高压氧治疗对慢性应激大鼠的影响

发布时间：2018-06-03 15:46

本文选题：慢性应激 + 束缚　；参考：《南方医科大学》2015年硕士论文

【摘要】：背景慢性应激(chronic stress)指的是机体长时间持续或间断暴露于应激源中,也就是日常生活工作中所存在的压力。长期应对压力刺激将造成人体的失代偿,全身功能失调,特别是中枢神经系统、神经内分泌系统、免疫系统功能低下,由此导致和(或)促进多种疾病的发生,严重影响人们的生活及工作质量。目前已经明确,慢性应激是多种心理疾病如抑郁症及创伤后应激障碍的病因之一,同时也是心脑血管疾病发生的一个中度危险因素。据报道,在美国慢性应激发生率约为50%,其中的25%为高强度的应激。而国内城市中慢性应激的发生率为42%-46%。近些年我们收治了大量突发性耳聋、神经性耳鸣、脑血管病的病人,其中有很大一部分存在生活、工作上的压力。这些患者初期往往出现心率、血压、血糖、血脂等指标的变化,后期出现各种器质性疾病,更有甚者发生严重的心、脑等严重不良事件。目前国内外慢性应激相关的研究大部分都集中在其致病机制上,而针对治疗方面的研究则相对薄弱。目前临床主要的干预方式为药物及心理干预上,但是它们或是副作用或花费过大等问题难以在大众中推广。高压氧治疗慢性器官或脏器缺血有特殊的效果,据白永杰等的介绍高压氧治疗的作用包括：提高人体内的氧分压、氧气的弥散距离、刺激侧枝循环的形成、抑制炎症反应等。在临床中我们发现,对于一些存在慢性应激的病人,经过一段时间的高压氧治疗后,发现这部分病人的主观症状,特别是因压力而来的轻度抑郁、焦虑状态有所减轻。我们设想高压氧治疗可能具有缓解慢性应激对机体损害的能力。为此我们设计了本实验,通过观察高压氧治疗对慢性应激处理大鼠各种生理指标的影响,探索高压氧治疗干预慢性应激的可行性。目的通过观察慢性应激和高压氧处理对Wistar大鼠体重、心率变异度、行为学、血清HPA轴激素水平、一氧化氮系统、TNF-α以及海马糖皮质激素受体表达的情况,探讨：(1)高压氧对动物慢性应激相关指标的影响；(2)慢性束缚是否能模拟动物慢性应激的生理状态；(3)高压氧治疗能否减轻慢性束缚对机体带来的影响。为此我们设计了本课题,旨在为高压氧治疗作为慢性应激的干预手段提供一定的实验依据。材料与方法1.实验材料1.1实验动物：选取健康雄性8月龄Wistar大鼠60只,体重200-250g,大鼠分笼饲养,5只/笼,饲养室内温度恒定于25-28℃,湿度50～60%,12h/d光照,给予自由进食饮水,垫料隔日更换。实验前进行适应性饲养1周。1.2高压氧治疗设备：单人医用高压纯氧舱,氧气由海军总医院中心气站提供。1.3十字高架迷宫材料：自制十字高架迷宫：高35cm,2个开臂及2个闭合臂宽10cm长50crm,闭合臂壁高25cm,内侧及底部涂黑。十字高架迷宫上方1米处设有照明用500W灯及摄像头。1.4旷场试验材料：旷场环境长100cm,宽100cm,高35cm,四周及底部涂黑,底部用白线分成20cm×20cm小格,共25个。旷场上方lm处设有照明用500W光源及摄像头。1.5免疫组织荧光材料：异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的羊抗兔IgG二抗,大鼠GR的单克隆抗体一抗。1.6心率变异度测量及计算：多导生理测量仪1.7 血清学检测：促肾上腺皮质激素释放激素(corticotropin releasinghormone, CRH)、促肾上腺皮质激素(adrenocor ticotropic hormore, ATCH)、糖皮质激素(glucocorticoids, GCS)、一氧化氮合酶(nitric oxide synthase,NOS)、一氧化氮(nitric oxide, NO)及肿瘤坏死因子-α(tumor necrosis factor alpha,TNF-a),使用MULTISKAN EX酶标仪检测。2.分组及实验方法2.1高压氧对大鼠十字高架试验的影响将20只雄性Wistar大鼠随机分成2组：单纯高压氧组和空白对照组,每组10只。单纯高压氧组给予2.0ATA高压氧治疗1次/d；空白对照组单纯饲养,共21d。2.2慢性束缚及高压氧处理对大鼠心率变异度及旷场试验的影响将40只雄性Wistar大鼠随机分成4组：单纯束缚组、单纯高压氧组、高压氧联合束缚组和空白对照组,每组10只。单纯束缚组给予行为限制3h/d；单纯高压氧组给予2.0ATA高压氧治疗1次/d；高压氧联合束缚组,每天先后给予高压氧处理与束缚处理；空白对照组单纯饲养,共21d。“慢性束缚及高压氧处理对大鼠海马区GR表达及血清HPA轴激素水平的影响”与“慢性束缚及高压氧处理对大鼠一氧化氮系统及促炎症因子表达的影响”分组方式同2.2。3.观察指标3.1高压氧对大鼠十字高架试验的影响以适应环境一周后的第1、7、14、21天对其进行十字高架迷宫(elevated plus maze)试验,观察其开臂(Open Arm, OA)逗留时间,统计采用两因素重复测量的方差分析。3.2慢性束缚及高压氧处理对大鼠心率变异度及旷场试验的影响以适应环境一周后观察大鼠在第1d、第11d、第21d旷场试验中运动能力(水平得分)及探索行为(垂直得分)的变化,心电的检测第21天大鼠自主神经的变化。3.3慢性束缚及高压氧处理对大鼠海马区GR表达及血清HPA轴激素水平的影响。21天处理后,利用免疫荧光染色检测大鼠海马区GR的表达水平。利用酶联免疫吸附测定法测定其血清中CRH、ACTH和GCS的含量。3.4慢性束缚及高压氧处理对大鼠一氧化氮系统及促炎症因子表达的影响利用酶联免疫吸附测定法测定血清中NOS、NO及TNF-α的含量。4.统计学处理利用SPSS13.0软件进行统计学分析,计量资料以均数±标准差(x±s)表示,计数资料以秩来表示,连续时间点上行为学的差异比较采用重复测量的方差分析。组间差异以及高压氧和束缚两因素相互关系的分析采用两因素析因分析设计资料的方差分析。组间两两比较采用LSD-t法。计数资料采用秩和检验,以P0.05表示差异有统计学意义。结果1.十字高架迷宫显示：分组因素不具有统计学意义(P=0.450),高压氧处理与空白对照组在焦虑程度上差异无统计学意义。2.旷场试验显示：与空白对照组比较,单纯束缚组在第11d水平得分(115.5±43.4)和垂直得分(23.9±10.1)均明显升高(P0.001)；与单纯束缚组比较,高压氧联合束缚组在第1d到第11d的得分升幅减小(P0.01)。3.心率变异度显示：4组间LF/HF、交感及副交感活性差异无统计学意义。但仍能看出以下趋势：束缚及高压氧治疗均可导致交感神经张力下降,而高压氧治疗可引起副交感神经张力上升。4.免疫组织荧光染色显示：单纯束缚组海马区GR表达(0.1546±0.0154)比空白对照组(0.2174±0.0123)减少(P0.001),高压氧联合束缚组与空白对照组比较无明显差异(P0.05)。5.HPA轴激素显示：4组血清CRH、ATCH及GCS差异无统计学意义。但可以得出以下趋势：单纯高压氧组,单纯束缚组,高压氧联合束缚组ACTH及GCS均较空白对照组有所升高,高压氧联合束缚组CRH水平较另三组下降。6.4组大鼠血清总NO、NOS及TNF-α差异无统计学意义。但仍能得出以下趋势：单纯束缚组NOS水平较各组下降,束缚联合高压氧处理后发现血清TNF-α较单纯束缚组下降。结论1.高压氧处理后a.大鼠焦虑程度无明显变化：b.大鼠出现轻度的交感神经张力下降及副交感兴奋；c.海马区GR表达无明显变化；d.大鼠血清HPA轴激素水平有所升高,而NO、NOS及TNF-α无明显变化。提示高压氧处理具有一般应激的特征,但应激强度不大。2.束缚处理后a.大鼠行为学试验表现出明显的焦虑状态；b.大鼠出现交感神经张力下降；c.海马区GR表达下降；d.大鼠血清GCS水平升高,但CRH及ACTH无明显变化；e.大鼠NOS水平下降。提示慢性束缚在一定范围内模拟慢性应激的生理状态。3.高压氧联合束缚处理后a.大鼠的焦虑状态较单纯束缚有所减轻；b.大鼠出现交感神经张力下降,但下降程度要小于单纯束缚组；c.海马区GR表达未见明显下降；d.大鼠血清ACTH及GCS均较空白对照组有所升高,但高压氧联合束缚组CRH水平较另三组有所下降。e.束缚联合高压氧处理后发现血清TNF-α较单纯束缚组下降。提示高压氧能减缓束缚对大鼠相关生理指标的影响。
[Abstract]:Background chronic stress (chronic stress) refers to the prolonged or intermittent exposure of the body to the stressor, which is the stress in the daily life. Long-term response to stress stimulates the body's decompensation and systemic dysfunction, especially the central nervous system, the neuroendocrine system, and the immune system, which leads to the low function of the immune system. It is now clear that chronic stress is one of the causes of many psychological diseases such as depression and post-traumatic stress disorder, and is also a moderate risk factor for the occurrence of cardiovascular and cerebrovascular diseases. It is reported that the incidence of chronic stress is about 50% in the United States. 25% of them were high stress. And the incidence of chronic stress in the domestic cities was 42%-46%. in recent years. We treated a large number of patients with sudden deafness, neurogenic tinnitus, and cerebrovascular disease, of which a large portion of these patients had life and stress. These patients were at the initial stage of heart rate, blood pressure, blood sugar, blood lipid and other indicators. There are various organic diseases in the later period and serious adverse events such as heart and brain. Most of the research on chronic stress at home and abroad is concentrated on its pathogenesis, but the research on treatment is relatively weak. The main clinical intervention methods are drug and psychological intervention, but they are the main methods of intervention. The effects of hyperbaric oxygen therapy on chronic organ or organ ischemia have special effects. The effect of hyperbaric oxygen therapy, such as Bai Yongjie and so on, includes improving the oxygen partial pressure in the body, the diffusion distance of oxygen, stimulating the formation of collateral branches and inhibiting the inflammatory reaction, and so on. We found that for some patients with chronic stress, after a period of hyperbaric oxygen therapy, we found that the subjective symptoms of the patients, especially the mild depression caused by pressure, were relieved. We conceive that hyperbaric oxygen therapy may have the ability to alleviate the damage to the body by chronic stress. The experiment was to observe the effect of hyperbaric oxygen therapy on the physiological indexes of rats treated with chronic stress, and to explore the feasibility of hyperbaric oxygen therapy to intervene chronic stress. Objective To observe the weight, heart rate variability, behavior, serum HPA axis hormone level, nitric oxide system, TNF- A and hippocampus of Wistar rats. The expression of glucocorticoid receptor: (1) the effect of hyperbaric oxygen on the related indicators of chronic stress in animals; (2) whether chronic restraint can simulate the physiological state of chronic stress in animals; (3) whether hyperbaric oxygen therapy can reduce the effect of chronic restraint on the body. The intervention means of chronic stress provide certain experimental basis. Material and methods 1. experimental materials 1.1 experimental animals: 60 healthy male 8 month old Wistar rats, weight 200-250g, cage rearing, 5 cage, feeding room temperature constant at 25-28, humidity 50 to 60%, 12h/ D light, free feeding drinking water, mat material daily replacement. .1.2 hyperbaric oxygen treatment equipment for 1 weeks before examination: single medical high pressure pure oxygen tank, oxygen by Navy General Hospital central gas station.1.3 cross elevated maze material: self-made cross elevated maze: high 35cm, 2 open arms and 2 closed arm width 10cm long 50crm, close arm wall high 25cm, inside and bottom black. Cross viaduct maze 1 meters above the top 1 meters with lighting and camera.1.4 open field test materials: open field environment long 100cm, wide 100cm, high 35cm, black around and bottom, the bottom is divided into 20cm x 20cm small lattice, a total of 25. Above the open field, there is a 500W light source and a camera.1.5 immunofluorescence material above the open field: fluorescein isothiocyanate (fluorescein I) Sothiocyanate, FITC) labeled Sheep anti rabbit IgG two resistance, rat GR monoclonal antibody one anti.1.6 heart rate variability measurement and calculation: multi lead physiological measuring instrument 1.7 serological detection: adrenocorticotropin releasing hormone (corticotropin releasinghormone, CRH), suprarenal cortical hormone (adrenocor ticotropic hormore, ATCH), sugar Corticosteroids (glucocorticoids, GCS), nitric oxide synthase (nitric oxide synthase, NOS), nitric oxide (nitric oxide, NO) and tumor necrosis factor - alpha (tumor necrosis factor). The effect of 2.1 hyperbaric oxygen on the cross viaduct test in rats was detected by the enzyme labeling method and the effect of hyperbaric oxygen on the cross Viaduct of rats. Rats were randomly divided into 2 groups: simple hyperbaric oxygen group and blank control group, 10 rats in each group. Pure hyperbaric oxygen group was given 2.0ATA hyperbaric oxygen therapy 1 times /d; blank control group was fed alone. The effect of chronic constriction of 21d.2.2 and hyperbaric oxygen treatment on heart rate variability and open field test in rats was divided into 4 groups of 40 male Wistar rats randomly: simple binding Group, simple hyperbaric oxygen group, hyperbaric oxygen binding group and blank control group, each group was 10. The simple binding group was given the behavior restriction 3h/d; the simple hyperbaric oxygen group was given the 2.0ATA hyperbaric oxygen therapy 1 times /d; the hyperbaric oxygen combined binding group was given hyperbaric oxygen treatment and binding treatment every day; the blank control group was simply fed, a total of 21d. "chronic shackles. Effect of hyperbaric oxygen treatment on the expression of GR and serum HPA axis hormone levels in the hippocampus of rats "and the effect of" chronic binding and hyperbaric oxygen treatment on the expression of nitric oxide and proinflammatory factors in rats "with 2.2.3. observation index 3.1 the effect of hyperbaric oxygen on the cross elevated test in rats to adapt to the environment one week after the 1,7,14 On the 21 day, the elevated plus maze test was carried out to observe the duration of the opening arm (Open Arm, OA), and the statistical analysis of the variance of the two factor repeated measurements was used to analyze the effect of.3.2 chronic binding and hyperbaric oxygen treatment on the heart rate variability and open field test in rats to observe the rats in the 1D, 11d, 21d open field after a week. Changes of exercise ability (level score) and exploratory behavior (vertical score) in the test, changes of electrocardiogram (vertical score), changes of autonomic nerve in twenty-first days of rats,.3.3 chronic binding and the effect of hyperbaric oxygen treatment on GR expression and serum HPA axis hormone levels in the hippocampus of rats.21 days after.21 day treatment, the expression level of GR in the hippocampus of rats was detected by immunofluorescence staining. Determination of CRH, ACTH and GCS in serum by enzyme linked immunosorbent assay the effect of.3.4 chronic binding and hyperbaric oxygen treatment on the expression of nitric oxide and proinflammatory cytokines in rats by using enzyme linked immunosorbent assay to determine the content of NOS, NO and TNF- alpha in serum.4. statistical treatment using SPSS13.0 software for statistical analysis The measurement data were expressed in the mean number of standard deviation (x + s), the counting data were expressed as rank, and the difference of behavior in the continuous time point was compared with the variance analysis of repeated measurements. The analysis of the interrelationship between the groups and the two factors of the hyperbaric oxygen and the binding were analyzed by the analysis of the variance of the two factors analysis of the factorial analysis. 22 of the groups were compared with LSD- T method. The count data were measured by the rank sum test, and the difference was statistically significant by P0.05. Results 1. the cross viaduct labyrinth showed that the group factors were not statistically significant (P=0.450). There was no significant difference between the hyperbaric oxygen treatment and the blank control group in the anxiety level. The.2. open field test showed that the simple binding group was compared with the blank control group. The score of 11d level (115.5 + 43.4) and vertical score (23.9 + 10.1) were significantly higher (P0.001). Compared with the simple binding group, the score of the HBO group from 1D to 11d decreased (P0.01).3. heart rate variability, and there was no statistical difference between the 4 groups of LF/HF, and there was no significant difference between the sympathetic and the parasympathetic activity. However, the following trend was still seen. The binding and hyperbaric oxygen therapy could lead to the decrease of sympathetic nerve tension, and hyperbaric oxygen therapy could cause the increase of parasympathetic nervous tension in.4. immunofluorescence staining. The expression of GR in the hippocampal area in the simple binding group was less than that of the blank control group (0.2174 + 0.0123) (P0.001), and the combination of hyperbaric oxygen binding group and blank control group was no more than that of the blank control group (P0.001). The significant difference (P0.05).5.HPA axis hormone showed that there was no statistical difference between the 4 groups of serum CRH, ATCH and GCS, but the following trend was found: simple hyperbaric oxygen group, simple binding group, hyperbaric oxygen binding group ACTH and GCS were higher than that of the blank control group, and the level of CRH in hyperbaric oxygen binding group was lower than that of the other three groups. There was no significant difference in O, NOS and TNF- alpha. However, the following trend was still found: the level of NOS in the simple binding group was lower than that of each group. The serum TNF- alpha was lower than that of the simple binding group after the binding combined hyperbaric oxygen treatment. Conclusion there was no significant change in the anxiety level of A. rats after hyperbaric oxygen treatment (1.): there was a slight decrease of sympathetic nervous tension and a pair of pairs in B. rats. There was no obvious change in the expression of GR in the hippocampal region of C.; the level of serum HPA axis hormone in D. rats increased, but NO, NOS and TNF- alpha had no obvious changes. It suggested that hyperbaric oxygen treatment had general stress characteristics, but the stress intensity was not significant in the behavior test of a. rats after the restraint of.2.; B. rats appeared sympathetic God. The tension decreased, the expression of GR in the C. hippocampus decreased, the level of GCS in the serum of D. rats increased, but there was no obvious change in the CRH and ACTH, and the level of NOS in E. rats decreased. It suggested that the chronic restraint in a certain range simulated the physiological state of chronic stress and the anxiety state of A. rats was less than that of the pure binding after the combined binding treatment of the.3. hyperbaric oxygen (HHP); B. rats The level of sympathetic nervous tension decreased, but the degree of decrease was less than that of the simple binding group; the expression of GR in the C. hippocampus was not significantly decreased; the serum ACTH and GCS in D. rats were higher than that in the blank control group, but the CRH level of the combined binding group of the hyperbaric oxygen group was lower than the other three groups and the serum TNF- a was found to be more than the simple binding group after.E. binding hyperbaric oxygen treatment. This indicates that hyperbaric oxygen can slow down the influence of restraint on Physiological Indexes in rats.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R459.6;R749

【共引文献】