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Ann Thorac Surg 2000;69:475-479
© 2000 The Society of Thoracic Surgeons

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Original Articles

Ischemic preconditioning protects against paraplegia after transient aortic occlusion in the rat

^a Gill Heart Institute at the University of Kentucky College of Medicine, Lexington, Kentucky, USA

Address reprint requests to Dr Abraham, Division of Cardiovascular and Thoracic Surgery, University of Kentucky College of Medicine, 800 Rose St, MN 276, Lexington, KY 40536-0084
e-mail: vsabra{at}pop.uky.edu

	Abstract

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Background. Paraplegia can result from operations requiring transient occlusion of the thoracic aorta. A rat model of paraplegia with the characteristics of delayed paraplegia and transient ischemic dysfunction was developed to determine whether ischemic preconditioning (IPC) improved neurologic outcome.

Methods. Rats underwent balloon occlusion of the upper descending thoracic aorta. One group (2 minute IPC, n = 19) underwent 2 minutes of IPC and a second group (5 minute IPC, n = 19) had 5 minutes of IPC 48 hours before 10 minutes of occlusion. The control group (n = 31) had no IPC prior to 10 minutes of occlusion.

Results. Paraplegia occurred in 68% of the control animals (21 of 31 paraplegic: 6 delayed and 15 immediate paraplegia). Both the 2-minute IPC and 5-minute IPC groups had a decreased incidence of paraplegia when compared to controls (32%, p = 0.011 and 26%, p = 0.009, respectively).

Conclusions. A rat model of spinal cord ischemia demonstrating both delayed paraplegia and transient ischemic dysfunction was characterized. Both 2-minute and 5-minute periods of IPC were found to protect against paraplegia.

	Introduction

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Spinal cord ischemia with resultant paraplegia occurs after operations on the descending thoracic aorta. The incidence of this severe complication is unpredictable and has been reported to be as high as 35% in some series [1]. Previous investigations have centered on modifying intraoperative techniques and have included the use of shunts, hypothermia, and CSF drainage [2–4]. Many of these techniques complicate the operative procedures and cannot be used in all cases.

Researchers have used many models of paraplegia secondary to aortic ischemia [5, 6]. Small animal models are particularly attractive for use in rapid screening of neuroprotective strategies. Several rat models of ischemia have been developed with variable neurologic results [7–9]. In the present study, a rat model of paraplegia was developed that did not result in such profound cord ischemia as to be impervious to protective strategies. Although the majority of animals were immediately paraplegic, many animals in this model demonstrated the clinical findings of delayed paraplegia or transient ischemic dysfunction. The production of transient ischemia that does not result in physiologic deficits, before a longer lethal period of ischemia, is termed ischemic preconditioning (IPC). The protective effects of ischemic preconditioning have been established in the heart [10] and the brain [11]. In the second part of the present study, the above-described rodent model was used to show that ischemic preconditioning of the spinal cord was neuroprotective and abolished delayed paraplegia.

	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 (NIH publication 85-23, revised 1985).

Retired breeder female Sprague-Dawley rats, approximately 250 to 400 grams (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). A 2 French Fogarty balloon catheter was introduced through the right femoral artery to a distance of 12 cm from the insertion site. Inflating the balloon with 0.2 cc of air produced upper thoracic aortic occlusion. The tail artery was cannulated with PE50 tubing to monitor distal aortic pressure in the paraplegia experiments to ensure complete aortic occlusion. For the short preconditioning occlusions, a Doppler ultrasonic probe (Parks Medical Electronics, Inc, Shaw Aloha, OR; model 810-A) for detection of the abdominal aortic flow signal verified aortic occlusion.

Temperature was measured with a rectal probe. The animals were kept at 37.0°C ± 5°C during the surgical preparation and ischemic period and housed in the laboratory postoperatively with an ambient temperature of approximately 21°C. Postoperative care of the paraplegic animals was conducted by established methods of good clinical care. The animals were euthanized after 8 days, or earlier if they demonstrated autophagia, by induction of general anesthesia followed by decapitation.

Paraplegia model
Three different occlusion times were tested; 20 minutes (9 animals), 15 minutes (11 animals), and 10 minutes (10 animals). These experiments were performed sequentially. When the 10-minute occlusion series produced paraplegia in the majority of rats, this occlusion time was selected to test the protective effects of IPC.

Neurologic evaluation
All animals underwent neurologic evaluations before both ischemic preconditioning and final ischemia, and then daily for 8 days after ischemia. 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 colleagues (Table 1) [9]. Observer variability is greater with this scoring system. The animals were also tested with the commonly used 4-point lower extremity walking score that examines gross motor function and has minimal interobserver variability. An observer blinded to treatment groups performed the neurologic scoring.

Ischemic preconditioning
Ischemic preconditioning was produced by either 2 minutes (n = 19) or 5 minutes (n = 19) of aortic occlusion. Forty-eight hours later the animals were reanesthesized and underwent 10-minute aortic occlusion as described above. The animals were randomly assigned either to the 2-minute or 5-minute IPC groups, or to the (no IPC) group (n = 31). Control experiments were done with all IPC experiments in order to assure equivalent postoperative environments for the animals.

Data analysis
Data are expressed as the mean ± standard error of the mean (SEM). Single factor analysis of variance (ANOVA) was used to determine differences in the time evolution of neurologic scores. Neurologic scoring was uncensored. Animals dying during the observation period maintained their scores of their last evaluation. Two sample t tests with assumed unequal variances were used for evaluating differences in mean scores at each time point of measurement. p-Values of less than 0.05 were considered significant.

	Results

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Model development
Development of the paraplegia model showed that 100% of the animals undergoing 15 minutes and 20 minutes of aortic occlusion had immediate paraplegia with hind limb motor scores of 0. Mortality for these periods of occlusion was 45% (5 of 11 animals undergoing 15 minutes of aortic occlusion) and 33% (3 of 9 animals undergoing 20 minutes of aortic occlusion). There was no mortality for 10 minutes of aortic occlusion and 9 of 10 animals were paraplegic.

Ischemic preconditioning
The incidence of neurologic deficit was significantly reduced in animals who had 2 minutes or 5 minutes of IPC followed by 10 minutes of aortic occlusion compared to control animals who had occlusion alone. These differences were present by grading with both the 15-point score and with the 4-point hind limb motor index. The 15-point score demonstrated a lesser neurologic deficit with 5 minutes of IPC when compared with either 2 minutes of IPC or with the control animal group (Fig 1). The 4-point hind limb motor index showed a significant difference between IPC animals and control but did not demonstrate a difference in neurologic protection between 2 and 5 minutes of IPC (Fig 2).

View larger version (20K): [in this window] [in a new window]   Fig 1. Control animals develop significantly lower neurologic scores (on a 15-point scale) following 10 minutes of aortic occlusion on day 0. The significant differences between control and ischemic preconditioned (IPC) animals (p < 0.05) are marked by an asterisk (*). At day 5, the difference in neurologic score between IPC groups is significant (++). Treatment with IPC leads to small reductions in neurologic score on days -1 and 0.

View larger version (18K): [in this window] [in a new window]   Fig 2. Control animals show reduced neurologic scores by the 4-point hind limb motor index in comparison to animals in either ischemic preconditioning (IPC) group. Significant differences between control and IPC animals (p < 0.05) are marked by an asterisk (*). There was no difference between scoring for 2-minute and 5-minute IPC groups.

Table 2 shows incidence of paraplegia subdivided into immediate and delayed (> 48 hours in onset) and the incidence of transient ischemic dysfunction. The incidence of transient ischemic dysfunction is not different between 2 minute and 5 minute IPC and control groups (p = 0.53). None of the IPC-treated animals in either group developed delayed paraplegia. In the control group, the incidence of paraplegia was 68% (21 of 31) with 19% (6 of 31) of the control animals demonstrating a delay in onset of greater than 48 hours (p < 0.05 control versus combined IPC groups). Delayed paraplegia accounts for most of the difference between treated and control groups. Although there was a trend towards reduced immediate paraplegia (49% versus 32% and 26%, respectively) it did not reach statistical significance.

	Comment

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The spinal ischemia model was developed to produce a less than 100% incidence of paraplegia, as is found in the clinical situation. By using the shortest duration of occlusion that produced paraplegia in the majority of animals, we created a degree of ischemia responsive to neuroprotective strategies. In this model, both neuroprotective and neurodestructive interventions can be demonstrated. The variability in the degree of neurologic injury, the occasional finding of a delay in paraplegia following ischemia, and the occurrence of transient ischemic dysfunction suggests that this experimental model possesses many of the sequelae found clinically. In addition, a rodent model permits screening larger numbers of neuroprotective strategies and facilitates the use of molecular biologic techniques to delineate the mechanisms of interventions.

This model has several limitations. Species differences in spinal cord sensitivity to ischemia, variability in the blood supply of the cord, and variability in the molecular mechanisms mediating neurologic injury may affect the application of these data to humans. Spinal cord blood supply in rats is, however, similar to humans. Rats do have significantly higher metabolic rates and require less ischemic time to produce neurologic injury. The similarities between the experimental model and clinical findings suggest that the model may indeed replicate the injury patterns seen in humans. Using a limited duration of ischemia to create a less than 100% incidence of paraplegia should allow the demonstration of protective and harmful interventions.

Ischemic preconditioning
Ischemic preconditioning is defined as a brief nonlethal period of ischemia before a lethal ischemic event. Several investigators have studied the protection of tissues sensitive to ischemia by preconditioning and IPC has been found to be protective in the heart and brain. The time course of IPC differs between different organ systems. In the heart, ischemic tolerance starts within 1 hour of preconditioning and lasts less than 3 hours, with a "second window" of tolerance at 24 hours [13]. The adenosine A1 receptor which has been implicated in mediating IPC effects in the heart is also thought to play a role in ischemic tolerance [14].

The protective effect of IPC has a different temporal onset for brain tissue, with a prolonged interval between initial ischemia and protective tolerance when compared to the heart. The minimal interval for production of tolerance in the brain is 24 hours and lasts for a poorly defined period beyond 48 hours [15]. The present study used a 48-hour interval because of the previous studies in the brain. In the present study, the 2-minute initial ischemic episode was chosen because this time has been found to be effective for brain IPC [16]. A longer period (5 minutes) also was tested in a successful attempt to augment protection.

The mechanism by which IPC protects brain tissues has not been delineated. The mechanism of protection may involve a stress response with the induction of heat shock protein (HSP70) [17] and other immediate-early gene products [18]. HSP70 is considered a "molecular chaperone" and maintains the tertiary configuration of proteins and may allow denatured proteins to regain their conformation, thereby preventing degradation. While these studies and others suggest that the generalized stress response is associated with the production of tolerance to ischemia, the exact mechanism of IPC in the brain continues to remain elusive.

Two previous studies have suggested that IPC is protective during spinal ischemia. In one study, IPC was produced in 6 dogs by 20 minutes of preconditioning, followed 48 hours later by 60 minutes of ischemia [6]. The animals were followed for only 24 hours after ischemia and the single neurologic evaluation showed 5 of 6 animals were normal. The control group had 3 of 6 paraplegic animals. In this study, all nonparaplegic animals in both groups demonstrated immunoreactivity to HSP70. Paraplegic animals consisting exclusively of animals from the control group failed to show heat shock protein (HSP) reactivity suggesting that HSPs are involved in tolerance from ischemia. In a rabbit study, Sakurai and associates showed that IPC is protective, and that both HSP70 mRNA and protein are induced following the initial sublethal ischemic insult [19]. In their study, 10 minutes of IPC was shown to protect from a subsequent 15-minute ischemic episode occurring 48 hours later. However, their study used a single neurologic examination at 7 days following injury and did not follow the development of neurologic injury and recovery.

Many operations associated with spinal ischemia such as repair of thoracoabdominal aneurysms are elective and may allow the opportunity for preoperative neuroprotective interventions, such as IPC. Our study has shown the protective effect of IPC using sensitive neurologic testing and suggests that the duration of preconditioning ischemia can affect the degree of neuroprotection. In addition, we show for the first time that IPC reduces ischemic injury by eliminating delayed neurologic injury. There was a trend towards reduction of immediate injury that did not reach statistical significance. The reduction in delayed paraplegia is surprising and may suggest a role for IPC in the protection from apoptosis after reversible spinal ischemia. A previous study has shown that loss of large motor neurons following transient ischemia is primarily because of apoptosis and not neuronal necrosis [20].

This study of spinal cord ischemia, demonstrating protection from paraplegia by IPC, is a first step in understanding the mechanism of neurologic injury and its attenuation by IPC. Ongoing studies in our laboratory are evaluating the histologic changes in the spinal cord after IPC and measuring the changes in mRNA and protein production of immediate-early genes in these animals. The goal of these studies is to be able to produce pharmacologic preconditioning of the spinal cord without an initiating ischemic insult, thereby increasing the opportunity for neuroprotection prior to operation.

In conclusion, a rat model of spinal ischemia was developed that demonstrates the variable neurologic course seen clinically after aortic operations. In this model, the prolonged neuroprotective effects of a short period of ischemic preconditioning performed 2 days before prolonged aortic occlusion was shown. These studies demonstrate that ischemic preconditioning ameliorates delayed neurologic injury but could not show a reduction in the rate of immediate paraplegia in our model. Further studies using this model will delineate the mechanism of IPC neuroprotection.

	Acknowledgments

 
The technical assistance of Katie Kraft and Mark Clements and the administrative assistance of Angela Fielder is appreciated. This study was supported by a regional 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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