OXIDATIVE STRESS UPON BILATERAL CAROTID ARTERY OCCLU- SION AND REPERFUSION IN RAT BRAIN: A BIOCHEMICAL STUDY

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1 Journal of Tissue Research Vol. 4 (2) (4) ISSN: (Printed in India) Original Article OXIDATIVE STRESS UPON BILATERAL CAROTID ARTERY OCCLU- SION AND REPERFUSION IN RAT BRAIN: A BIOCHEMICAL STUDY NAGA, K.K., PANIGRAHI, M. 1 AND BABU, P.P.? Department of Animal sciences, School of Life Sciences, University of Hyderabad, Hyderabad-46 India. 1 Department of Neurosurgery, Nizam s institute of Medical Sciences, Punjagutta, Hyderabad-82 India. Received May 15, 4 ; Accepted May 25, 4 Abstract: Bilateral carotid artery occlusion results in a wide spread ischemic episode in rat brain. Moreover, reperfusion causes oxidative damage to the tissue by massive production of free radicals. Therefore, present study was designed to understand the extent of oxidative stress upon bilateral carotid artery occlusion and reperfusion injury in the rat brain. The levels of reduced glutathione, oxidized glutathione and lipid peroxidation were estimated in cortex, cerebellum and hippocampus of rat, which underwent min carotid artery occlusion followed by various time periods of reperfusion. A significant decrease in reduced glutathione, an increase in oxidized glutathione and an increase in lipid peroxidation were observed after min carotid artery occlusion and 1 hour reperfusion. Most of the biochemical alterations were reverted back to normal after 24 hours of reperfusion. These data indicates oxidative stress is induced during reperfusion, although this does not cause early cell death but might results in more prolonged cell dysfunction and eventual neuronal loss. Key words: Oxidative stress, Carotid artery, Occlusion, Rat brain INTRODUCTION Stroke is one of the main causes of adult disability and death. This disorder most commonly is due to the occlusion of one of the major blood vessels in the brain (Ameriso and Sahai, 1997) resulting in localized reductions in blood flow. The affected tissue typically contains an ischemic core surrounded by penumbra regions ( Jacewicz et al., 1992; Memezawa et al., 1992). Reversal of the arterial occlusion can occur through spontaneous or treatment with thrombolytic agents. Treatments initiated well in advance can block cell death in tissue that was contained within the penumbral region (Chopp et al., 1996; Matsumoto et al., 1999; Yoshimoto and Siesjo, 1999). This indicates that at least majority of the cells, which are still viable at the onset of reperfusion are under a risk of damage by the molecular events that occur during reperfusion period. Many factors play an important role in pathogenesis of stroke, viz., energy failure, exitotoxicity, pre-infarct depolarization wave, oxidative stress and alteration of gene expression play important roles in pathogenesis of stroke (Takeshi et al., 1999). Among these factors, the role of oxidative stress becomes much greater in the case where cerebral blood flow (CBF) is restored, because reflow to the previous ischemic tissue results in an increase in oxygen level, and consequently causes severe oxidative damage to the tissue by massive production of oxygen free radicals (Wei and Kontos, 1987). However, CBF restoration is necessary in order to restrict the infarct area in patients of stroke (Hiramoto et al., 1994). Therefore, elucidation of the extent and role of oxidative stress induced brain damage after ischemia/ reperfusion is of great importance. The nervous system, particularly brain is vulnerable to the free radical damage for a number of reasons. Membrane lipids in the brain contain high levels of polyunsaturated fatty acid side chains, which are 189

2 J. Tissue Research prone to free radical attack (Rao and Balachandran, 2). Thus, measuring the extent of lipid peroxidation is an important parameter to study oxidative stress. In addition to the presence of readily peroxidisable fatty acids, brain also consumes large quantities of oxygen contributing to the formation of ROS (Halliwell, 1992). However, brain contains low or moderate levels of enzymes such as catalase, superoxide dismutase and glutathione peroxidase that play an important role in the metabolism of ROS (Cohen, 1988). Glutathione is a central component in the antioxidant defenses of cells, acting directly to detoxify reactive oxygen species and also as a substrate for several peroxidases (Dringen, ). Thus alterations in the availability of this metabolite during ischemia or early reperfusion are likely to influence tissue damage. Various animal models have been developed to study the reperfusion injury. The brain receives its blood supply through a pair of carotid arteries and the vertebral arteries. These two pairs of arteries are interconnected at the circle of willis at the base of the brain, allowing for extensive collateral flow between the two cerebral hemisphere and between the two pairs of arteries. However, the occlusion of the bilateral carotid artery alone in the rat leads to reduction of cerebral blood flow by % (Eklof and Siesjo, 1972). Therefore, this model has been chosen to study the glutathione levels and level of oxidative stress by lipid peroxidation in brain regions during ischemia and various periods of reperfusion after ischemia. MATERIALS AND METHODS Animals: The experiments were performed on Wistar rats weighing -1 grams and aged 1-2 months, obtained from National institute of Nutrition, Hyderabad. Animals had free access to pelleted diet (NIN, INDIA) and water, ad libitum. To avoid sex related considerations we used only male rats. Surgical procedure: Rats were anesthetized with ether and a small midline neck incision was made on the ventral side of the animal, the carotid artery which lies medial to and below the jugular vein was exposed by blunt dissection between the sterno hyoid, sterno mastoid and omo hyoid muscles. The carotid arteries were then occluded for minutes using temporary aneurysm clips to create ischemia and the clips are slowly removed to allow reperfusion. This model employs bilateral carotid-artery occlusion to significantly reduce blood flow to the brain. The surgery was performed in four groups of animals: 1. Sham operated: Animals were anesthetized, carotid arteries were traced out but occlusion was not done. 2. min Ischemia: Animals were anesthetized; carotid arteries were traced out and occluded for minutes. Animals were sacrificed immediately without allowing any reperfusion hour reperfusion: Animals were anesthetized, carotid arteries were traced out and occluded for minutes. These animals were sacrificed after allowing reperfusion for 1 hour hour reperfusion: Animals were anesthetized, carotid arteries were traced out and occluded for minutes. These animals were sacrificed after 24 hours of reperfusion. Chemicals: Reduced and oxidized glutathione (Sigma Chemicals Co.). N-ethyl maleimide (Sigma Chemicals Co.), o-phthaldialdehyde (OPT) (analytical grade), thiobarbituric acid (Hi Media), 1,1,3,3- tetramethoxy propane TMP (Sigma Chemical Co.). All other chemicals were reagent grade and obtained locally. Estimation of glutathione levels: Glutathione levels were estimated according to the protocol of Hissin and Hilf (1976). Briefly, a portion of tissue (2 mg) was homogenized in 3.75 ml of the phosphate-edta buffer and 1ml of 25% HPO 3 which was used as a protein precipitant. The total homogenate was centrifuged at 4 C at,g for 3 min to obtain the supernatant for the assay of GSSG and GSH. For GSH assay the assay mixture contained ml of the diluted tissue supernatant, 1.8 ml of phosphate- EDTA buffer, and ml of OPT solution, containing mg of OPT. After thorough mixing and incubation at room temperature for 15 min, the solution was transferred to quartz cuvette, fluorescence at 4 nm was determined with the activation at 3 nm. Various concentrations of pure glutathione (GSH) were used as a standard. For GSSG assay, a.5 portion of original,g supernatant was incubated at room temperature with ml of.4 M NEM for 3 min to interact with GSH present in the tissue. To this mixture, 4.3 ml of.1n NaOH was added. A ml portion of this mix- 19

3 Naga et al. ture was taken for measurement of GSSG, using the procedure outlined above for GSH assay, except that.1 N NaOH was employed as diluent rather then phosphate EDTA buffer. Various concentrations of pure glutathione (GSSG) were used as a standard. Estimation of lipid peroxides: Lipid peroxides were measured according to the protocol of Ohkawa et al. (1978). The tissue homogenates were prepared in a ratio of 1gram of wet tissue to 9 ml of 1.15% KCl by using a glass potter elvehjem homogeniser. The homogenate was then centrifuged at g for 1 min. The resultant supernatant was used for further assay. To samples less than.2 ml of 1% (w/v) tissue homogenate were added.2 ml of 8.1% SDS,1.5 ml of % acetic acid solution adjusted to ph 3.5 with NaOH, and 1.5 ml of aqueous solution of thiobarbituric acid. The mixture was made up to 4. ml with distilled water, and then heated in an oil bath at 95 C for minutes using a glass ball as a condenser. After cooling with tap water, 1. ml of distilled water and 5. ml of the mixture of n-butanol and pyridine (15:1, v/v) were added and shaken vigorously. After centrifugation at rpm for 1 min, the organic layer was taken and its absorbance at 532 nm was measured. TMP was used as an external standard, and level of lipid peroxides was expressed as nm of MDA (malondialdehyde) per gram wet tissue. Estimation of proteins: Protein estimation was done by the method of Lowry et al. (1951). Data Analysis: Data were analysed by one-way analysis of variance (ANOVA) according to Sokal and Rohlf (1969). When a significant F value was found, Fisher s least difference (LSD) multiple comparison was performed to test the differences between the means. P<.5 was considered statistically significant. RESULTS Glutathione content in cerebral cortex: Reduced glutathione (GSH) content did not show significant variation in cerebral cortex after ischemia /reperfusion when compared to that of sham operated samples (Fig 1A; Table 1). Oxidised glutathione (GSSG) content increased after hr, 1hr and 24 hr reperfusion following min ischemia occlusion in cerebral cortex samples compared to that of sham operated samples (Fig 1B; Table 1). The increase was 23.87%, 26.2% and 34.5% respectively to that of sham operated samples. Lipid peroxidation in cerebral cortex: Lipid peroxidation reveals a significant increase after 1hr reperfusion (57.55% to sham operated) following min ischemia. hr and 24 hr samples did not show significant variation when compared to that of sham operated cerebral cortex samples (Fig 1C; Table 1). Table 1: Levels of GSH, GSSG and lipid peroxidation in cerebral cortex. Data is expressed as Mean SEM (n=3) as well as percentage. Significance of the data was evaluated by ANOVA. Sample Sham Operated min ischemia 1hr reperfusion 24 hr reperfusion GSH GSSG MDA % (ng/mg) changes (ng/mg) % changes (nm/g) % changes LSD Value P=.5; *Significant * ** *** 23.87* 26.2** 34.5 *** * * 4.8 Table 2: Levels of GSH, GSSG and lipid peroxidation in cerebellum. Data is expressed as Mean SEM (n=3). As well as percentage.significance of the data was evaluated by ANOVA. Sample Sham Operated min ischemia 1 hr reperfusion 24 hr reperfusion GSH GSSG MDA % (ng/mg) changes (ng/mg) % changes (nm/g) % changes * * LSD Value P=.5; *Significant * * * 15.7* 12.4* 15.1* * * * 43.6* 69.9* 69.4 * Glutathione content in cerebellum: GSH content did not show much variation after min ischemia without reperfusion. Whereas min ischemia followed by 1 hr reperfusion showed a small but significant decrease (6.2% to sham operated) in GSH content when compared to sham operated cerebellum (Fig 2A; Table 2). min ischemia followed by 24 hr reperfusion increased GSH content by 37.4% to that of sham operated. GSSG content increased after 1 hr and 24 hr reperfusion following min ischemia. 191

4 J. Tissue Research The increase was 12.4% and 15.1% respectively to that of sham operated cerebellum samples (Fig 2B; Table 2). Lipid peroxidation in cerebellum: Lipid peroxidation increased significantly after 1hr and 24 hr reperfusion (69.9% and 69.4% respectively to sham operated) following min ischemia compared to sham operated cerebellum samples (Fig 2C; Table 2). Glutathione content in hippocampus: min ischemia without any reperfusion (31.1% to sham operated) decreased GSH level in hippocampus when compared to sham operated. This decrease continued for 1 hr reperfusion samples whereas GSH level in 24 hr reperfusion following min ischemia were Table 3: Levels of GSH, GSSG and lipid peroxidation in hippocampus. Data is expressed as Mean SEM (n=3) as well as percentage significance. Significance of the data was evaluated by ANOVA. Sample Sham Operated min ischemia 1hr reperfusion 24 hr reperfusion GSH GSSG MDA % (ng/mg) changes (ng/mg) % changes (nm/g) % changes * * * 31.5* 5. LSD Value P=.5; *Significant * * Fig.1A Fig.1B Fig.1C 39 CC IC 1C 24C CC IC 1C 24C Fig. 1A, 1B, 1C: Cerebral cortex, CC - Control (Sham operated); IC - Ischemic ( min occlusion no reperfusion); 1C -1 hour reperfusion ( min occlusion and 1 hour reperfusion); 24C -24 hour reperfusion ( min occlusion and 24 hour reperfusion). CC IC 1C 24C 4 Fig.2A Fig.2B 3 2 Fig.2C CB IB 1B 24B CB IB 1B 24B CB IB 1B 24B Fig. 2A, 2B, 2C: Cerebellum, CB - Control (Sham operated); IB - Ischemic ( min occlusion no reperfusion); 1B -1 hour reperfusion ( min occlusion and 1 hour reperfusion); 24B -24 hour reperfusion ( min occlusion and 24 hour reperfusion) Fig.3A Fig.3B Fig.3C Fig. 3A, 3B, 3C: Hippocampus, CH - Control (Sham operated); IH - Ischemic ( min occlusion no reperfusion); 1H -1 hour reperfusion ( min occlusion and 1 hour reperfusion); 24H -24 hour reperfusion ( min occlusion and 24 hour reperfusion) Hippocampus. 192

5 Naga et al. same as compared to sham operated (Fig 3A; Table 3). GSSG levels did not show any significant variation in hr, 1 hr and 24 hr reperfusion following min ischemia when compared to sham operated hippocampus samples (Fig 3B; Table 3). Lipid peroxidation in hippocampus: A significant increase of lipid peroxides was observed in min ischemia without reperfusion in hippocampus samples when compared to that of sham operated (Fig 3C; Table 3). 1hr and 24 hr reperfusion after min ischemia showed not much alteration in lipid peroxidation when compared to that of control. DISCUSSION The neuropathological changes observed during brain injury, trauma, stroke, and epileptic associated brain damage have all been ascribed to enhanced oxidative stress and related lipid protein and DNA molecules (Srinivasan, 2). One of the mechanisms responsible for the delayed neuronal injury after global cerebral ischemia is the membrane alteration. The most important cause of membrane alteration is the lipid peroxidation due to a relative excess of free radicals. Many mechanisms involving free radical production have been documented both during ischemia and reperfusion but their actual importance is still controversial (Nita et al., 1). When free radical production exceeds the buffering capabilities, LPO levels increase while non-enzymatic scavengers (GSH) and antioxidant enzymes decrease. A decrease in free radical production was followed by a gradual normalization of LPO and both GSH and antioxidant enzymes (Nita et al., 1). Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and cumulative evidence suggests involvement of oxygen radicals in the pathogenesis of ischemic lesions (Sugawara et al., 4). Reoxygenation during reperfusion provides a substrate for numerous enzymatic oxidation reactions. Lipid peroxidation is a major contributor to ischemic damage, but the underlying mechanisms are poorly understood (Eileen et al., 1). The rate of lypolysis during ischemia in selectively vulnerable zones is significantly greater than in other areas of the brain (White et al., ). Studies involving animal models for ischemia reperfusion have been extensively done in focal Ischemia and global Ischemia (Sugawara et al., 4). But global ischemic models involved the occlusion of carotid arteries and vertebral arteries (complete global ischemia) or occlusion of carotid arteries along with hypotension (incomplete global ischemia). To our knowledge this kind of study is not reported earlier. Many contradictory reports have emerged regarding the status of GSH during reperfusion injury in brain. Investigations by Folbergrova et al. (1979) led to the observation that the GSH and GSSG levels were unaltered in brain during Hypoxia. It was observed that the brain GSH levels decreased during reperfusion after complete or incomplete Ischemia without any concomitant accumulation of GSSG in the brain or cerebrospinal fluid (Cooper et al., 198; Rehncrona et al., 198). The authors concluded that decreased synthesis of GSH during reperfusion might account for these observations. The results of the present study have shown that reversible incomplete brain ischemia lead to a reduction of concentrations of GSH in cortex, cerebellum and hippocampus accompanied by small but significant increase in GSSG. The alteration in the ratio of GSH and GSSG suggests that there is a significant amount of oxidative stress in rat brain upon min bilateral carotid artery occlusion. The maximum alteration is observed in brains after 1 hr of reperfusion following min ischemia, suggesting maximum oxidative stress at that time period. These changes were found to be normalized after 24 hr of reperfusion following min ischemia. In our study we addressed the problem of relative contribution of ischemia and reperfusion to the free radical production estimated by the level of lipid peroxidation (LPO). The amount of MDA was found to be increased in all brain regions after 1 hr of reperfusion following min ischemia. Further, maximum LPO occurred in hippocampus suggesting that it is a region for the production of more free radicals and more vulnerable region for damage when compared to cortex and cerebellum. Result of this study clearly shows that a small reduction in blood flow could lead to oxidative stress and can have deleterious effects on brain cells. Hemoglobin shows a reduced oxygen carrying capability at higher altitudes, so people living at higher altitudes may experience a reduction of oxygen to brain (hypoxia) and normal oxygen when on ground (reperfusion). This kind of small reductions in blood volume to brain can also be observed in case of pa- 193

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