Effects of Cyclosporin A on Neurological Outcome and Serum Biomarkers in the Same Setting of Spinal Cord Ischemia Model
Article Outline
Spinal cord ischemic injury is one of the feared complications during aortic cross-clamping. The aim of this study was to investigate whether cyclosporin A (CsA) has a protective effect on spinal cord during ischemia in a rabbit model. A total of 22 New Zealand white rabbits were studied in three groups. One of the groups served as a sham group (n = 7), in which only laparatomy was performed and dosed. One group served as a control group (n = 7), in which rabbits had their abdominal aortas cross-clamped for 40 min following median laparatomy. The last group was the CsA group (n = 8), in which rabbits underwent the same procedure as the control group as well as CsA infusion at 20 mg/(kg · hr) over 60 min starting with aortic cross-clamping and continuing in the first 20 min of reperfusion. Neurological outcome of rabbits was evaluated according to Johnson's scale at postoperative hours 24 and 48 in all groups, and then they were killed. Their spinal cords were harvested, and segments corresponding to L4-L6 were prepared for pathological examination. Serum neuron-specific enolase (NSE) and nitric oxide (NO) levels were measured prior to and following aortic occlusion, and comparisons were made. Physiological data were similar in all groups. Rabbits in the sham group did not have any neurological deficit. However, all rabbits in the control group showed severe neurological deficits, including total paraplegia in five. According to Johnson's scale, neurological status of the rabbits at postoperative hour 48 was better in the CsA group compared to controls (p < 0.01). Pathological examination of spinal cord specimens revealed a higher viability index in the CsA group compared to controls (p < 0.01). Serum NSE and NO levels were lower in CsA-treated animals compared to controls. Our results demonstrate that CsA, when administered during ischemia and in the early period of reperfusion, may reduce neuronal damage in the spinal cord in a rabbit model of transient spinal cord ischemia.
INTRODUCTION
Spinal cord ischemic injury following aortic cross-clamping continues to be a devastating complication, with a paraplegia incidence rate of 4–33%.1 Several mechanisms are believed to be responsible in ischemic neuronal injury, including glutamate-mediated excitotoxicity, nitric oxide (NO) overproduction, inflammation, apoptosis, and free radical generation.2, 3 Administration of various pharmacological agents as well as other adjuncts, such as distal perfusion, hypothermia, cerebrospinal fluid drainage, and reimplantation of major intercostal and lumbar arteries, have been investigated to minimize this devastating complication.4 However, the application of these techniques has decreased but not eliminated postoperative spinal cord dysfunction, and continuing investigations are necessary to further reduce the rate this complication.
Cyclosporin A (CsA), a cyclic undecapeptide, has been used as an effective immunosuppressant to prevent allograft rejection and treat autoimmune disorders.5, 6, 7 CsA is a specific inhibitor of phosphatase-calcineurin, an enzyme abundant in nervous tissue.8 Calcineurin is believed to be involved in cell death through its increasing effects on free radical generation following ischemia and reperfusion.8
In an effort to improve spinal cord protection by pharmacological means, we aimed to investigate the cytoprotective effects of CsA on the spinal cord during ischemia in an experimental model.
MATERIALS AND METHODS
Animal Care
A total of 22 male New Zealand white rabbits (mean weight 2,200 ± 308 g, range 1,700–2,800 g), which survived throughout the procedure, were included in the study. All animals were given a 5-day period to adapt to their environment by remaining in the same unit at 24°C room temperature and being fed the same nutrition protocol, and then they were studied. This work was approved by the Animal Care Committee of the Adnan Menderes University. All animals received humane care, in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication 5377-3, 1996).
Anesthesia and Monitoring
Anesthesia was induced by intramuscular ketamine (50 mg/kg) and xylazine (5 mg/kg), then maintained by an additional one-quarter single dose of this combination given intravenously during the operation. Animals were allowed to breathe spontaneously. Core temperature was monitored with a rectal probe. Each procedure was performed in the same operating room at ambient temperature. A catheter (24 gauge) was placed in an ear vein to give maintenance fluid (0.9% saline solution at a rate of 20 mL/hr). An arterial catheter (24 gauge) was placed in an ear artery for monitoring the blood pressure and heart rate (monitor model KMA 275; Petas, Ankara, Turkey). Both catheters were removed after completion of the procedure.
Operative Technique
Animals were placed in the supine position. After sterile preparation, a 10 cm midline incision was made, and the infrarenal abdominal aorta was exposed through the transperitoneal approach, with the abdominal contents reflected to the right. The aorta was isolated from the left renal artery down to the aortic bifurcation. Each animal was given 150 U/kg heparin intravenously 5 min prior to aortic occlusion. Heparin was not reversed at the end of the procedure. The aorta was cross-clamped at two sites using arterial bulldog clamps (Vascustatt; Scanlan, Saint Paul, Minnesota, 55107, USA): one was just below the left renal artery, and the other was just proximal to the aortic bifurcation. The inferior mesenteric artery was also clamped at its origin from the aorta, and loss of aortic pulse was confirmed by palpation. All animals were subjected to 40 min of cross-clamp time. Aortic clamps were removed after 40 min, and it was verified that satisfactory aortic pulse returned. The abdomen was closed in layers, and the catheters were removed. Sham-operated animals underwent the same operative conditions but without aortic occlusion. Animals were allowed to recover in their cages, with free access to food and water in the postoperative period.
Study Groups
All animals were randomly assigned to one of three study groups. Rabbits that survived the entire procedure were included in the study. A total of three animals (one in the control, two in the study group) died during the procedure. All animals received a similar volume of maintenance fluids (20 mL/hr saline) for the whole procedure.
Biochemical Analysis
Blood samples were drawn after the induction of anesthesia (preischemia) and 2 hr following release of aortic cross-clamping (postischemia) via the ear artery catheter. Blood was allowed to clot for 20–30 min at room temperature and then centrifuged and kept frozen at −70°C until studied. Samples were analyzed for neuron-specific enolase (NSE) and NO (nitrite + nitrate).
NSE was measured using a commercial kit (Roche, Mannheim, Germany; catalog 12133121-122, via Moduler E170 hormone autoanalyzer). This method was based on chemiluminescent enzyme immunoassay, and results were expressed as micrograms per liter.
NO (nitrite + nitrate) was assayed by a modification of the cadmium-reduction method as described by Navarro-Gonzalvez et al.9 The nitrite produced was determined by diazotization of sulfanilamide and coupling to naphthylethylene diamine. For the measurement of NO (nitrite + nitrate), a 400 μL sample was denatured by adding 80 μL 30% ZnSO4 solution, stirring, and then centrifuging at 10,000g for 20 min at 4°C. First, we activated Cd granules using CuSO4 solution in glycine-NaOH buffer. Then, 100 μL of deproteinized samples and standards were added. This reaction uses pretreatment of samples to reduce nitrate to nitrite, which can be accomplished by catalytic reactions using enzyme or Cd. The samples were analyzed spectrophotometrically using a microplate reader and quantified automatically against a KNO3 standard curve, and the results were expressed as micromoles per liter.
Neurological Assessment
Neurological function was evaluated at 24 and 48 hr after the operation. Animals were graded on a 5-point scale according to the method of Johnson and associates10: 0, hindlimb paralysis; 1, severe para-paresis; 2, functional movement, no hop; 3, ataxia, disconjugate hop; 4, minimal ataxia; 5, normal function. One member of the research team without knowledge of the treatment graded neurological function. Neurological status of rabbits in the study group was compared with that in the control group cumulatively, not at a specific paraplegia score.
Sacrifice and Tissue Preparation
All animals were killed at 48 hr postoperatively by a lethal cardiac injection of pentobarbital (100 mg/kg). Spinal cords were harvested immediately, and the L3–L6 segments (6–8 cm in length) were fixed in 10% neutrally tamponaded formalin solution and stored for 24 hr before being embedded in paraffin blocks for sectioning and histological examination.
Histopathological Examination
Sections 4–7 μm in thickness were affixed to glass slides, then stained with hematoxylin-eosin and examined by light microscopy. The extent of ischemic damage and leukocyte infiltration in motor neurons in the ventral horns of the spinal cord were assessed. Cells with eosinophilic cytoplasm that had lost the nucleus were considered injured. Neurons with prominent nucleolus with fine chromatins and cytoplasmic Nissl bodies were considered viable cells. The neurons from each spinal cord section were examined and categorized as injured or viable by a blinded observer. Viability index was then calculated by dividing the number of viable cells by the total number of neurons counted within the entire microscopic section for each animal (viability index = number of viable cells/total number of neurons). In addition, the inflammatory response was graded semiquantitatively by counting the number of leukocytes in randomly selected fields and scored according to the number leukocytes infiltrating the field as follows: 0, none; 1, fewer than 20; 2, 20–50; 3, more than 50 leukocytes.
Statistical Analysis
The Mann-Whitney U-test was used to compare the neurological and pathological findings. All other data are presented as means ± standard deviation (SD). Intergroup and intragroup mean values were compared by repeated measures analysis of variance and post hoc Tukey's test. Differences were considered significant at p < 0.05.
RESULTS
Physiological Parameters
There was no significant difference in the physiological parameters between the study and control groups at any time measured (Table I). No untoward effects in CsA-treated animals were observed during the study period.
Table I. Physiological and hemodynamic measurements
| Sham (n = 7) | Control (n = 7) | CsA (n = 8) | ||
|---|---|---|---|---|
| Weight | 2,385 ± 365 | 2,157 ± 258 | 2,075 ± 242 | |
| Mean arterial pressure (mm Hg) | ||||
| Before laparotomy | 67.2 ± 12.9 | 62.7 ± 6.9 | 60.4 ± 9.2 | |
| During ischemia | – | 69.4 ± 4.6 | 66.4 ± 7.9 | |
| After operation, 5 min | 60.9 ± 7.08 | 58.5 ± 7.3 | 58.8 ± 8.0 | |
| Rectal temperature (°C) | ||||
| Before laparotomy | 38.9 ± 0.33 | 38.9 ± 0.2 | 39.0 ± 0.40 | |
| During ischemia | – | 38.7 ± 0.39 | 39.1 ± 0.19 | |
| After operation, 5 min | 38.8 ± 0.18 | 38.6 ± 0.38 | 38.6 ± 0.43 | |
| Heart rate (beat/min) | ||||
| Before laparotomy | 180.5 ± 6.5 | 178.2 ± 9.1 | 189.6 ± 21 | |
| During ischemia | – | 186.4 ± 10.8 | 187.2 ± 18.2 | |
| After operation, 5 min | 182.2 ± 10.2 | 186.7 ± 8.7 | 184.5 ± 12.9 | |
Neurological Outcome
Rabbits in the sham-operated group did not have any neurological deficit. However, all rabbits in the control group showed severe neurological deficits, including total paraplegia in five (Table II). According to Johnson's scale, neurological status of the CsA-treated rabbits at postoperative hours 24 and 48 was better compared to controls (p < 0.01, Table II).
Table II. Neurological outcome of rabbits according to Johnson's scale
| Sham (n = 7) | Control (n = 7) | CsA (n = 8) | ||||
|---|---|---|---|---|---|---|
| Johnson's score | 24 hr | 48 hr | 24 hr | 48 hr | 24 hr* | 48 hr* |
| 0 | 5/7 | 5/7 | 3/8 | |||
| 1 | 2/7 | 1/7 | 1/8 | 4/8 | ||
| 2 | 1/7 | 3/8 | 1/8 | |||
| 3 | 1/8 | 2/8 | ||||
| 4 | 1/8 | |||||
| 5 | 7/7 | 7/7 | ||||
* p < 0.01 compared to control, cumulatively. |
Histopathologic Examination
Sham-operated animals had spinal cords with a high viability index. Exclusive ischemic damage was observed in the ventral horns of spinal cords harvested from animals in the control group, which was consistent with the low viability index. All animals in the CsA group had spinal cords with mild neuronal damage and better viability index values compared to controls (p < 0.01, Fig. 1, Fig. 2).
Fig. 1.
Comparison of cytoarchitecture of spinal cords expressed by viability index values. Higher viability index indicates lesser neuronal damage. Bar = mean, error bar = mean ± SD). *p < 0.01 for CsA versus control and for CsA versus sham.
Fig. 2.
Representive photographs of spinal cord sections with hematoxylin and eosin. Neuronal cells are well preserved in the sham operation group (A), necrotic neurons in the control group (B), grossly normal neurons in the CsA-treated group (C). Arrows, normal motor neuron; arrowhead, necrotic neuron.
The mean grading score of inflammatory response in CsA animals was close to that in the sham group and significantly less than in controls (Fig. 3).
Fig. 3.
Grading of inflammatory score in the histopathological examination of spinal cords. Lower grade indicates lesser inflammatory response. *p < 0.01 for CsA versus controls.
Serum NSE levels
There was a significant rise in postischemia serum NSE levels in all control animals compared to CsA-treated animals (p < 0.01). However, NSE levels were similar when CsA-treated animals were compared to sham-operated animals (Table III).
Table III. Comparison of changes in biochemical markers
| Sham (n = 7) | Control (n = 7) | CsA (n = 8) | ||
|---|---|---|---|---|
| NSE (μg/L) | ||||
| Preischemia | 5.341 ± 1.16 | 5.42 ± 1.08 | 5.39 ± 1.65 | |
| Postischemia | 4.99 ± 1.33 | 7.87 ± 2.16 | 5.09 ± 1.70* | |
| NO (μM/L) | ||||
| Preischemia | 33.45 ± 5.91 | 31.68 ± 11.18 | 30.48 ± 10.13 | |
| Postischemia | 33.48 ± 6.30 | 47.88 ± 16.40 | 32.14 ± 9.27* | |
* p < 0.01 for CsA versus control. |
Serum NO (nitrite + nitrate)
The mean postischemia values of serum NO (nitrite + nitrate) were significantly lower in CsA-treated animals compared to controls (p < 0.01). NO (nitrite + nitrate) levels in CsA-treated animals did not show any difference from those in sham-operated animals (Table III).
DISCUSSION
Neuronal injury due to spinal cord ischemia and reperfusion is believed to result from several interrelated processes, including free radical generation and lipid peroxidation, increased intracellular calcium, NO overproduction, and inflammation.3 Free radical production was reported to cause mitochondrial dysfunction leading to rapid loss of adenosine triphosphate stores and eventually cell death.11 CsA, an immunosuppressive compound acting via cyclophilin and inhibition of the phosphatase calcineurin, is directly linked to the maintenance of mitochondrial homeostasis, which is believed to act by inhibiting mitochondrial permeability transition.12
In the present study, CsA was found to be effective at diminishing the extent of neuronal damage as evidenced by the findings of histopathological examination of spinal cords revealing higher viability index values in CsA-treated animals (p < 0.01, Fig. 1). This was also supported by the fact that blood NSE levels were significantly lower in CsA-treated animals compared to controls which underwent aortic occlusion without any pharmacological measure of spinal cord protection (p < 0.01, Table III). NSE is a glycolytic enzyme that is found mainly in the cytoplasm of neurons and cells of neuroendocrine origin. Clinical as well as experimental studies have provided evidence of increased levels of serum NSE titers in focal and global cerebral or spinal cord ischemia.13, 14, 15
The efficacy of CsA was reported to be dependent on its dose of administration and its ability to freely cross the intact blood-brain barrier (BBB). Hypoxia induces permeability in porcine brain microvascular endothelial cells via vascular endothelial growth factor and NO.16 The BBB is breached immediately following injury17 and appears to fluctuate in a dynamic fashion thereafter. The optimal dose and timing of CsA is of critical value since in high doses CsA itself may cause neuronal damage. In the present study, the 20 mg/(kg · hr) dose was chosen to be the most effective at preserving neurons against injury.18 We administered the drug peripherally during ischemia and early after ischemia, when the BBB is believed to develop openings, a unique opportunity to administer drugs that are normally BBB-impermeant before neurons are damaged.
The findings of our study were consistent with the findings of recent reports which used an experimental model of ischemia-reperfusion in rabbits to study CsA for its neuroprotective effect. Tachibana et al.19 showed that chronic CsA administration for 9 days in the preischemic period had a protective effect on spinal cord from ischemia-reperfusion injury. CsA administration in the postischemic period was also shown to decrease delayed motor neuron injury following spinal cord ischemia.20 Our study differs in terms of timing and dosing of CsA in that CsA was administered not only during ischemia but also in the early period of reperfusion. Furthermore, to the best of our knowledge, this study was the first to assess the clinical outcome and changes in the cytoarchitecture as well as biochemical markers in the same setting of experimental spinal cord transient ischemia in order to evaluate the neuroprotective effect of CsA.
For the purposes of immunosuppression, CsA has been given to patients for long periods of time, expressed in months or years. However, spinal cord ischemia in the clinical setting of thoracoabdominal aortic surgery, which necessitates aortic cross-clamping that causes diminution of the blood supply to the spinal cord, requires that immediate measures be taken in order to prevent spinal cord ischemic injury. It has been suggested in an in vitro study on the isolated spinal cord injury model that CsA is effective at reducing hypoxic spinal cord injury.21 Although our study did not directly measure the effect of CsA on the spinal cord, it represented a model for in vivo application of CsA during and immediately after spinal cord ischemia caused by aortic cross-clamping.
A recent study on the ischemic rabbit spinal cord showed that spinal neurons in the ventral horn respond to ischemic injury with increased NO production.22 Our finding in the present study that postischemia NO levels were highest in the control group where no pharmacological protection was used during spinal cord ischemia is also consistent with these findings. In our study, this increase in NO levels may originate from any tissue, neuronal or endothelial, affected by aortic occlusion; it may be both. Due to the setting of the experimental model, it is not possible to differentiate the origin of this increased synthesis of NO. However, what is clear is that CsA somehow inhibited this stimulation on NO synthesis during ischemia. The role of NO in the pathophysiology of spinal cord injury is not clear. It is appealing to speculate on the possible altering mechanisms of the Ca2+-dependent neuronal and endothelial NO synthase activity by the phosphatase calcineurin inhibitor CsA.23 It is also a matter of speculation as to the impact of this inhibition of NO synthesis on lesser neuronal damage observed in this study as evidenced by the relatively good structural integrity observed in spinal cord tissue of animals treated with CsA, as well as better neurological outcome.
In conclusion, this study provides clear evidence that CsA, when administered during ischemia and in the early period of reperfusion, may reduce neuronal damage in the spinal cord in a rabbit model of transient spinal cord ischemia.
This work was supported in part by Adnan Menderes University Foundation.
REFERENCES
- Experience with 1509 patients undergoing thoracoabdominal aortic operations . J Vasc Surg . 1993;17:357–370
- Spinal cord protection during surgical procedures on the descending thoracic and thoracoabdominal aorta: review of current techniques . Chest . 1996;109:799–809
- Pharmacologic interventions for prevention of spinal cord injury caused by aortic crossclamping . J Thorac Cardiovasc Surg . 1992;104:256–261
- Prevention of spinal cord ischaemia during descending thoracic and thoracoabdominal aortic surgery . Eur J Cardiothorac Surg . 2001;19:203–213
- Successful treatment of pure red cell aplasia in systemic lupus erythematosus with cyclosporin A . Clin Exp Rheumatol . 2003;21:759–762
- . Cyclosporine, FK-506 and other drugs in organ transplantation . Curr Opin Immunol . 1991;3:748–751
- . Cyclosporine heart transplantation . Transplant Proc . 1998;30:1881–1884
- . Molecular mechanisms of neuroprotective action of immunosuppressants - facts and hypotheses . J Cell Mol Med . 2004;8:45–58
- . Semi-automated measurement of nitrate in biological fluids . Clin Chem . 1998;44:679–681
- . Effects of flunarizine on neurological recovery and spinal cord blood flow in experimental spinal cord ischemia in rabbits . Stroke . 1993;24:1547–1553
- Oxygen and glucose deprivation induces mitochondrial dysfunction and oxidative stress in neurones but not in astrocytes in primary culture . J Neurochem . 2002;81:207–217
- Powerful cyclosporin inhibition of calcium-induced permeability transition in brain mitochondria . Brain Res . 2003;960:99–111
- S-100 protein and neuron-specific enolase in cerebrospinal fluid and serum: markers of cell damage in human central nervous system . Stroke . 1987;18:911–918
- Serum neurone specific enolase (NSE) levels as an indicator of neuronal damage in patients with cerebral infarction . Eur J Clin Invest . 1991;21:497–500
- Serum biomarkers for experimental acute spinal cord injury: rapid elevation of neuron-specific enolase and S-100beta . Neurosurgery . 2005;56:391–397
- Hypoxia induces permeability in brain microvessel endothelial cells via VEGF and NO . Am J Physiol . 1999;276:812–820
- Peripheral markers of brain damage and blood-brain barrier dysfunction . Restor Neurol Neurosci . 2003;21:109–121
- Dose-response curve and optimal dosing regimen of cyclosporin A after traumatic brain injury in rats . Neuroscience . 2000;101:289–295
- Immunophilin ligands FK506 and cyclosporine A improve neurologic and histopathologic outcome after transient spinal cord ischemia in rabbits . J Thorac Cardiovasc Surg . 2005;129:123–128
- Cyclosporin A reduces delayed motor neuron death after spinal cord ischemia in rabbits . Ann Thorac Surg . 2003;75:1294–1299
- . Role of calcineurin in calcium-mediated hypoxic injury to white matter . Spine J . 2003;3:11–18
- Regulation and localization of neuronal nitric oxide synthase in the ischemic rabbit spinal cord . Mol Cells . 2003;15:406–411
- Cyclosporin A inhibits constitutive nitric oxide synthase activity and neuronal and endothelial nitric oxide synthase expressions after spinal cord injury in rats . Neurochem Res . 2005;30:245–251
This study was presented at the Fourth European Association for Cardio-thoracic Surgery/The European Society of Thoracic Surgeons Joint Meeting, Barcelona, Spain, September 24–28, 2005.
PII: S0890-5096(06)60038-1
doi:10.1007/s10016-006-9022-2
© 2006 Annals of Vascular Surgery, Inc. Published by Elsevier Inc All rights reserved.
