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Increased Expression of HO-1 Induced by Intermittent Hypobaric Hypoxia Preconditioning in the Hippocampus and Its Protective Effect on the Hippocampus Neurons of Rats with Global Cerbral Ischemia/Reperfusion

Author: LiuWeiLi
Tutor: LiQingJun
School: Hebei Medical University
Course: Pathology and Pathophysiology
Keywords: Cerebral ischemia / reperfusion Intermittent hypobaric hypoxia preconditioning Hippocampal HO-1 Immunohistochemical staining
CLC: R743.3
Type: Master's thesis
Year: 2011
Downloads: 32
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Abstract

Objective: cerebrovascular disease serious harm to human health, and the incidence rate has been high. Most of cerebrovascular disease, ischemic encephalopathy, for the treatment of ischemic encephalopathy cerebrovascular recanalization and restore the blood supply of ischemic brain tissue. However, this process will inevitably produce cerebral ischemia-reperfusion injury (ischemia. / Reperfusion, I / R). Currently looking for mitigation or prevention of cerebral ischemia-reperfusion injury is the major goal of researchers. In 1986, Murry et al found that heart resist more severe ischemia-induced damage to the heart once or several times after transient ischemia, can induce, called ischemic preconditioning, pulled from the prelude to the pretreatment studies. So far, the pre-processing method in addition to ischemic preconditioning, as well as the distal limb ischemic preconditioning and hypoxia preconditioning and other methods. In the 1990s, studies have reported that rats pre-hypoxia for 3 min, followed by more severe hypoxia produce a certain degree of tolerance. In recent years, people began to study the protective effect of hypoxic preconditioning on ischemic tissue hypoxia preconditioning in different ways, and the current lack of generally accepted ideal pretreatment model, the protective effect of hypoxic preconditioning on ischemic The mechanism is not very clear. In this study, using the method of intermittent hypobaric hypoxia (Intermittent Hypobaric Hypoxia Preconditioning, IHHP) pretreatment of animals using the four-vessel occlusion rat global cerebral ischemia model, first observed IHHP cerebral ischemia reperfusion big the protective effect of rat hippocampal neurons, and further observed impact of IHHP HO-1 expression in the hippocampus explore IHHP protection mechanisms of ischemia-reperfusion hippocampal tissue protective effects of cerebral ischemia and reperfusion organizations, HO-1. The first part: IHHP on ischemia / reperfusion protective effect of hippocampal CA1 neurons: 30 healthy male Wistar rats, weighing 280 ~ 300g, were randomly divided into five groups, each group of six animals. Were: (1) normal group (normal group): no treatment, the brains were removed directly. (2) sham group (sham group): bilateral vertebral artery occlusion of bilateral common carotid artery 48 hours after exposure, but not clipping. ③ the simple intermittent hypobaric hypoxia preconditioning group (IHHP group): Rats were exposed to low pressure (atmospheric pressure for 70.66-70.93kp) and anoxic environment, once a day for a total of 4 times. ④: ischemia / reperfusion group (I / R group): occluded bilateral vertebral artery 48 hours after exposure bilateral carotid artery occlusion 8min then restore blood flow reperfusion. ⑤ intermittent hypobaric hypoxia preconditioning global cerebral ischemia / reperfusion (IHHP I / R group): hypobaric hypoxia pretreatment 4 days, once a day, five days bilateral vertebral arteries were occluded 48 hours after exposure to double side of the carotid artery and clipping 8min, then recovery reperfusion. 7 days brains were removed in all animals after reperfusion, thionine staining hippocampal CA1 region surviving neurons density value. Hippocampal CA1 region at high optical microscopy were counted under per mm2 within intact membranes, nucleus full, the nucleolus clear the number of pyramidal cells, each slice bilateral hippocampal count each of three regions, averaged as each case of survival of nerve elements density value (neuronal density, ND). Results: Normal rat hippocampal CA1 pyramidal cells arranged in neat rows, encapsulated clear nucleus, survival, neuronal density value of 142.67 ± 13.30 (cells/mm2, the same below). sham rats hippocampal CA1 pyramidal cells arranged in neat rows, almost Absence of pyramidal cells, cell membrane integrity, cell nucleus and nucleolus is complete and clear, high density of surviving neurons was 121.33 ± 12.04, compared with the normal group, no significant differences (P gt; 0.05). IHHP rats hippocampal CA1 region morphology with normal group did not change significantly, the density of neurons to 135.00 ± 13.25, compared with the normal group, no significant difference (P gt; 0.05), but individual pyramidal cell edema and cell The gap widened phenomenon. I / R group rat hippocampal CA1 area organizations obvious damage, membrane integrity, clear nucleus pyramidal cells sparse, disorganized, obviously missing pyramidal cells, retained the part of the cell body irregular cells, were polygonal or shuttle type, membrane shrinkage, pyknotic nuclei stain, ND 39.30 ± 9.90 ND significantly reduced compared with the sham group (P lt; 0.01). Hippocampal CA1 pyramidal cell IHHP I / R group arrangement of cell morphology than those I / R group significantly improved survival of neuronal density value significantly increased (P lt; 0.01) for 89.33 ± 13.50. These results suggest that, The simplex IHHP hippocampal neurons did not cause obvious damage, but can hippocampal neurons its severe ischemia produces a certain degree of tolerance. The second part: IHHP HO-1 expression in rat hippocampal CA1 region of HO-1 expression in ischemia / reperfusion brain tissue protective effect: experimental male Wistar rats 90, weight 280 ~~ 300g. The experiment was divided into two parts: the first part of the observation IHHP of normal rats and ischemia / reperfusion hippocampal HO-1 protein expression. The second part of the observed protective effect of HO-1 protein expression in ischemia-reperfusion brain tissue. In observed IHHP of normal rats and ischemia / reperfusion rat hippocampal HO-1 protein expression in experiments (the first part), 30 animals were randomly divided into five groups: ① normal group (normal group) ② sham group (sham group), and (3) simple intermittent hypoxia preconditioning group (IHHP group) ④: ischemic / reperfusion (I / R group), the ⑤ intermittent hypobaric hypoxia preconditioning whole brain missing blood / reperfusion group (IHHP I / R group). Each group of animals is dealing with the first portion. In ischemia-reperfusion 24 hours the brains were removed to observe the HO-1 protein expression in the hippocampus. In the second part of the experiment, 60 animals were randomly divided into five groups. 12 animals in each group. Were: ① intermittent hypobaric hypoxia preconditioning global cerebral ischemia / reperfusion (IHHP I / R group): rats in the four days of the bilateral vertebral arteries were occluded preoperative exposed to low pressure (atmospheric pressure 70.66-70.93 kp) hypoxic environment, five days line vertebral arteries were occluded, 48 hours carotid artery occlusion, the brains were removed after 24 hours. (2) intermittent hypobaric hypoxia preconditioning global cerebral ischemia / reperfusion solvent group (IHHP I / R DMSO group): rats in four days of bilateral vertebral arteries were occluded preoperative hypobaric hypoxia preconditioning, 5 days line vertebral arteries were occluded, 30 minutes before intraperitoneal injection the solvent of HO-1 inhibitor Znpp DMSO50μg vertebral artery occluded the 48 hours carotid artery occlusion, the brains were removed after 24 hours. ③ intermittent hypobaric hypoxia preconditioning global cerebral ischemia / reperfusion the Znpp50μg group (IHHP the I / R Znpp50μg group), the ④ intermittent hypobaric hypoxia preconditioning global cerebral ischemia / reperfusion Znpp100μg group (IHHP I / R Znpp100μg group ) and ⑤ intermittent hypobaric hypoxia preconditioning global cerebral ischemia / reperfusion the Znpp150μg group (IHHP the I / R Znpp150μg group), three groups of animals in the 30 minutes before the occluded vertebral arteries were injected intraperitoneally with HO-1 inhibitor Znpp50μg 100μg and 150μg, dealing with IHHP the I / R DMSO group. Each group of 12 animals, six brains were removed 24 hours after ischemia-reperfusion hippocampal HO-1 protein expression using immunohistochemistry; 6 in 7 days reperfusion brains were removed for thionin staining density values ??of the observed survival of neurons. Results: 1 IHHP HO-1 expression in rat hippocampal CA1 region Normal rats hippocampal CA1 region of HO-1 positive cells were scarce positive cells were stained with shallow positive staining area of ??146.96 ± 10.94 (μm2, the same below), average optical density value of 0.05 ± 0.03; compared with the normal group, the sham group, no significant change in hippocampal CA1 region of HO-1 immune response positive staining area and mean optical density value, positive staining area of ??211.02 ± 17.00, the average optical density A value of 0.10 ± 0.02. Compared with the normal group, have IHHP rats hippocampus HO-1 immunoreactive staining area and positive cells mean optical density increases, the positive staining area as 328.52 ± 220.48 average optical density of 0.12 ± 0.07 (P lt; 0.05) . Compared with sham group, I / R group hippocampal CA1 region of HO-1 positive cells increased significantly to 660.50 ± 63.12 positive staining area, the mean optical density value was significantly higher, 0.25 ± 0.17, compared to both the sham group were significant difference (P LT; 0.01). IHHP I / R rats hippocampus HO-1 immune response stain area and the mean optical density values ??were 1655.32 ± 289.26 and 0.61 ± 0 49, compared with simple I / R group, the positive staining area and the mean optical density values were significantly higher (P lt; 0.01). 2 HO-1 expression on the protective effect of ischemic reperfusion brain tissue 2.1 Znpp cerebral ischemia reperfusion in rat hippocampal CA1 region of the HO-1 protein expression IHHP I / R group hippocampal HO-1 immune response in the positive area 1655.32 ± 289.26, the positive cells mean optical density value of 0.61 ± 0.49 IHHP I / R DMSO group rat hippocampal HO-1 immunoreactive area to 1667.00 ± 121.77, mean optical density value of 0.72 ± 0.33, compared the two groups, positive area and the positive cell average optical density values ??were not significantly different (P gt; 0.05). Hippocampus rats IHHP I / R Znpp50μg HO-1 immunoreactive cells in an area of ??617.96 ± 37.00, the average optical density of 0.21 ± 0.13, compared with IHHP I / R group, both significantly lower (P lt; 0.05) . IHHP I / R Znpp100μg group of HO-1 immune response in the positive area and the mean optical density values ??were 204.99 ± 121.51 and 0.07. ± 0.04, compared with IHHP I / R Znpp50μg group were lower (P lt; 0.05); Znpp150μg group HO-1 immune response positive area of ??161.11 ± 68.25, and the average optical density of 0.06 ± 0.03, compared with Znpp100μg group, the two were not statistically different (P gt; 0.05). These results suggest that the intraperitoneal injection Znpp significantly inhibited IHHP and brain ischemia-reperfusion-induced hippocampal HO-1 protein expression. 2.2 Znpp reperfusion of ischemic hippocampal CA1 neurons density value IHHP I / R group surviving neurons density value of 89.33 ± 13.50 IHHP I / R DMSO group the neuronal density hippocampus survival value of 86.00 ± 16.34 compared with IHHP I / R group was no significant difference (P gt; 0.05); survival of hippocampal CA1 region IHHP I / R Znpp50μg neuronal density is 63.33 ± 9.26, is reduced compared with IHHP I / R group ( P lt; 0.05). Znpp100μg hippocampus CA1 region visible large areas of neuronal necrosis, a significant reduction in the survival of neurons, is 39.33 ± 9.93, compared with IHHP I / R Znpp50μg group further reduce; The hippocampal CA1 IHHP I / R Znpp150μg surviving neurons density value of 40.00 ± 9.79, compared with Znpp100μg group, no significant change (P gt; 0.05). These results suggest that inhibition of the hippocampus Znpp HO-1 expression, enables further reduce the number of surviving neurons hippocampal tissue ischemia and reperfusion, to prompt IHHP and ischemia-reperfusion induced HO-1 expression has a protective effect on hippocampal neurons. Conclusion: a simple intermittent hypobaric hypoxia preconditioning on rat neuronal elements no obvious damage, but it can reduce global cerebral ischemia / reperfusion induced neuronal damage in hippocampal CA1 region of global cerebral ischemia / re hippocampus of rats after perfusion has a protective effect. The 2 intermittent hypobaric hypoxia preconditioning raised ischemia / reperfusion hippocampus HO-1 protein expression. 3 HO-1 has a protective effect for ischemia reperfusion brain tissue, which may be an important mechanism of intermittent hypobaric hypoxia preconditioning ischemia / reperfusion brain tissue.

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