氢气盐溶液对大鼠脑缺血再灌注后炎症反应和认知功能的影响
发布时间：2018-10-08 17:35
【摘要】：研究背景与目的： 脑缺血再灌注损伤是指大脑血流阻断一定时间后血流复通造成原缺血区脑组织损伤加重的一种病理现象，在临床上常见于脑卒中、心跳骤停、心脏手术、外伤性脑损伤，严重影响患者的预后、增加住院时间。脑缺血再灌注损伤的机制涉及炎症反应、线粒体损伤、氧自由基、兴奋性氨基酸的产生、细胞内钙离子超载、细胞凋亡等，这些过程相互影响，共同造成组织损伤，而炎症反应在其中发挥的作用越来越受到重视。 氢气是自然界中含量最丰富的化学元素，一度被认为是不能和生物体内物质发生反应的惰性气体，随着实验技术和检测方法的不断提高，其医学效应逐渐被发现。目前研究结果表明，氢气可以通过清除过氧离子、抗炎症反应、抑制细胞凋亡、增加突触可塑性等途径产生神经保护作用。因此我们建立大鼠脑缺血再灌注损伤模型，观察氢气盐溶液对大鼠短暂性脑缺血再灌注损伤炎症反应以及认知功能的影响，为氢气用于脑缺血再灌注损伤的预防和治疗提供更多的实验基础。 方法 健康SD大鼠45只，实验动物随机分为脑缺血组（Bccao组）、氢气盐溶液治疗组（Bccao+HRS组）和假手术组（Sham组），每组15只。采用Morris水迷宫检测认知功能，包括造模前连续5天的定位航行实验和造模后第1天和第3天的空间探索实验。定位航行实验结束后，建造模型，Bccao+HRS组采用结扎双侧颈总动脉（bilateral common carotid artery occlusion, Bccao）伴低血压的方法建立全脑缺血模型，并在缺血15min时开放双侧颈总动脉再灌注，此时按照5ml/kg的剂量快速腹腔注射氢气盐溶液(hydrogen rich saline,HRS)；Bccao组采用同样的方法建造模型同时腹腔注射5ml/kg的生理盐水；Sham组只暴露双侧颈总动脉不结扎，腹腔注射等量的生理盐水。分别从各组中随机抽取5只大鼠，于造模后第1、3天行空间探索实验（n=5），空间探索实验结束后经心脏灌注固定并取脑组织制作石蜡切片，行HE染色法和尼氏染色法观察海马CA1区神经元的形态学改变；再分别从各组中随机抽取5只大鼠，于造模后第1天心脏灌注固定并取脑作石蜡切片，采用免疫组化检测核转录因子-κB (nuclear factor-kappaB,NF-κB)的阳性表达；另外15只于造模后第1天断头取脑，采用酶联免疫吸附测定(enzyme-linked immune sorbentassay, ELISA)检测脑组织海马区肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)、白介素-1β(interleukin-1β, IL-1β)的表达。 结果 1.行为学测试：在术前定位航行实验中，随着训练天数的增加，各组大鼠的逃避潜伏期逐渐缩短，在相同的训练时间每组动物之间逃避潜伏期无差别（p0.05）；在术后空间探索实验中，术后第1天，与Sham组比较，Bccao组目标象限停留时间百分比降低，差异有统计学意义(p0.05),与Bccao组相比，Bccao+HRS组目标象限停留时间百分比增加,差异有统计学意义(P0.05)。术后第3天,与Sham组比较,Bccao+HRS组的目标象限停留时间百分比的差异无统计学意义(p0.05)。而Bccao组的目标象限停留时间百分比低于Sham组,差异有统计学意义(p0.05)。 2. ELISA法检测大鼠海马区TNF-a、 IL-1β的浓度:缺血再灌注24h后，与Sham组相比，Bccao组海马区TNF-a、 IL-1β的浓度均增加，差异有统计学意义（p0.05）；Bccao+HRS组海马区IL-1β的浓度增加，，差异有统计学意义（p0.05）。与Bccao组相比，Bccao+HRS组TNF-a、 IL-1β的浓度均减少，差异有统计学意义（p0.05）。 3.免疫组化检测NF-κΒ阳性表达：与Sham组相比，Bccao组和Bccao+HRS组再灌注24h后阳性表达明显增多，差异有统计学意义（p0.05），与Bccao组相比，Bccao+HRS组阳性细胞显著减少，差异有统计学意义(p 0.05)。 4.HE染色和尼氏染色观察海马CA1区神经元的形态学:再灌注72h时，与Sham组相比， Bccao组和Bccao+HRS组未损伤的神经元显著较少，差异有统计学意义(p 0.05);与Bccao组相比, Bccao+HRS组未损伤的神经元数目显著增多，差异有统计学意义(p 0.05)。 结论 1.5ml/kg剂量的氢气盐溶液可以有效的改善大鼠短暂性全脑缺血再灌注损伤造成的记忆能力减退。 2.动物行为学结果的改善可能和氢气盐溶液有效的抑制缺血再灌注损伤引起的炎症反应有关。
[Abstract]:Background and purpose of study: Cerebral ischemia-reperfusion injury refers to a pathological phenomenon in which cerebral blood flow is blocked by blood flow after a certain period of time, which causes serious injury to the brain tissue of the original ischemic region. In clinic, it is common in stroke, sudden cardiac arrest, cardiac surgery, traumatic brain. Injury, serious impact on the patient's prognosis, increased hospitalization The mechanism of cerebral ischemia reperfusion injury is related to inflammatory response, mitochondrial damage, oxygen free radical, excitable amino acid production, intracellular calcium ion overload, apoptosis and so on. Damage, and the role of inflammatory response in it is becoming more and more affected Hydrogen is the most abundant chemical element in nature, once considered to be an inert gas that cannot react with the substance in the organism. With the increasing of experimental technology and detection method, its medical effect It has been found that hydrogen can be produced by scavenging peroxy ions, anti-inflammatory responses, inhibiting apoptosis, increasing synaptic plasticity, and the like. Therefore, we established model of cerebral ischemia reperfusion injury in rats, observed the effect of hydrogen salt solution on inflammatory response and cognitive function of transient cerebral ischemia reperfusion injury in rats, and provided more for the prevention and treatment of cerebral ischemia-reperfusion injury. real Methods Forty-five healthy SD rats were randomly divided into cerebral ischemia group (Bccao group), hydrogen salt solution treatment group (Bccao + HRS group) and sham operation group (S The cognitive function was detected by Morris water maze, including the first day after modeling and the first day after modeling. After the end of the experiment, the model was constructed. The Bccao + HRS group established the model of the whole cerebral ischemia with the method of ligation of the double-sided common carotid artery (Bccao) with hypotension, and at the time of ischemia 15min. Open double-sided common carotid artery reperfusion, at which time the hydrogen-rich salt (HRS) was injected intraperitoneally at a dose of 5ml/ kg; the Bccao group used the same method to construct the model while injecting 5ml/ kg of normal saline at the same time; the sham group only exposed the double-side neck total artery not ligated, Five rats were randomly extracted from each group. In the first and third day after modeling (n = 5), the space exploration experiment was completed and the brain tissue was fixed at the end of the experiment, paraffin sections were made from brain tissue, HE staining and Niche staining were performed to observe the hippocampal CA. The morphological changes of neurons in 1 region were randomly extracted from each group. 5 rats were randomly extracted from each group. The positive expression of nuclear transcription factor-Sepharose B (nchar factor-kappaB, NF-Sepharose B) was detected by immunohistochemistry, and the positive expression of nuclear transcription factor-Sepharose B (nchar factor-kappaB, NF-Sepharose B) was detected by immunohistochemistry. The brain was broken by decapitation on the first day of the model, and the tumor necrosis factor in the hippocampus of the brain was detected by enzyme-linked immunosorbent assay (ELISA). The tumor necrosis factor (TNF-AOC), interleukin-1 (IL-1) in the hippocampus of brain were detected by ELISA. for example, IL-1 gene expression. Results 1. Behavior testing: With the increase of number of training days, the escape latency of each group decreased gradually with the increase of number of training days in the pre-operative positioning navigation experiment, and there was no difference between each group of animals at the same training time (P0.05). In the post-op space exploration experiment, the percentage of residence time in the target quadrant of the Bcao group was decreased compared with the sham group after the first day of operation. The difference was statistically significant (P0.05). Compared with the Bccao group, the residence time of the target quadrant of the Bccao + HRS group was increased. In addition, the difference was statistically significant (P0.05). In the 3rd day after operation, the target quadrant dwell time of the Bccao + HRS group was compared with the sham group. The difference in the ratio was not statistically significant (Table. 05) whereas the percentage of the target quadrant dwell time for the Bccao group was lower than the Shaam The concentrations of TNF-a, IL-1 and TNF-a, IL-1 and TNF-a, IL-1 in hippocampus were increased in the hippocampus of rats compared with sham group (P0.05). The concentration of TNF-a and IL-1 in Bccao + HRS group were higher than that of Bccao group. Compared with the sham group, the positive expression in the Bccao group and the Bccao + HRS group increased significantly after 24h, and the difference was statistically significant (P0.05). Compared with the Bccao group, the Bccao + HRS group was positive. The morphology of neurons in hippocampal CA1 region was observed by HE staining and Niche staining. Compared with the sham group, the neurons in the Bccao group and the Bccao + HRS group were significantly less than that of the sham group (p 0.05). No damage to the Bccao + HRS group. ..the God of God. The difference was statistically significant (p 0.05). Conclusion 1. 5ml/ kg of hydrogen salt The solution can effectively improve the memory ability caused by transient whole cerebral ischemia and reperfusion injury in rats.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741
本文编号：2257666
[Abstract]:Background and purpose of study: Cerebral ischemia-reperfusion injury refers to a pathological phenomenon in which cerebral blood flow is blocked by blood flow after a certain period of time, which causes serious injury to the brain tissue of the original ischemic region. In clinic, it is common in stroke, sudden cardiac arrest, cardiac surgery, traumatic brain. Injury, serious impact on the patient's prognosis, increased hospitalization The mechanism of cerebral ischemia reperfusion injury is related to inflammatory response, mitochondrial damage, oxygen free radical, excitable amino acid production, intracellular calcium ion overload, apoptosis and so on. Damage, and the role of inflammatory response in it is becoming more and more affected Hydrogen is the most abundant chemical element in nature, once considered to be an inert gas that cannot react with the substance in the organism. With the increasing of experimental technology and detection method, its medical effect It has been found that hydrogen can be produced by scavenging peroxy ions, anti-inflammatory responses, inhibiting apoptosis, increasing synaptic plasticity, and the like. Therefore, we established model of cerebral ischemia reperfusion injury in rats, observed the effect of hydrogen salt solution on inflammatory response and cognitive function of transient cerebral ischemia reperfusion injury in rats, and provided more for the prevention and treatment of cerebral ischemia-reperfusion injury. real Methods Forty-five healthy SD rats were randomly divided into cerebral ischemia group (Bccao group), hydrogen salt solution treatment group (Bccao + HRS group) and sham operation group (S The cognitive function was detected by Morris water maze, including the first day after modeling and the first day after modeling. After the end of the experiment, the model was constructed. The Bccao + HRS group established the model of the whole cerebral ischemia with the method of ligation of the double-sided common carotid artery (Bccao) with hypotension, and at the time of ischemia 15min. Open double-sided common carotid artery reperfusion, at which time the hydrogen-rich salt (HRS) was injected intraperitoneally at a dose of 5ml/ kg; the Bccao group used the same method to construct the model while injecting 5ml/ kg of normal saline at the same time; the sham group only exposed the double-side neck total artery not ligated, Five rats were randomly extracted from each group. In the first and third day after modeling (n = 5), the space exploration experiment was completed and the brain tissue was fixed at the end of the experiment, paraffin sections were made from brain tissue, HE staining and Niche staining were performed to observe the hippocampal CA. The morphological changes of neurons in 1 region were randomly extracted from each group. 5 rats were randomly extracted from each group. The positive expression of nuclear transcription factor-Sepharose B (nchar factor-kappaB, NF-Sepharose B) was detected by immunohistochemistry, and the positive expression of nuclear transcription factor-Sepharose B (nchar factor-kappaB, NF-Sepharose B) was detected by immunohistochemistry. The brain was broken by decapitation on the first day of the model, and the tumor necrosis factor in the hippocampus of the brain was detected by enzyme-linked immunosorbent assay (ELISA). The tumor necrosis factor (TNF-AOC), interleukin-1 (IL-1) in the hippocampus of brain were detected by ELISA. for example, IL-1 gene expression. Results 1. Behavior testing: With the increase of number of training days, the escape latency of each group decreased gradually with the increase of number of training days in the pre-operative positioning navigation experiment, and there was no difference between each group of animals at the same training time (P0.05). In the post-op space exploration experiment, the percentage of residence time in the target quadrant of the Bcao group was decreased compared with the sham group after the first day of operation. The difference was statistically significant (P0.05). Compared with the Bccao group, the residence time of the target quadrant of the Bccao + HRS group was increased. In addition, the difference was statistically significant (P0.05). In the 3rd day after operation, the target quadrant dwell time of the Bccao + HRS group was compared with the sham group. The difference in the ratio was not statistically significant (Table. 05) whereas the percentage of the target quadrant dwell time for the Bccao group was lower than the Shaam The concentrations of TNF-a, IL-1 and TNF-a, IL-1 and TNF-a, IL-1 in hippocampus were increased in the hippocampus of rats compared with sham group (P0.05). The concentration of TNF-a and IL-1 in Bccao + HRS group were higher than that of Bccao group. Compared with the sham group, the positive expression in the Bccao group and the Bccao + HRS group increased significantly after 24h, and the difference was statistically significant (P0.05). Compared with the Bccao group, the Bccao + HRS group was positive. The morphology of neurons in hippocampal CA1 region was observed by HE staining and Niche staining. Compared with the sham group, the neurons in the Bccao group and the Bccao + HRS group were significantly less than that of the sham group (p 0.05). No damage to the Bccao + HRS group. ..the God of God. The difference was statistically significant (p 0.05). Conclusion 1. 5ml/ kg of hydrogen salt The solution can effectively improve the memory ability caused by transient whole cerebral ischemia and reperfusion injury in rats.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741
【参考文献】
相关期刊论文 前2条
1 Guo-Dan Liu;Hong Zhang;Lin Wang;Qing Han;Shi-Feng Zhou;Ping Liu;;Molecular hydrogen regulates the expression of miR-9,miR-21 and miR-199 in LPS-activated retinal microglia cells[J];International Journal of Ophthalmology(English Edition);2013年03期
2 李伟;滕云鹏;薛荣亮;张瑛;;不完全性脑缺血后大鼠认知功能的变化及其病理学机制的研究[J];山西医科大学学报;2012年05期
本文编号：2257666
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