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Ann Thorac Surg 2003;75:1294-1299
© 2003 The Society of Thoracic Surgeons

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Original article: cardiovascular

Cyclosporin A reduces delayed motor neuron death after spinal cord ischemia in rabbits

^a Department of Anesthesiology, Sendai, Japan
^b Department of Cardiovascular Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan

Accepted for publication November 1, 2002.

^* Address reprint requests to Dr Sakurai, Department of Cardiovascular Surgery, Tohoku University Graduate School of Medicine, 1-1, Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan
e-mail: sakuraim{at}mail.cc.tohoku.ac.jp

	Abstract

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BACKGROUND: Spinal cord ischemia has varied etiologies, and in some cases, may develop into paraplegia. This is attributable to the vulnerability of spinal motor neurons to ischemia. We evaluated the potential of the immunosuppressant cyclosporin A for treatment of spinal motor neuron damage caused by ischemia.

METHODS: Twenty-eight rabbits were randomized into four groups of 7 animals each: group A (cyclosporin A not administered), group B (2.5 mg/kg cyclosporin A), group C (25 mg/kg cyclosporin A), and group S (sham-operated). The spinal cord ischemia model was created by a 15-minute occlusion of the aorta just caudal to a renal artery with a balloon catheter. Administration of cyclosporin A began 30 minutes after restoration of blood flow. The spinal cords were removed after 7-day monitoring of neurologic function. Pathology specimens were prepared, and after staining them with hematoxylin-eosin, viable motor neurons in the ventral spinal cord were counted under light microscopy.

RESULTS: At 7 days after reperfusion, recovery of motor function was seen at varying degrees in groups B and C, whereas all animals in group A continued to exhibit paraplegia. In group C, most of the animals recovered to the baseline level, before creation of the ischemia model. A significant difference in numbers of viable neurons was found in group A (cell count, 10.1 ± 4.7) and group C (cell count, 22.2 ± 8.0) (p < 0.05). Higher numbers of viable motor neurons corresponded to a greater recovery of motor function.

CONCLUSIONS: These results suggest that cyclosporin A administration is effective against neuronal damage caused by spinal cord ischemia.

	Introduction

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There are various reports noting the incidence of paraplegia after operation on the thoracic or abdominal aorta [1,2], and it is thought to be caused by spinal cord infarction from the surgical procedure. Spinal cord infarction, which causes paraplegia, is thought to result from a lowering or blockage of blood flow from the intercostal arteries to the spinal cord. However, Kats and associates [3] reported that even patients who do not exhibit motor neuron damage immediately after replacement of the thoracic aorta with an artificial vascular graft may develop delayed damage with paraplegia in some cases. However, the mechanisms have not been fully elucidated. There have been a number of reports on methods of treatment or prevention of ischemic spinal cord injury [4–6], but to date, there is no scientifically established method. We have previously reported that hyperbaric oxygenation performed at an early period after ischemic injury reduces delayed motor neuron death caused by spinal cord ischemia [6]. In addition, in recent years, the neuroprotective effect of cyclosporin A, widely used as an immunosuppressant in clinical practice, has received attention and has been studied in the area of basic medicine [7, 8]. Cyclosporin A inhibits the activity of calcineurin, a protein phosphatase believed to be involved in cell death, by binding to it by intracellular cyclophilin D.

The purpose of this study is to ascertain the appropriate dosage for and investigate the effectiveness of cyclosporin A administration on motor neuron death after transient spinal cord ischemia.

	Material and methods

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During the experiment, animals were cared for in compliance with the "Principles of Laboratory Animal Care" formulated by the National Institutes of Health (National Institutes of Health publication no. 96 to 23, revised, 1996). The experiment and animal care protocol was approved by the Animal Care Committee of Tohoku University School of Medicine. Twenty-eight Japanese white rabbits weighing 2 to 3 kg were used in this study. They were randomly divided into four subgroups of 7 animals each. The animals in group S were catheterized without inflation of the balloon (sham-control group). In groups A, B, and C, spinal cord ischemia was induced by inflation of the catheter balloon. A reproducible model for spinal cord ischemia was created by modifying the method of Zivin and associates [9], following the procedures described below. The animals were anesthetized with intramuscular administration of 50 mg/kg ketamine. Anesthesia was maintained with 1% to 2% halothane inhalation through a mask under spontaneous respiration, without endotracheal intubation.

After a sufficient depth of anesthesia was obtained, a 5F pediatric pulmonary artery balloon catheter (Baxter International Inc, Deerfield, IL) was inserted through the femoral artery. The tip of the catheter was advanced 15 cm cranially, and led to the abdominal aorta. We confirmed thruogh preliminary investigations by laparotomy that the balloon in the distal end of the catheter was positioned 0.5 to 1.5 cm just distal to the left renal artery. Additional catheters were inserted in the auricular artery and vein, and in the femoral artery contralateral to the balloon catheter insertion site. Preparations were made for continuous monitoring of aortic pressure and heart rate, and collection of arterial blood. Pharyngeal and rectal temperatures were monitored through temperature sensors, which were inserted in the pharynx and rectum. Rectal body temperature was maintained at more than 37°C with the aid of a heating pad during the study.

The balloons were inflated until the pressure in the femoral artery contralateral to the balloon catheter insertion site became near 0 mmHg, ensuring that ischemia had been induced. After 15 minutes of transient ischemia, the balloons were deflated, allowing reperfusion to the spinal cord. Administration of physiologic saline or cyclosporin A was initiated after 30 minutes of reperfusion.

The animals in group A were administered 20 mL of physiologic saline alone, and the animals in groups B and C were administered 20 mL of cyclosporin A solution in physiologic saline by 1-hour intravenous infusion to the auricular vein. The cyclosporin A dosages were 2.5 mg/kg for group B and 25 mg/kg for group C.

After administration was completed, the catheters were removed. The wounds were treated carefully, the inhalation anesthetic was stopped, and the animals recovered from anesthesia.

The experiment and measurements were begun 10 minutes after respiration and circulation were stabilized. Heart rate (HR), femoral artery pressure, auricular artery pressure, pharyngeal temperature, and rectal temperature were measured. Arterial blood was collected, and arterial blood gas analysis (pH, PO₂, PCO₂, hematocrit, and blood glucose) and measurement of blood urea nitrogen (BUN) and creatinine (Cr) were conducted. Measurement was performed immediately before inflation of the balloon (before ischemia), 10 minutes after spinal cord ischemia was induced by inflation of the balloon (during ischemia), and 10 minutes after reperfusion was initiated by deflation of the balloon. In the sham-control group, measurement was performed using the same time schedule as for the ischemia groups, without the balloon inflated. BUN and Cr were measured using blood collected before ischemia and at 7 days after reperfusion.

Neurologic function was monitored and evaluated for all animals for 7 days after reperfusion. The animals were euthanized by intravenous administration of a high concentration of pentobarbital on the 7th day, and the spinal cords were quickly removed. For groups A, B, and C, blood was collected upon removal of the spinal cords, and BUN and Cr were measured. The spinal cords were immersed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer and stored at 4°C for 2 weeks. The specimens for microscopy were prepared by obtaining spinal cord cross sections from the L2 or L3 vertebra. The specimens were then embedded in paraffin, cut into sections of 5 µm thickness, and stained with hematoxylin-eosin (H&E).

Neurologic evaluation
Neurologic evaluation was performed at 2 and 7 days after reperfusion according to Johnson’s score [10] (grade 0, hind limb paralysis; grade 1, severe paraparesis; grade 2, functional movement, no hop; grade 3, ataxia, disconjugate hop; grade 4, minimal ataxia; grade 5, normal function). Evaluation was performed by 2 observers unaware of the protocol.

Histopathological evaluation
The neurons present in the anterior horn of the spinal cord in each slide of H&E-stained specimens were counted using a light microscope. With H&E staining, the cells were considered "died" if the cytoplasm was diffusely eosinophilic and "viable" if the cells demonstrated basophilic stippling (that is, if they contained Nissle substance). Measurement was performed at the ventral side of a line drawn through the central canal of the anterior horn. To avoid bias due to measurement of a single area, cells in specimens created from three different sites of the lumbar vertebra of each animal were counted by 2 observers unaware of the protocol, and the mean was used as the representative value.

Statistical analysis
Unless specified, all values are given as mean ± standard deviation. The results were analyzed by t test, Mann Whitney U test, and one-way analysis of variance, with a significance level of 5%.

	Results

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Changes in hemodynamics and body temperature are shown in Table 1, and changes in the arterial blood gas analysis and blood glucose are shown in Table 2. When the catheter balloon was inflated, the femoral artery pressure both ipsilateral and contralateral to the balloon insertion site was decreased to approximately 15 mm Hg, and conversely, the auricular artery pressure increased. When the balloon was deflated after 15 minutes, the femoral artery pressure began to increase, with the pressure gradually recovering to baseline level (before ischemia). Heart rate decreased as the balloon was deflated, and remained lowered with little change after reperfusion. Body temperature decreased after ischemia in comparison with before ischemia in groups A and C, and remained lowered after reperfusion, but no animals exhibited a body temperature less than 37°C during the monitoring. In groups B and C, arterial blood pH decreased due to ischemia and continued to decrease with progression of acidosis after restoration of blood flow; whereas in group A, no changes were observed in pH over time. No changes in PaO₂ were found after reperfusion in groups B and C, whereas a decrease was found in group A. Decreases were found in PaCO₂ after reperfusion in groups A, B, and C. A significant increase was found in blood glucose after restoration of blood flow in groups A, B, and C, with higher values in comparison with group S. A significant increase in BUN was found between before ischemia and at 7 days after reperfusion in group C only, but the difference is minor (preischemia, 21.4 ± 3.9; 7 days after ischemia, 24.2 ± 2.4). No significant change in Cr was found in any of the three groups.

Histopathology study
The motor neuron count in the anterior horn of the spinal cord is shown in Table 4. At 7 days after reperfusion, in comparison with group S, significant decreases in cell count were found in groups A and B, but no significant difference was found in group C. In addition, the cell count was significantly higher in group C in comparison with group A. A representative photograph of an H&E-stained spinal cord specimen is shown in Figure 1. In group A, increases in the number of glial cells were observed around the anterior horn, but few were observed in groups C and S. No neuronal damage was found in the posterior horn of the spinal cord in all three groups. These histopathological findings were consistent with neurologic function observations.

View larger version (160K): [in this window] [in a new window]   Fig. 1. Representative photographs of spinal cord sections stained with hematoxylin & eosin. There was no neuronal damage to any motor neuron cells in groups S (panel S) and C (panel C), whereas in groups A (panel A) and B (panel B), almost all motor neuron cells in ventral gray matter were lost (original magnification, x200).

	Comment

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We have previously demonstrated delayed and selective motor neuron death in the lumbar region of the rabbit spinal cord with this same reproducible model [11]. Although the 15-minute ischemia model has a relatively short ischemic period in comparison with other reported models [10], it allows observation of delayed and selective motor neuron damage at 7 days after reperfusion. This phenomenon is known as selective motor neuron death after transient spinal cord ischemia [11, 12], and is similar to the delayed and selective neuronal death seen in hippocampal CA1 cells after cerebral ischemia [13]. Despite restoration of blood flow [12], motor neurons that appear to have survived in the early period after ischemic injury will die a few days later. This suggests that motor neurons are vulnerable to ischemia.

Recent studies have suggested that delayed neuronal death after transient ischemic injury in rat and gerbil brains displays some apoptotic features [15]. Apoptosis is defined as programed cell death, regulated by genes and distinguished from accidental cell death [16]. Apoptosis is characterized by condensation of the cytoplasm and internucleosomal DNA fragmentation. Cyclosporin A induces conformational changes to the catalytic site of the A subunit of calcineurin by binding to the B subunit of calcineurin by cyclophilin, an immunophilin, thereby inhibiting the dephosphorylation activity of calcineurin.

In T-cells, calcineurin exerts an immunosuppressive effect through the inhibition of transcription and activation of nuclear factor of activated T-cell (NFAT) [17]. Antiinflammatory effects of cyclosporin A are different from the immunosuppressive ones, and Gripepp and associates [18] reported that it might be implicated in the neuroprotection afforded by cyclosporin A after hypothermic circulatory arrest. Calcineurin has other, diverse functions as well, and its neuronal death–related target substrates include neuronal nitric oxide synthase (nNOS) [19] and pro-apoptotic protein Bcl-xi/Bcl-2 associated death promoter (BAD) [20]. It has been reported that dephosphorylated (activated) nNOS induces the overproduction of nitric oxide (NO), which reacts with oxygen radicals to produce peroxynitrate, and directly inhibits mitochondrial functions, thereby inducing cell death [21]. In addition, BAD is dephosphorylated by calcineurin, forming a heterodimer with Bcl-xL, which is anchored on the mitochondrial membrane, and maintaining patency of MPT pores, thereby allowing the release of mitochondrial proteins into the cytoplasm [22]. Cytochrome C is an enzyme that is normally found between the inner and outer membranes of the mitochondria, and is involved in ATP production in the electron transport system. However, once released into the cytoplasm, it activates caspase-9 with Apaf and other compounds, serving as a key protein in the process of cell death [23]. Sasaki and associates [24] have reported the release of cytochrome C into cytoplasm, and subsequent caspase-3 and TUNEL-positive neurons beginning 3 hours after occlusion in the penumbra region in a rat model of permanent middle cerebral artery occlusion, suggesting a strong possibility that the cytochrome C–mediated pathway plays an important role in neuronal death resulting from ischemia. In this study, the neurologic prognosis and histologic findings markedly improved after 7 days in the cyclosporin A administration group, suggesting that a similar mechanism plays a central role in delayed motor neuron death in the anterior horn of the spinal cord after reperfusion, and was efficiently inhibited by cyclosporin A.

There are several reports concerning the effectiveness of cyclosporin A administration in neuronal ischemia-reperfusion models [25], but many of them concern studies conducted in the brain as the target organ, or studies in which cyclosporin A is administered before ischemia. In addition, cyclosporin A normally does not cross the blood-brain barrier [26], and therefore, it is difficult to deliver it to the site of ischemia. There are many unknowns concerning the blood-brain barrier of the spinal cord, but it was revealed in this study that cyclosporin A migrates to the spinal cord in a dose-dependent manner, because cyclosporin A inhibited delayed cell death at a high dosage. And it might be associated with improvement of tissue oxygen consumption [27]. The study also revealed that administration of a single dose did not cause serious complications such as renal damage and the effectiveness of intravenous administration after ischemia. These findings provide valuable information with respect to clinical applications for treatment of paraplegia resulting from spinal cord ischemia caused by major abdominal or thoracic vascular surgery.

In conclusion, cyclosporin A administration is effective in the spinal cord ischemia-reperfusion model. However, some details, including effectiveness with ischemia of longer duration, the time window for administration after ischemia, and the optimum dosage, are still unknown, and these problems remain for future study [14].

	Acknowledgments

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This work was supported in part by a grant-in-aid for scientific research (C13671561) from the Ministry of Education, Science and Culture of Japan. We also thank Mr Shyouichi Obara for his excellent technical support.

	References

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