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Ann Thorac Surg 2000;70:1490-1495
© 2000 The Society of Thoracic Surgeons

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Original articles: cardiovascular

Hyperthermic preconditioning protects against spinal cord ischemic injury

^a Gill Heart Institute and Sanders-Brown Center on Aging, University of Kentucky College of Medicine, Lexington, Kentucky, USA

Address reprint requests to Dr Swain, National Aeronautics and Space Administration, Headquarters, Code U, 300 E St SW, Washington, DC 20536-0001
e-mail: Jswain{at}hq.nasa.gov

	Abstract

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Background. Paraplegia can result from operations requiring transient occlusion of the descending thoracic aorta. The present study tested whether inducing hyperthermia in rats before aortic ischemia would be neuroprotective.

Methods. Rats were randomly assigned to hyperthermic preconditioning (n = 27) or control (n = 32) groups. Eighteen hours before ischemia, the hyperthermic preconditioned rats were heated at 41°C for 15 minutes. Ten minutes of spinal ischemia were produced by balloon occlusion of the thoracic aorta. Neurologic performance scores were evaluated daily to 7 days after ischemia. The lumbar region of the spinal cord was removed for histologic grading.

Results. The hyperthermic preconditioned animals had less permanent spinal cord injury compared with controls (29.6% versus 59.4%, p = 0.02), and the incidence of immediate paraplegia in the hyperthermic preconditioned group was significantly less than that in the control group (3.7% versus 28.1%, p = 0.03). Histologic scores correlated with the neurologic outcome at the time of sacrifice in rats with permanent spinal cord injury but not in those walking normally.

Conclusions. We used a rat model of spinal cord ischemia and found that hyperthermic preconditioning before spinal cord ischemia resulted in improved clinical outcome.

	Introduction

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One of the most serious complications of operations on the descending aorta is paraplegia, with an incidence as high as 35% [1]. Paraplegia is caused by perioperative spinal cord ischemia and can be aggravated by reperfusion, leading to spinal neuronal cell necrosis and apoptosis [2]. The occurrence of paraplegia is unpredictable. Several strategies, such as the use of shunts, hypothermia, cerebral spinal fluid drainage, and ischemic preconditioning before spinal cord ischemia, have been developed to decrease the incidence of paraplegia after spinal cord ischemia [3–5]. Many of these techniques complicate the operative procedures and cannot be used in all cases.

It has been reported that whole-body hyperthermia can induce synthesis of heat shock proteins (HSPs), which have been shown to provide protection against subsequent stress conditions in the liver, small intestine, heart, forebrain, hippocampus, and kidney [6, 7]. We hypothesized that whole-body hyperthermia might also protect against spinal cord ischemic injury.

Previously we developed [8] a model of spinal cord ischemia secondary to thoracic aortic occlusion, which showed the spectrum of clinical findings after aortic operations, including immediate paraplegia, delayed paraplegia, partial injuries, and no injury. We showed that ischemic preconditioning afforded spinal cord neuroprotection [8]. In the present study, the rat model of paraplegia was used to test the hypothesis that whole-body hyperthermia before transient spinal cord ischemia would be neuroprotective in rats.

	Material and methods

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Animal preparation
The animal protocol was approved by the Institutional Animal Care and Use Committee and was in accordance with the "Guide for the Care and Use of Laboratory Animals," published by the National Institutes of Health (National Institutes of Health publication no. 85-23, revised 1985).

Retired breeder female Sprague-Dawley rats, weighing 250 to 325 g (Harlan, Madison, WI, colony #205) were allowed free access to food and water before and after the procedures. General anesthesia was induced by intraperitoneal administration of a mixture of ketamine (45 mg/kg), acepromazine (0.75 mg/kg), and xylazine (2.5 mg/kg). Spinal cord ischemia was produced by introducing a 2F Fogarty balloon catheter (Baxter Healthcare Corp, Irvine, CA) through the left femoral artery approximately 12 cm to the proximal descending thoracic aorta. This distance had been measured as the distance to the left subclavian artery. The tail artery was cannulated with PE50 tubing to monitor distal aortic pressure in order to ensure complete aortic occlusion. Inflating the balloon with 0.2 mL of air produced upper thoracic aortic occlusion. The distal blood pressure decreased rapidly after balloon inflation from 80 to 110 mm Hg to 10 to 20 mm Hg. The rats were not heparinized. There was no evidence of thrombus on the withdrawn balloon catheter, and all distal blood pressures returned to baseline. Temperature was measured with a rectal probe and controlled by feedback to the heating lamp. The rats were kept at 37.0 ± 0.5°C during the ischemic period. Temperature was not allowed to vary during the preischemic, intraischemic, or postischemic periods.

The rats were randomly assigned to one of the following groups [1]. Control rats (n = 32) had thoracic aortic occlusion for 10 minutes followed by 7 days of recovery [2]. In the hyperthermic preconditioning group (n = 27), the rectal temperature was elevated for approximately 40 minutes to 41°C, and the rats were maintained at this temperature for 15 minutes. After that, they were removed from the heating lamp and allowed to cool to normal body temperature and awaken. Eighteen hours later, the rats were reanesthetized and underwent 10 minutes of descending thoracic aortic occlusion followed by 7 days of recovery.

In a separate study, an additional 33 rats had 10 minutes of thoracic aortic occlusion, as described above, to exclude the neuroprotective role of anesthesia. They were randomly divided into three groups according to the preconditioning types [1]. In the anesthesia preconditioning group (n = 12), rats were anesthetized by intraperitoneal administration of a mixture of ketamine (45 mg/kg), acepromazine (0.75 mg/kg), and xylazine (2.5 mg/kg) 18 to 24 hours before the thoracic aorta occlusion [2]. in the ketamine preconditioning group (n = 11), 80 mg/kg of ketamine was administered intraperitoneally 24 hours before the thoracic aortic occlusion [3], and in the control group (n = 12), there was no preconditioning before thoracic aortic occlusion.

Neurologic evaluation
All rats underwent neurologic evaluation before hyperthermic or anesthetic preconditioning and spinal ischemia and daily until 7 days after ischemia. The neurologic evaluations were done in a blinded fashion. Data were analyzed using two scoring systems. The 15-point scale is a detailed motor and sensory evaluation of lower extremity function and was modified from that developed by Lemay and associates [9] (Table 1). The rats were also tested with the commonly used four-point lower extremity walking score that examines gross motor function and has minimal interobserver and intraobserver variability [9].

Spinal cord tissue processing
Rats were anesthetized on the day 7 after ischemia. The left ventricle was cannulated and perfused with 0.1 mol/L phosphate-buffered saline (pH 7.4) followed by 4% paraformaldehyde in phosphate-buffered saline. The spinal cord (T12 to L3) was dissected immediately and postfixed in paraformaldehyde for 4 hours, then rinsed in 0.2 mol/L phosphate buffer solution overnight before cryoprotecting it in 20% sucrose phosphate-buffered saline solution at 4°C. Spinal cords were placed side by side into cryomolds containing embedding medium consisting of gum tragacanth (Sigma, Hopkinton, MA) mixed in 25% sucrose with the ventral sides and rostral ends of each cord evenly aligned. The entire mold was snap-frozen in acetone chilled to -40°C and stored at -80°C until it was sectioned on a cryostat (Microm Laborgerate, Bad Schwalbach, Germany). Starting from the rostral ends of each mold, serial 40-µm longitudinal horizontal cryosections cut from ventral to dorsal were mounted onto 10 sequential gelatin-coated slides before the next series of sections were similarly mounted, such that each slide contained sections separated by approximately 400 µm. Tissue sections were stored at -20°C until histologic analysis was done.

Histology
All slides for each animal were stained with cresyl violet, dehydrated, and cleared for coverslipping with Permount (Fisher, Pittsburgh, PA). Individual sections throughout the rostrocaudal extent of the spinal cords were viewed under an Olympus microscope to histologically assess gliosis and necrosis. Histologic score was determined using the following scale: 0 = normal, 1 = mild injury, 2 = severe injury, and 3 = necrosis. All slides were graded histologically by two independent observers in a blinded fashion.

Data analysis
Statistical analyses of 15-point and four-point scale neurologic scores were performed by Kruskal-Wallis nonparametric analyses of variance and the Mann-Whitney U test. A ² test was used to determine the differences in the incidence of each type of clinical outcome, with p values less than 0.05 considered significant. Neurologic performance scores are graphed using median values with 25th to 75th quartile values indicated.

	Results

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The rectal temperatures at the time of occlusion and throughout the occlusion period were not statistically different. Both the 15-point neurologic performance score and the four-point hind limb walking score showed smaller deficits in the hyperthermic preconditioned animals compared with the control group (p = 0.02 and p = 0.007, respectively, Fig 1). Table 2 shows the incidence of events subdivided into normal, transient ischemic defect, immediate paraplegia, delayed paraplegia, persistent ischemic defect, and unexpected death. In the control and hyperthermic preconditioned groups, the incidence of normal and transient ischemic defect was 13 of 32 (40.6%) and 19 of 27 (70.4%), respectively (p = 0.02). The incidence of permanent spinal cord injury, which includes immediate paraplegia, delayed paraplegia, persistent ischemic defect, and unexpected death, was significantly less in hyperthermic preconditioned animals than in controls (29.6% and 59.4%, respectively, p = 0.02). Only one rat (3.7%) in the hyperthermic preconditioned group had immediate paraplegia, whereas immediate paraplegia occurred in 9 rats (28.1%) in the control group (p = 0.03).

View larger version (32K): [in this window] [in a new window]   Fig 1. (A) Data from the 15-point scale of neurologic performance scores are presented as a box plot. The Mann-Whitney rank-sum test demonstrated significant differences between the two groups at all time points after spinal cord ischemia. (B) Data from the four-point scale of hind limb motor score are shown as a box plot. Mann-Whitney rank-sum test demonstrated significant differences between the two groups at all time points after spinal cord ischemia. (box area = 25th to 75th percentile; T-bar = 10th and 90th percentiles; bar = median value of group; square = mean score of group.)

View larger version (149K): [in this window] [in a new window]   Fig 2. (A) Histologic photograph of normal lumbar spinal cord (injury score = 0) in a hyperthermic preconditioned animal with normal neurologic score at the time of sacrifice. (B), Histologic photograph of lumbar spinal cord necrosis (injury score = 3) in a hyperthermic preconditioned animal with persistent ischemic defect manifested by knuckle-walking at the time of sacrifice. Calibration bar = 500 µm.

View larger version (15K): [in this window] [in a new window]   Fig 3. Histologic scores of different neurologic patterns. Animals with immediate paraplegia (P), delayed paraplegia (DP), or persistent ischemic defect (PID) had severe spinal cord injury or necrosis. The histologic scores for normal (N) and transient ischemic defect (TID) animals were not correlated with neurologic performance.

	Comment

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We developed a spinal cord ischemia model that produced a less than 100% incidence of paraplegia. The spectrum of neurologic outcomes, including no injury, immediate paraplegia, delayed paraplegia, transient ischemic defect, and persistent ischemic defect was observed during the follow-up period. In our previous study, we used this model to show that ischemic preconditioning protected the spinal cord [8]. In the present study, whole-body hyperthermia before ischemia protected against ischemic injury. There were fewer permanent spinal cord defects and the incidence of normal spinal cord function was higher with preconditioning using whole-body heat stress.

One possible neuroprotective mechanism of whole-body hyperthermic preconditioning is the induction of HSPs. Heat shock proteins are a group of stress proteins that are induced under various conditions, such as ischemia, trauma, elevated body temperature, hypothermia, neurodegenerative disease, and drug administration [10–13]. They are crucial for the maintenance of cell integrity during normal growth and development as well as during pathologic conditions [14, 15]. They correlate with the ability of neuronal cells to survive under unfavorable conditions [16, 17]. However, the mechanism of the neuroprotective action of HSP has not been well explained. Recent studies have shown that HSP70 has the function of a molecular chaperone in protein processing [14]. Under stressful conditions, HSP can denature or disrupt proteins until the abnormal proteins are refolded or degraded. That mechanism might explain the protection against ischemic neuronal injury and could have an important effect in the acquisition of ischemic tolerance [14].

Different tissues have varied levels of HSP synthesis after whole-body hyperthermia [7]. In the nervous system, some tissues show a higher expression of HSP70 mRNA and HSP synthesis after heat shock [16]. In one study, ischemic damage was not found in either the CA3 or CA4 hippocampus or the medial-ventral striatum after hyperthermic preconditioning to 41°C [11]. Another study showed that optimal neuroprotective effects of a single hyperthermic treatment were obtained when hyperthermia occurred 18 hours before forebrain ischemia [17]. This transient neuroprotective effect of hyperthermia correlated with the temporal profile of HSP production after hyperthermia. In the rat spinal cord, whole-body hyperthermia selectively induced HSP72 in the ventral horn motor neurons [18]. Because of these studies, we hypothesized that whole-body hyperthermic pretreatment would afford protection against spinal cord ischemic defect.

Ketamine acts as N-methyl-D-aspartate receptor antagonist, which can have a neuroprotective effect [19]. The present study tested preconditioning with ketamine alone or ketamine + acepromazine + xylazine. No neuroprotective effect was found, and the trend was toward greater injury in the ketamine-treated animals.

The causes of sudden death after spinal cord ischemia are not known. Most deaths occurred 30 to 48 hours after spinal cord ischemia, and some were associated with severe lower extremity pain with muscle spasm. Most of these animals were found dead in their cages on the morning of the second day, so their spinal status before death is not known. This phenomenon needs further investigation.

The dramatic protection from immediate paraplegia in the hyperthermic pretreated group demonstrates the early protection from necrosis, which correlated with improved histologic findings in animals with some degree of spinal defect. A previous study [20] found that in the rat spinal cord ischemic preconditioning model, neurologic function scores were consistent with histologic injury. However, the rats were only followed up for 2 days after spinal cord ischemia. In the present study, we followed up the animals for 7 days after spinal cord ischemia. Several animals had a decrease in score between days 2 and 5 which would be consistent with apoptosis. A few animals had improved clinical status in the later days. The histologic score appeared to be consistent with neurologic outcomes in the animals with permanent neurologic deficit; but in the animals without permanent defects, there was no obvious correlation between histologic scores and neurologic scores. Five rats with spinal cord necrosis had normal neurologic performance. More sensitive measures of neurologic performance in the rat model may be needed to detect these anatomic abnormalities.

	Acknowledgments

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This study was supported by an Ohio Valley American Heart Association grant (#9806289), by the Linda and Jack Gill Foundation, by the Cardiothoracic Research and Education Foundation, and by a University of Kentucky Institutional grant.

	References

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