Temporary aortic occlusion in surgical repair of thoracic and thoracoabdominal aneurysms can cause different extents of spinal cord ischemia/reperfusion (I/R), which leads to spinal cord functional impairment and then to acute or delayed paraplegia. Despite many strategies having been developed to increase the ischemic tolerance of the spinal cord and minimize the incidence of neurological complications, spinal cord ischemia occurs in approximately 100% of patients undergoing thoracic aorta or thoracoabdominal aorta aneurysm surgery. The quoted figure of 3%–18% is the incidence of residual permanent injury (acute and/or delayed paraplegia) caused by the ischemic episode, depending on the type of aneurysm and other comorbidities.1 Therefore, other novel measures to protect against spinal cord ischemic injury require further development.
Inhaled anesthetics are used widely in anesthetic practice. Sevoflurane, as a novel inhaled anesthetic with minimal pungency, low solubility, and less hepatic toxicity, is a preferable clinical choice.2 Preconditioning with inhaled anesthetics can induce ischemic tolerance in the brain and spinal cord.3–6 Recently, studies demonstrated that sevoflurane preconditioning could induce ischemic tolerance in the brain and alleviate hypoxic and ischemic cerebral injury.7,8 However, there are few studies that have described the protective effect of this drug on spinal cord I/R.
In addition, extracellular signal-regulated kinase (ERK), one of the three major members of the mitogen-activated protein kinases (MAPKs) family, plays a crucial role in regulating neuron survival after ischemia/hypoxemia in in vitro studies of isoflurane preconditioning.9–12 Recent studies in heart and kidney cells showed that ERK was involved in the protection by sevoflurane preconditioning.13,14
Therefore, this study, using a well-established model of the spinal cord I/R in rabbits induced by infrarenal aorta occlusion, was designed to investigate whether sevoflurane preconditioning could induce rapid ischemic tolerance to the spinal cord and the role of ERK in this process.
The experimental protocol used in this study was approved by the Ethics Committee for Animal Experimentation and was conducted according to the Guidelines for Animal Experimentation of the Fourth Military Medical University. The animals were studied in Xijing Hospital, Fourth Military Medical University (Xi'an, China).
The design of Protocol 1 is illustrated in Figure 1A. To test whether preconditioning with sevoflurane induces rapid ischemic tolerance, 25 New Zealand White male rabbits were randomly assigned to three groups. Animals in the Sev group (n = 10) received preconditioning with 3.7% sevoflurane (1.0 minimum alveolar anesthetic concentration) in 96% oxygen by breathing spontaneously through a face mask for 30 min.15–17 Animals in the O2 group (n = 10), as control, inhaled only 96% oxygen for 30 min. The concentration of sevoflurane and oxygen inside the mask was measured continuously by a monitor for volatile anesthetics (Philips IntelliVue G5-M1019A, MP60; Philips Electronics N.V., The Netherlands). After preconditioning, animals were allowed to recover freely in room air. Spinal cord I/R was induced at 60 min after the end of preconditioning. Five rabbits in the Sham group received the same anesthesia and surgical preparation but no preconditioning or spinal cord I/R.
The design of Protocol 2 is illustrated in Figure 1B. To evaluate the role of ERK activation in neuroprotection by sevoflurane preconditioning, 32 New Zealand White male rabbits were randomly assigned to four groups (n = 8, each): the vehicle + O2, vehicle + Sev, U0126 + O2, and U0126 + Sev groups. In the vehicle + O2 and vehicle + Sev groups, 50 μL/kg dimethylsulfoxide (Sigma Chemical Co., St. Louis, MO) was injected IV to the rabbits 20 min before the beginning of preconditioning. In the U0126 + O2 and U0126 + Sev groups, 50 μL/kg 0.4% U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-amino-phenylthio] butadiene; Promega Co., Madison, WI), a MAPK (MEK) inhibitor which has demonstrated potent inhibitory activation on ERK, dissolved in dimethylsulfoxide, was administered IV at the same time.18,19 The methods of preconditioning and spinal cord I/R were the same as Protocol 1. A catheter was inserted into the ear vein, and lactated Ringer's solution (4 mL · kg−1 · h−1) was administered IV during preconditioning in all groups to attenuate the hypotensive action of sevoflurane. To analyze the levels of phosphor-ERK1/2 (p-ERK1/2) at 48 h after reperfusion, 25 additional rabbits were randomly assigned to five groups (n = 5, each): the Sham, vehicle + O2, vehicle + Sev, U0126 + O2, and U0126 + Sev groups.
Animals and Surgical Preparation
Fifty-seven New Zealand White male rabbits (weight 2.0–2.9 kg) were used in this study. All rabbits were neurologically intact before preconditioning and instrumentation. After an overnight fast with unrestricted access to water, the rabbits were anesthetized with pentobarbital sodium (30 mg/kg, IV),20 allowed to breathe spontaneously, and inhaled oxygen by face mask at a flow rate of 2 L/min. The lactated Ringer's solution (4 mL · kg−1 · h−1) was infused IV. A 22-gauge catheter was inserted into the ear artery to measure the distal blood pressure and sample arterial blood. Another catheter was inserted into the left femoral artery to measure the femoral blood pressure. Arterial blood pressure and heart rate were monitored continuously by using a calibrated pressure transducer connected to an invasive pressure monitor (Colin BP-508; Colin, Japan). Rectal temperature was maintained between 38°C and 39°C by an overhead lamp. Arterial blood was sampled at baseline, the end of preconditioning, preischemia, 10 min after ischemia, and 10 min after reperfusion, respectively, for the determination of Pao2, Paco2, pH, and blood glucose. Arterial blood gases were measured by means of the Rapid Lab 1260 (Bayer HealthCare AG, Germany).
Spinal Cord Ischemia/Reperfusion
Spinal cord I/R was induced by infrarenal aorta occlusion in the rabbits as described in our previous studies.16,20,21 Briefly, animals were placed in supine position. A 3- to 4-cm medial incision was made in the abdominal skin to expose the abdominal aorta at the level of the left renal artery after being infiltrated with 0.2% lidocaine. Spinal cord I/R was induced by aorta clamping with an artery forceps just below the left renal artery for 20 min, and 150 U/kg heparin was administered 5 min before the aortic occlusion. The artery forceps were then removed and the abdominal wall was closed with wound clips. After these procedures, all catheters were removed and 0.25% bupivacaine was infiltrated around the incision sites to minimize pain. An antibiotic (40,000 IU gentamicin) was administered IM immediately after the operation. Rabbits recovered from anesthesia at room temperature, then were returned to their home cages and survived for 2 days. Bladder content was compressed manually as required.
At 4, 8, 12, 24, and 48 h after reperfusion, rabbits were neurologically assessed by an observer who was unaware of the grouping, using the modified Tarlov criteria22: 0, no voluntary hindlimb function; 1, movement of joints perceptible; 2, active movement but unable to stand; 3, able to stand but unable to walk; 4, complete normal hindlimb motor function.
Hematoxylin and Eosin Staining and Histopathologic Evaluation
After completion of the evaluation of hindlimb motor function 48 h after reperfusion, the animals were reanesthetized. Transcardiac perfusion and fixation were performed with 1000 mL heparinized saline followed by 500 mL 4% paraformaldehyde. The lumbar spinal cord was removed and refrigerated at 4°C in 4% paraformaldehyde for 3 days. After dehydration in graded concentrations of ethanol and butanol, the spinal cord was embedded in paraffin. Coronal sections of the spinal cord (L5 segment) were cut to a thickness of 5 μm.
Three sections selected randomly from the rostral, middle, and caudal levels of the L5 segment of each rabbit were stained with hematoxylin and eosin. The sections were transformed into digital images (Olympus BX51 reflected system, the digital camera DP 70, and the visual communication suite Olympus DP-control, Olympus, Japan). An investigator, unaware of the grouping and outcomes, counted the numbers of viable motor neurons in the anterior spinal cord (anterior to an imaginary line drawn through the central canal perpendicular to the vertical axis). Averages were used and compared among groups. The remaining normal neurons were judged by their morphological appearance, whereas injured neurons were identified by intensely eosinophilic cytoplasm with loss of Nissl substance and by the presence of pyknotic homogenous nuclei.
TUNEL Staining and Quantification of Apoptosis
For deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining, three sections selected as described above were examined. TUNEL staining was performed to detect DNA fragmentation in cell nuclei using an In Situ Cell Death Detection Kit, POD (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. The sections were counterstained with hematoxylin.23 In this study, if a motor neuron had a nucleus stained dark brown, with or without chromatin condensation, it was considered a positive cell. By counterstaining with hematoxylin (a blue dye), the nonapoptotic neurons were also demonstrated. The numbers of TUNEL-positive motor neurons in the anterior spinal cord were calculated by the same investigator, and the average of the three sections was used to compare among the groups.
Western Blot Analysis
At 48 h after reperfusion, the animals were deeply reanesthetized. The lumbar spinal cords (L5 segment) were harvested, homogenized in lysis buffer, then centrifuged, and supernatants were used for sodium dodecylsulfate-polyacrylamide gel electrophoresis. After being heat treated at 100°C for 11 min in equal amount of 6× protein loading buffer, the protein samples were loaded on each lane, separated by 10% gel for electrophoresis. The protein bands were transferred to nitrocellulose membranes. The membranes were blocked with 5% skimmed milk for 1 h at room temperature, and then incubated overnight with p-ERK (1:2500, Cell signaling Technology, Beverly, MA) or β-actin (1:5000, Sigma Chemical Co.) antibody at 4°C. After washing with Tris-buffered saline, the membranes were incubated at room temperature for 1 h with secondary antibodies (1:2000, Santa Cruz Biotechnology, Santa Cruz, CA). The specific protein bands were visualized using the enhanced chemoluminescence reagents. The protein concentration was analyzed densitometrically, corrected with values determined on anti-β-actin blots, and expressed as relative values compared in five groups.
The software, SPSS 11.0 for Windows (SPSS, Chicago, IL), was used to conduct statistical analysis. Physiological data and Western blot data were presented as mean ± sem, and the scores of hindlimb motor function were expressed as median (minimum-maximum value). Physiologic values at baseline and the end of sevoflurane preconditioning in Protocol 1 were compared by independent samples t-test. Other physiologic variables were analyzed using one-way analysis of variance followed by Student–Newman– Keuls test. The scores of hindlimb motor function, the number of normal and TUNEL-positive neurons in the anterior spinal cord, and the ratio of p-ERK1/2 were analyzed using a nonparametric method (Kruskal– Wallis test) followed by the Mann–Whitney U-test with Bonferroni correction. A P value <0.05 was considered statistically significant.
Physiologic values during preconditioning and surgery are presented in Table 1. There were no physiologically relevant differences among groups for all monitored variables except distal mean arterial blood pressure (MAP) at the end of preconditioning, whereas no hypotension (MAP <60 mm Hg) or hypoxemia (Pao2 <60 mm Hg) was found during sevoflurane preconditioning. The femoral MAP decreased to 10–15 mm Hg during ischemia in the O2 and Sev groups.
Sevoflurane Preconditioning Improved Neurologic Outcomes
All animals survived until the final neurological assessment at 48 h after reperfusion. The time course of neurological function scores after reperfusion in groups is shown in Figure 2A. The neurological function scores in the Sev group were higher than the O2 group at 48 h after reperfusion (P = 0.008; Fig. 2B).
Sevoflurane Preconditioning Increased the Numbers of Viable Neurons
The histopathologic results at 48 h after reperfusion are shown in Figure 3. Compared with the O2 group, there were more normal neurons in the anterior spinal cord of the Sev group (P = 0.002).
Physiologic values during preconditioning and surgery are presented in Table 2. There were no significant differences among groups for all monitored variables, and the femoral MAP was approximately 10–15 mm Hg during ischemia.
Sevoflurane Preconditioning-Induced Improvement of Neurologic Outcome was Reversed by U0126
All animals survived until the final neurological assessment at 48 h after reperfusion. The time course of neurological function scores after reperfusion is shown in Figure 4A. The vehicle + Sev group had higher hindlimb function scores than the vehicle + O2 group and U0126 + Sev group at 48 h after reperfusion (P = 0.009 versus vehicle + O2 group, P = 0.009 versus U0126 + Sev group), whereas there were no significant differences between the vehicle + O2 group and U0126 + O2 group (Fig. 4B).
Sevoflurane Preconditioning-Induced Enhancement of Normal Neurons was Abolished by U0126
The histopathologic results at 48 h after reperfusion are shown in Figure 5. The vehicle + Sev group had more viable neurons in the anterior spinal cord than vehicle + O2 group and U0126 + Sev group (P = 0.009 versus vehicle + O2 group, P = 0.010 versus U0126 + Sev group). There were no significant differences between the vehicle + O2 group and U0126 + O2 group (Fig. 5B).
Sevoflurane Preconditioning-Induced Reduction of Neuronal Apoptosis was Attenuated by U0126
Neuronal apoptosis caused by spinal cord I/R was examined by TUNEL staining at 48 h after reperfusion (Fig. 6). The vehicle + Sev group had fewer apoptotic neurons in the anterior spinal cord than vehicle + O2 group and U0126 + Sev group (P = 0.001 versus vehicle + O2 group, P = 0.002 versus U0126 + Sev group), and there were no significant differences between the vehicle + O2 group and U0126 + O2 group (Fig. 6B).
Sevoflurane Preconditioning-Induced Increase of p-ERK was Inhibited by U0126
ERK activation caused by spinal cord I/R was analyzed by Western blot at 48 h after reperfusion (Fig. 7). The phosphorylation level of ERK1/2 in the spinal cord was higher in the vehicle + Sev group than the vehicle + O2, U0126 + Sev, and Sham groups (P = 0.009 versus vehicle + O2 group, P = 0.009 versus U0126 + Sev group, P = 0.009 versus Sham group), and there were no significant differences between the vehicle + O2 group and U0126 + O2 group (Fig. 7B).
This study demonstrated that, at 1 h before spinal cord I/R, sevoflurane preconditioning for 30 min improved neurological outcome, preserved viable motor neurons, and upregulated the phosphorylation level of ERK1/2 in the spinal cord at 48 h after reperfusion. These beneficial effects were abolished by pretreatment with U0126, a specific MEK inhibitor. These results suggest that preconditioning with sevoflurane induces rapid ischemic tolerance to the spinal cord, and the tolerance is possibly mediated through the activation of ERK.
The phenomenon of ischemic tolerance can be induced by pretreatment of sublethal stresses other than ischemia.3,20,24 The window of protection from preconditioning is bimodal. The first window of protection (rapid tolerance), lasting no longer than 3 h, appears immediately after the preconditioning maneuvers, after which protection wears off; the second window of protection (delayed tolerance) reappears 24 h after preconditioning, lasting 12–72 h.24,25 Sevoflurane, as a widely used clinical volatile anesthetic, has demonstrated the ability of inducing hypoxia/ischemic tolerance in cultured neuronal cells26,27 and brain.7,8 However, Zvara et al.28 described that neither acute sevoflurane preconditioning nor chronic sevoflurane preconditioning reduced neurological injury or improved survival in their rat model of spinal cord I/R. In our study, at 1 h before spinal cord I/R, sevoflurane preconditioning for 30 min reduced neurological injury induced by spinal cord I/R in rabbits as manifested by higher neurological scores and more normal neurons in the anterior spinal cord. The most likely reason may be the differences between the dose, administration method, and the duration of sevoflurane used in the preconditioning.
We monitored heart rate, distal MAP, and rectal temperature continuously during preconditioning and examined the arterial blood gases at baseline and the end of sevoflurane preconditioning. There were no significant differences of these physiologic variables among groups. Because our previous study demonstrated that exposure to oxygen for 24 h could induce ischemic tolerance in the brain,29 in this study, rabbits receiving oxygen preconditioning for 30 min were used as control. Therefore, the neuroprotection induced by preconditioning with 3.7% sevoflurane in oxygen was mainly caused by the pharmacologic effects of sevoflurane rather than the effects secondary to the disturbance of physiologic variables and oxygen exposure. Previous studies have proved the worsening of neurological function 14–48 h after spinal cord I/R in the rabbit model.30,31 Therefore, the final assessments for neurological and histological outcomes were performed at 48 h after reperfusion in our experiments.
Although some studies indicated that U0126, the inhibitor of MEK, protected the central nervous system from hypoxia or I/R injury,32,33 several investigators have suggested that inhaled anesthetics given before or during ischemia/hypoxemia are neuronally protective through modulating the ERK pathway and activating its downstream apoptotic regulator. Bickler et al.9,10 demonstrated that isoflurane preserved neurons in hippocampal slice cultures by transiently enhancing phosphorylation of the Ca2+-dependent MAPK p42/44 (ERK) and attenuating the activation of the pro-apoptotic cofactors Bad and p90RSK. Zou et al.12 verified that isoflurane preconditioning protected human neuroblastoma SH-SY5Y cells against in vitro simulated I/R through the activation of ERK and upregulation of its downstream Egr-1 and Bcl-2. Recently, several studies showed that sevoflurane preconditioning could protect heart and kidney cells through ERK phosphorylation in vitro.13,14 Therefore, we hypothesized that the early neuroprotection induced by sevoflurane preconditioning on the rabbit spinal cord was also dependent on phosphorylation of ERK. U0126, a MEK inhibitor, has demonstrated potent inhibitory activation on ERK. The dose and administration method of U0126 in our experiment were chosen based on previous studies.18,19 Our results showed that sevoflurane preconditioning upregulated the level of p-ERK1/2 in the spinal cord at 48 h after reperfusion, which was in accordance with the previous study of erythropoietin on cardiac repair by Kobayashi et al.34 Administration of U0126 at 20 min before preconditioning reversed the neuroprotection and activation of ERK1/2 induced by sevoflurane preconditioning, whereas U0126 itself had no effect on neurological and histopathologic outcomes. These suggest that the sevoflurane-induced early-phase neuroprotection against spinal cord I/R is mediated, at least partly, through the activation of the ERK-related pathway.
Previous studies showed U0126 could inhibit the activation of both ERK1/2 and ERK5.35,36 Some investigators also indicated that ERK5 was activated in the hippocampal CA1 region after ischemic preconditioning, and the inhibition of ERK5 activation resulted in a significant increase in the number of TUNEL-positive apoptotic cells.37,38 Therefore, the role of ERK5 in the protection of sevoflurane preconditioning is still not exactly clear. Obviously, this problem and the pathway of ERK mediated for antiapoptosis in sevoflurane preconditioning should be elucidated in future studies. A number of other factors have been reported in several studies to be responsible for ischemic tolerance, such as adenosine triphosphate-regulated potassium channels,3 intracellular Ca2+ release,9 release of free radicals,16 and upregulation of antioxidant enzymes.20
In summary, the current study demonstrates that sevoflurane preconditioning induces rapid ischemic tolerance to spinal cord ischemic injury in rabbits, and the tolerance is possibly mediated through the activation of ERK. These data suggest that sevoflurane preconditioning may provide a new practical method for protecting perioperative spinal cord I/R.
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