs may have a tremendous impact on contemporary aortic surgery.
In summary, neither aSEVO nor cSEVO reduced neurologic injury or improved survival in this rat model of spinal cord ischemia. The results of histologic evaluation up to seven days after the injury were consistent with neurologic outcome. In light of the strong protective benefit of IPC for the spine, brain, and heart, it was surprising that APC was not also as protective of the spinal cord as it is for the brain and heart. That outcome is a reminder that the spinal cord and brain have different responses to the same type of injury. Thus, it will be important to elucidate spinal cord-specific mechanisms responsible for the protective effect of IPC.
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Footnotes |
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  H. Huang, L. Zhang, Y. Wang, J. Yao, H. Weng, H. Wu, Z. Chen, and J. Liu Effect of ischemic post-conditioning on spinal cord ischemic-reperfusion injury in rabbits: [Effet du post-conditionnement ischemique sur les lesions d'ischemie-reperfusion de la moelle epiniere chez le lapin] Can J Anesth, January 1, 2007; 54(1): 42 - 48. [Abstract] [Full Text] [PDF]
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