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Department of Anesthesiology-Resuscitology, Yamaguchi University School of Medicine, Yamaguchi, Japan
Address correspondence and reprint requests to Mishiya Matsumoto, MD, Department of Anesthesiology-Resuscitology, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi, 755-8505, Japan. Address e-mail to mishiya{at}yamaguchi-u.ac.jp.
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Abstract |
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Introduction |
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There has been good evidence that mitochondrial dysfunction plays a pivotal role in the pathophysiology of central nervous system ischemia (6). Under ischemic conditions, cytochrome c, apoptosis-inducing factor, and calcium ion may be released through the mitochondrial permeability transition (MPT) pore, leading to neuronal death (7). Therefore, preserving mitochondrial integrity may be essential for brain and spinal cord protection. Indeed, cyclosporin A (CsA) has been shown to have a robust protective effect against cerebral ischemia, especially in delayed neuronal death in the hippocampus (8,9). In the spinal cord, two recent studies have shown the protective or ameliorating effect of CsA, which was given either chronically before (10) or after the ischemic event (11). However, these studies were not concerned with blood glucose concentrations. Wahlstrom et al. (12) showed that administration of CsA at 20 mg/kg per day for 1 wk resulted in a combination of decreased endogenous insulin secretion and peripheral insulin resistance in dogs. It is possible that CsA may increase blood glucose concentrations. In the cerebral ischemia models, the protective effect of CsA was diminished when the animals were maintained hyperglycemic (approximately 20 mM) (13,14). Therefore, the effect of CsA should be carefully evaluated under the strict control of blood glucose concentrations.
In the present study, we sought to examine the effects of CsA on ischemic spinal cord injury with or without control of blood glucose concentrations. The effect of insulin was also examined. As the poor penetration of CsA through the blood-brain (spinal cord) barrier (15) is another problem, we attempted to open the barrier to facilitate drug penetration into the tissue.
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Methods |
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The experimental design is shown in Figure 1. To open the blood-spinal cord barrier and to facilitate the translocation of CsA across the barrier, we made a mild lesion in the lumbar spinal cord 4 days before spinal cord ischemia. Rabbits were anesthetized in a plastic box with 5% sevoflurane in oxygen. A catheter was inserted in an ear vein, and pentobarbital (30 mg) was administered to facilitate tracheal intubation. After placing a 3-mm cuffed endotracheal tube, the inspired gas mixture was changed to isoflurane 2%-3% in 40% oxygen/60% nitrogen, and the rabbits’ lungs were mechanically ventilated. With the rabbits in the prone position, midline skin and subcutaneous fascia were incised between the fourth and sixth lumbar spinous process after infiltration with 0.25% bupivacaine. Muscles were dissected; the fifth and sixth processes, ligamentum flavum, and epidural fat were sequentially removed; and the underlying dura was exposed. Using a technique adopted from Uchino et al’s study (8,9), blood-spinal cord barrier opening (BO) was made by insertion of a 0.35 mm steel needle into the spinal cord (2 mm in depth) bilaterally at the levels of L4-5 and L5-6 interlamina space. For the sham BO, the dura was exposed but the needle was not inserted. After suturing subcutaneous fascia and skin, isoflurane was discontinued and the lungs were ventilated with 100% oxygen. Extubation of the trachea was performed when adequate spontaneous ventilation occurred. The rabbits were allowed to recover for 6 h in a warmed plastic box that contained supplemental oxygen. IV fluid was provided until the rabbits began to drink. Antibiotic (cephazolin 30 mg/kg, IM) was administered once daily.
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Rabbits were randomly assigned to one of the following groups (n = 6 in each): a sham BO group, a BO group, a BO-CsA group, a BO-insulin group (BO-I group), or a BO-CsA-insulin group (BO-CsA-I group). The animals in the BO-CsA and BO-CsA-I groups received CsA (30 mg/kg, total), whereas the animals in the sham BO, BO, and BO-I groups received its vehicle (castor oil). The animals in the BO-I and BO-CsA-I groups received insulin IV 30 min before ischemia.
On days 3 and 4, rabbits were briefly anesthetized with 5% sevoflurane with a nonsealing facemask device and CsA (10 mg/kg; Novartis Pharma, Japan) or its vehicle, diluted with 20 mL of saline, administered IV via an ear vein catheter over 30 min.
On day 5, anesthesia was induced and maintained in the same manner as that for opening the barrier. Temperatures were monitored with a calibrated esophageal thermistor (Model MG -Type 209; Nihon Koden, Tokyo, Japan) and a needle-type thermistor (Model PTC-201; Unique Medical, Tokyo, Japan) inserted into the paravertebral muscle at the level of L4-5. The paravertebral muscle temperature was controlled throughout the study at approximately 38.0°C with a heating lamp and warming pad. PE-60 catheters were inserted into both femoral arteries to measure arterial blood pressure above and below the aortic occlusion. The right-side catheter was advanced 3 cm into the abdominal aorta, the left one was advanced 17 cm.
In all groups, spinal cord ischemia was produced as previously reported (5,16–18). In brief, in the right lateral position, the abdominal aorta was exposed retroperitoneally at the level of the left renal artery. A PE-60 catheter was placed around the aorta immediately distal to the left renal artery for later occlusion of the aorta. Then, an occluder tube (16F rubber tube) was tunneled to the skin.
After completion of surgery, end-tidal isoflurane concentration was maintained at 2%. CsA (10 mg/kg, the third dose) or its vehicle was administered IV over 60 min starting at 30 min before aortic occlusion. In the BO-I and BO-CsA-I groups, insulin was administered IV 30 min before aortic occlusion to control the blood glucose concentrations. Insulin 1.5 U, 1 U, and 0.5 U was administrated when blood glucose concentrations were >200 mg/dL, 150-200 mg/dL, and <150 mg/dL, respectively. Heparin 400 U was administered immediately before aortic occlusion. Ischemia was induced by pulling the PE catheter and clamping an occluder tube for 13 min. Segmental spinal cord evoked potentials (SSCEPs) were monitored, stimulating the left sciatic nerve with square-wave pulses of 0.1 ms duration and 0.6 mA intensity delivered at 3 Hz, and recording in a bipolar fashion (L5 and L6) with silver needle electrodes. The typical recording of SSCEP demonstrated 2 positive waves and 4 negative waves (N1-N4). We measured amplitude of N3 that represents the postsynaptic component (19). SSCEPs were recorded every 1 min for 7 min after aortic occlusion, and then every 2 min until 15 min after reperfusion. After the final recording of SSCEP, all catheters were removed, and then all incisions were sutured. Emergence from anesthesia and postanesthetic care were the same as described before (5,16–18).
The rabbits were neurologically assessed at 12, 24, 48, 72, and 96 h after reperfusion by an observer unaware of the treatment group using the 5-point score system proposed by Drummond and Moore (20): 4 = normal motor function, 3 = ability to draw legs under body and hop but not normally, 2 = some lower extremity function with good antigravity strength but inability to draw legs under body and/or hop, 1 = poor lower extremity function but weak antigravity movement only, 0 = paraplegic with no lower extremity function.
After the final neurological assessment (96 h after reperfusion), the rabbits were reanesthetized with 2% isoflurane in oxygen. Transcardiac perfusion and fixation were performed with 10% phosphate-buffered formalin. Coronal sections of the spinal cord at the level of L5 were cut at a thickness of 8 µm, and stained with hematoxylin and eosin. Normal neurons in the anterior spinal cord (anterior to a line drawn through the central canal perpendicular to the vertical axis) were counted in two sections for each rabbit and averaged. Ischemic neurons were identified by cytoplasmic eosinophilia with loss of Nissl substances and the presence of pyknotic homogenous nuclei.
Parametric data are presented as mean ± sd. Physiological variables were analyzed by a repeated-measures analysis of variance followed by factorial analysis of variance. Where differences were identified, Scheffé’s post hoc test for intergroup comparisons was performed. The time for N3 wave of SSCEPs to disappear after aortic occlusion or to appear after reperfusion were analyzed by a factorial analysis of variance. Hindlimb motor function and the number of normal neurons in the anterior spinal cord were analyzed with a nonparametric method (Kruskal-Wallis test followed by the Mann-Whitney U-test). P < 0.05 was considered statistically significant.
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Results |
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All rabbits survived until the final neurological assessment (96 h after reperfusion). BO did not induce neurological deficit in any animal. In all groups, the time required for N3 wave of SSCEPs to disappear was 5 to 6 min, whereas the time required for N3 wave to appear in each group was 13.0 ± 1.8, 13.3 ± 2.7, 11.0 ± 4.9, 9.0 ± 3.1, and 9.7 ± 2.7 min in the sham BO, BO, BO-CsA, BO-I, and BO-CsA-I groups, respectively. There were no significant differences in the time required for the N3 wave of SSCEPs to disappear and to appear among the groups.
The time course of changes in motor function score in each group is shown in Figure 2. There were no significant differences in motor function score between the sham BO, BO, and BO-CsA groups at 12, 24, 48, 72, and 96 h after reperfusion. The scores in the BO-I and BO-CsA-I groups were significantly better than in the sham BO, BO, and BO-CsA groups at 48, 72, and 96 h after reperfusion. In the BO-I group, four rabbits showed normal motor function and two rabbits could not hop, whereas all rabbits in the BO-CsA-I group showed normal motor function at 96 h after reperfusion. However, the difference between the BO-I and BO-CsA-I groups did not reach statistical significance.
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The number of morphologically normal appearing neurons 96 h after reperfusion is shown in Figure 3. The number of normal-appearing neurons in the BO-I and BO-CsA-I groups was significantly larger than that in the sham BO group. The number of normal-appearing neurons in the BO-CsA-I group was also significantly larger than in the BO and BO-CsA groups. Figure 4 shows microphotographs of the spinal cord in each group. In the animals of the sham BO, BO, BO-CsA groups, the structure of the spinal cord gray matter was destroyed, the majority of motor neurons disappeared, and the prominent inflammatory cell infiltration was observed (Fig. 3 A, B, C). In contrast, those changes were not observed in the BO-I and BO-CsA-I groups (Fig. 3 D, E).
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Discussion |
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We demonstrated in the present study that CsA (30 mg/kg in total) alone increased blood glucose concentrations and failed to protect against ischemic spinal cord injury produced by 13 min of aortic occlusion. Insulin alone and insulin with CsA significantly improved the neurological and histopathological outcome.
Two studies have been published concerning the effects of CsA on spinal cord ischemic damage. Sato et al. (11) demonstrated in rabbits subjected to 15 min ischemia that a single IV administration of 25 mg/kg, but not 2.5 mg/kg, of CsA 30 min after restoration of blood flow improved neurological and histopathological outcome 7 days after reperfusion. Tachibana et al. (10) also demonstrated in rabbits subjected to 15 min ischemia that 20 mg/kg per day of CsA administered IV for 9 days before ischemia significantly improved the neurological and histopathological outcome 2 days after reperfusion. However, they failed to show a protective effect with a single preischemic dose of 30 mg/kg.
In the present study, CsA alone given before ischemia did not improve neurological or histopathological outcome, although the duration of aortic occlusion (ischemia time) in the present study was shorter (13 min) than in the studies of Sato et al. (11) or Tachibana et al. (15 min). The reason for these differences is not clear. The methods used to induce ischemia were different; the aorta was occluded with intravascular balloon inflation in the studies of Sato et al. (11) and Tachibana et al. (10), whereas the aorta was occluded by a snare in the present study and ischemia was verified with a decrease in distal pressure to approximately 8 mm Hg, with disappearance of the N3 component in the SSCEP. With regard to the dose of CsA, our dose (30 mg/kg in total) might be insufficient. However, in our preliminary study, rabbits showed severe diarrhea and loss of appetite with 20 mg/kg per day of CsA for 3 days. Therefore, simply increasing the dose of CsA was deemed inappropriate. Indeed, Tachibana et al. (10) reported that 2 of 8 rabbits died during chronic administration of CsA (20 mg/kg per day for 9 days).
Failure to demonstrate a protective effect of CsA alone in the present study might be attributed to hyperglycemia. In rat models of forebrain ischemia and focal cerebral ischemia (middle cerebral artery occlusion), hyperglycemia is reported to decrease the protective effects of CsA (13,14). Also, an increase in mean preischemic blood glucose concentrations of 40 mg/dL by infusing dextrose has been reported to increase frequency of postischemic paraplegia in rabbits (20).
In the present study, insulin alone (BO-I group) and combined use of CsA and insulin (BO-CsA-I group) improved the neurological and histopathological outcome. Because there were no significant differences in neurological and histopathological outcome between BO-I and BO-CsA-I groups, the improvement may have partly resulted from the decrease in blood glucose concentrations per se. Voll and Auer (22) have demonstrated that insulin acts directly on the brain, independent of inducing hypoglycemia (3 mM), and reduces ischemic brain necrosis in a rat transient forebrain ischemia model. Nakao et al. (23) examined the effects of insulin-like growth factor 1 (IGF-1) and insulin in rabbits receiving 15 min spinal cord ischemia. IGF-1, an equipotent dose to insulin in decreasing plasma glucose (approximately 50 mg/dL), given 30 min before spinal cord ischemia significantly improved neurological function and histopathological changes in association with increased Bcl-xL in the motor neurons 24 h after reperfusion. Insulin itself only modestly increased Bcl-xL and inhibited Bax expression. The number of normal motor neurons was preserved with insulin, but the neurological outcome was not significantly improved. It was suggested that IGF-1 has a protective effect, but the protective effect of insulin is incomplete and insulin may have the effect of delaying motor neuron death. Nevertheless, both IGF-1 and insulin appeared to modify the Bcl-xL and Bax, although the effect of insulin was much weaker than IGF-1. Both an increase in BcL-xL and a decrease in Bax expression have been suggested to inhibit the permeability transition in the isolated mitochondria from the liver (24). If this is the case in the spinal cord, the inhibition of MPT may be the common target for insulin and CsA in spinal cord protection, although the effects of CsA were at best minimal in the present study.
There are some limitations of the present study. The sample size in each group (n = 6) was small and the difference between BO-I and BO-CsA-I groups may have been undetected. The study was not perfectly performed in a blind fashion because of the design that sham BO and BO groups received different treatments.
In summary, pretreatment with insulin improved neurological and histopathological outcome after spinal cord ischemia in a model of delayed motor dysfunction, whereas CsA alone did not. Failure to improve outcome with the use of CsA alone might have been attributable to hyperglycemia induced by CsA.
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Footnotes |
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Supported, in part, by the Ministry of Education, Science, Sports, and Culture grant No. 15591635.
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This article has been cited by other articles:
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  D. Nagamizo, S. Tsuruta, M. Matsumoto, H. Matayoshi, A. Yamashita, and T. Sakabe Tight Glycemic Control by Insulin, Started in the Preischemic, but Not Postischemic, Period, Protects Against Ischemic Spinal Cord Injury in Rabbits Anesth. Analg., November 1, 2007; 105(5): 1397 - 1403. [Abstract] [Full Text] [PDF]
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: significant difference (P < 0.05) from sham barrier-opened (sham BO), barrier-opened (BO), and barrier-opened-cyclosporin (Cs) A (BO-CsA) groups, respectively.
