Is subdiaphragmatic aortic cross-clamping a suitable model for spinal cord ischemia / reperfusion injury study in rats ?

Purpose: To evaluate the efficacy of subdiaphragmatic aortic cross-clamping in an experimental model of ischemia/ reperfusion injury of the spinal cord in albino rats. Methods: Thirty-six male Wistar rats were randomized in two groups (n=18): G-1 (Sham) and G-2 (Ischemia/Reperfusion, I/R). G-2 rats were submitted to 30 min subdiafragmatic aortic crossclamping. G-1 rats served as controls and were submitted to surgical trauma (laparotomy) without ischemia. Samples (spinal cord and arterial blood) were collected at the end of ischemic period and 10 (T-10) and 20 (T-20) min later in G-2 rats. Sham rats (G-1) samples were collected at the same time-points. Blood and tissue metabolites concentrations of pyruvate, lactate, glucose and medullary adenosine triphosphate (ATP) were assayed. Results: Blood and tissue concentrations of pyruvate and glucose as well as lactate and medullary ATP were not different when comparing G1 to G2. Lactacemia was significantly elevated in G-2 compared with G-1 rats during reperfusion (T-10). Conclusion: Subdiaphragmatic aortic cord cross-clamping is not a suitable rat model for spinal cord ischemia/reperfusion injury study as it does not ensure changes in in vivo tissue metabolites concentrations similar to those found in tissues subjected to ischemia/reperfusion.


Introduction
Temporary interruption of spinal cord blood supply may result in irreversible ischemic injury and neurological deficits.Surgical repair of some arterial diseases including thoracoabdominal aortic aneurysm is a challenging task to the surgeon's technical skills.Devastating complications may take place during the surgical procedure, leading to paraplegia, renal failure or cardiac arrest and death 1 .
Fortunately, infra-renal aortic cross-clamping is not frequently accompanied by paraplegia in humans due the blood supply provided by the greater radial artery (formerly known as Adamkiewicz artery) 2 .There is an urgent need to develop new surgical techniques and drugs aimed at reducing operating time and increasing the tolerable duration of ischemia as a priority in medical research 3 .
Experimental models of spinal cord ischemia have been developed in many animals, including dog 4 , rabbit 5 , baboon 6 , pig 7 , and rat 8 .In these models thoracic or abdominal aorta is cross-clamped, causing significant but incomplete reduction in spinal cord blood flow 9 .Schievink et al. 10 studied the arterial supply of the spinal cord of the Wistar rat and did not observe a single large artery at the thoracolumbar level supplying a large part of the spinal cord.They concluded that the equivalent of the greater radial artery is not invariably present in the rat; therefore, the vascular anatomy of the rat spinal cord is not as similar to that of man as it has previously been reported 10 .The aim of the study was to evaluate the efficacy of subdiaphragmatic aortic cross-clamping in a model of ischemia/reperfusion injury of the spinal cord in Wistar rats, as judged by changes in in vivo concentrations of metabolites in spinal cord tissue and in blood.

Methods
Thirty-six male albino Wistar (Mammalia Rodentia, Muridae, Rattus Norvegicus Albinus) rats obtained from Faculty of Medicine (Federal University of Ceará) Small Animals Laboratory weighing 300-350 g (average 330 g) were used in this study.All animals were of the same breed and were raised in controlled environment for research use only.The study was conducted in compliance with the guidelines of the Council for International Organization of Medical Sciences (CIOMS) ethical code for animal experimentation 11 and the rules on animal breeding and use in studies and research set forth by Colégio Brasileiro de Experimentação Animal (COBEA). 12Rats were subjected to a standard 12 h light-dark cycle (6:00 AM to 6:00 PM) and room temperature was maintained at 25°C.All animals were housed in polypropylene cages in groups of 5 rats and allowed free access to food and water.Rats were randomized in 2 groups (n=18).Additionally, each group was distributed in 3 subgroups (T-0, T-10 and T-20).G-2 rats were submitted to 30 min subdiafragmatic aortic crossclamping.G-1 rats served as controls and were submitted to surgical trauma (laparotomy) without ischemia.Samples (spinal cord and arterial blood) were collected at the end of ischemic period (T-0) and 10 (T-10) and 20 (T-20) min later in G-2 rats.Sham rats (G-1) samples were collected at the same time-points, starting collection of samples 30 min after laparotomy.All surgical procedures were performed under inhalatory diethyl ether anesthesia.Following sample harvesting all animals were killed under general inhalatory anesthesia.4][15] Values are expressed as mean±S.E.M. Student's t test or nonparametric Mann-Whitney U-test as appropriate were used to analyze the significance of the differences between control and experimental groups.Kruskal-Wallis test (with Dunn's post test) was used for multiple comparisons.

Discussion
Acute hypoxic tissue injury has been studied in a variety of rat tissues [16][17][18][19][20] .When a tissue is deprived of its oxygen supply, the mitochondrial electron transportoxidative phosphorylation sequence is inhibited resulting in decline of cellular levels of ATP and creatine phosphate.As cellular ATP levels diminish, anaerobic glycolysis is in an attempt to maintain normal cellular functions.Glycogen levels are rapidly depleted and lactic acid in cytosol increases, reducing intracellular pH 21 .The absence of significant differences in spinal cord concentrations of pyruvate lactate, glucose and ATP suggests that there was no effective spinal cord ischemia in this experimental model.The increase in lactacemia during reperfusion (T-10) in rats subjected to aortic cross-clamping could be explained to release of lactate into the blood stream after aortic clamp removal.As lactate production within the cells results from anaerobic glycolysis, with conversion of pyruvate to lactate when oxygen supply is not adequate, the increase in lactacemia could be related to a certain degree of tissue ischemia, in organs and tissues, other than the spinal cord, such as kidneys and limb muscles, that have suffered reperfusion lesion due to aortic arterial clamping .

Conclusion
Subdiaphragmatic aortic cord cross-clamping is not a suitable rat model for spinal cord ischemia/reperfusion injury study as it does not ensure complete spinal cord ischemia or changes in in vivo tissue metabolites concentrations, for example lactate and ATP, similar to those found in tissues subjected to ischemia/reperfusion.