氫氣鹽溶液對大鼠腦缺血再灌注后炎癥反應和認知功能的影響
發(fā)布時間：2018-10-08 17:35
【摘要】：研究背景與目的： 腦缺血再灌注損傷是指大腦血流阻斷一定時間后血流復通造成原缺血區(qū)腦組織損傷加重的一種病理現(xiàn)象，在臨床上常見于腦卒中、心跳驟停、心臟手術(shù)、外傷性腦損傷，嚴重影響患者的預后、增加住院時間。腦缺血再灌注損傷的機制涉及炎癥反應、線粒體損傷、氧自由基、興奮性氨基酸的產(chǎn)生、細胞內(nèi)鈣離子超載、細胞凋亡等，這些過程相互影響，共同造成組織損傷，而炎癥反應在其中發(fā)揮的作用越來越受到重視。 氫氣是自然界中含量最豐富的化學元素，一度被認為是不能和生物體內(nèi)物質(zhì)發(fā)生反應的惰性氣體，隨著實驗技術(shù)和檢測方法的不斷提高，其醫(yī)學效應逐漸被發(fā)現(xiàn)。目前研究結(jié)果表明，氫氣可以通過清除過氧離子、抗炎癥反應、抑制細胞凋亡、增加突觸可塑性等途徑產(chǎn)生神經(jīng)保護作用。因此我們建立大鼠腦缺血再灌注損傷模型，觀察氫氣鹽溶液對大鼠短暫性腦缺血再灌注損傷炎癥反應以及認知功能的影響，為氫氣用于腦缺血再灌注損傷的預防和治療提供更多的實驗基礎(chǔ)。 方法 健康SD大鼠45只，實驗動物隨機分為腦缺血組（Bccao組）、氫氣鹽溶液治療組（Bccao+HRS組）和假手術(shù)組（Sham組），每組15只。采用Morris水迷宮檢測認知功能，包括造模前連續(xù)5天的定位航行實驗和造模后第1天和第3天的空間探索實驗。定位航行實驗結(jié)束后，建造模型，Bccao+HRS組采用結(jié)扎雙側(cè)頸總動脈（bilateral common carotid artery occlusion, Bccao）伴低血壓的方法建立全腦缺血模型，并在缺血15min時開放雙側(cè)頸總動脈再灌注，此時按照5ml/kg的劑量快速腹腔注射氫氣鹽溶液(hydrogen rich saline,HRS)；Bccao組采用同樣的方法建造模型同時腹腔注射5ml/kg的生理鹽水；Sham組只暴露雙側(cè)頸總動脈不結(jié)扎，腹腔注射等量的生理鹽水。分別從各組中隨機抽取5只大鼠，于造模后第1、3天行空間探索實驗（n=5），空間探索實驗結(jié)束后經(jīng)心臟灌注固定并取腦組織制作石蠟切片，行HE染色法和尼氏染色法觀察海馬CA1區(qū)神經(jīng)元的形態(tài)學改變；再分別從各組中隨機抽取5只大鼠，于造模后第1天心臟灌注固定并取腦作石蠟切片，采用免疫組化檢測核轉(zhuǎn)錄因子-κB (nuclear factor-kappaB,NF-κB)的陽性表達；另外15只于造模后第1天斷頭取腦，采用酶聯(lián)免疫吸附測定(enzyme-linked immune sorbentassay, ELISA)檢測腦組織海馬區(qū)腫瘤壞死因子-α(tumor necrosis factor-α, TNF-α)、白介素-1β(interleukin-1β, IL-1β)的表達。 結(jié)果 1.行為學測試：在術(shù)前定位航行實驗中，隨著訓練天數(shù)的增加，各組大鼠的逃避潛伏期逐漸縮短，在相同的訓練時間每組動物之間逃避潛伏期無差別（p0.05）；在術(shù)后空間探索實驗中，術(shù)后第1天，與Sham組比較，Bccao組目標象限停留時間百分比降低，差異有統(tǒng)計學意義(p0.05),與Bccao組相比，Bccao+HRS組目標象限停留時間百分比增加,差異有統(tǒng)計學意義(P0.05)。術(shù)后第3天,與Sham組比較,Bccao+HRS組的目標象限停留時間百分比的差異無統(tǒng)計學意義(p0.05)。而Bccao組的目標象限停留時間百分比低于Sham組,差異有統(tǒng)計學意義(p0.05)。 2. ELISA法檢測大鼠海馬區(qū)TNF-a、 IL-1β的濃度:缺血再灌注24h后，與Sham組相比，Bccao組海馬區(qū)TNF-a、 IL-1β的濃度均增加，差異有統(tǒng)計學意義（p0.05）；Bccao+HRS組海馬區(qū)IL-1β的濃度增加，，差異有統(tǒng)計學意義（p0.05）。與Bccao組相比，Bccao+HRS組TNF-a、 IL-1β的濃度均減少，差異有統(tǒng)計學意義（p0.05）。 3.免疫組化檢測NF-κΒ陽性表達：與Sham組相比，Bccao組和Bccao+HRS組再灌注24h后陽性表達明顯增多，差異有統(tǒng)計學意義（p0.05），與Bccao組相比，Bccao+HRS組陽性細胞顯著減少，差異有統(tǒng)計學意義(p 0.05)。 4.HE染色和尼氏染色觀察海馬CA1區(qū)神經(jīng)元的形態(tài)學:再灌注72h時，與Sham組相比， Bccao組和Bccao+HRS組未損傷的神經(jīng)元顯著較少，差異有統(tǒng)計學意義(p 0.05);與Bccao組相比, Bccao+HRS組未損傷的神經(jīng)元數(shù)目顯著增多，差異有統(tǒng)計學意義(p 0.05)。 結(jié)論 1.5ml/kg劑量的氫氣鹽溶液可以有效的改善大鼠短暫性全腦缺血再灌注損傷造成的記憶能力減退。 2.動物行為學結(jié)果的改善可能和氫氣鹽溶液有效的抑制缺血再灌注損傷引起的炎癥反應有關(guān)。
[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.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R741
本文編號：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.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R741
【參考文獻】
相關(guān)期刊論文 前2條
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 李偉;滕云鵬;薛榮亮;張瑛;;不完全性腦缺血后大鼠認知功能的變化及其病理學機制的研究[J];山西醫(yī)科大學學報;2012年05期
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