应激反应与抑郁症中下丘脑的性别差异：针对人脑样本和应激动物的研究

发布时间：2018-06-03 22:04

本文选题：抑郁症 + 下丘脑　；参考：《浙江大学》2016年博士论文

【摘要】：第一部分抑郁症患者下丘脑视交叉上核GABA改变的研究背景：抑郁症又称情感障碍,主要包括重型抑郁障碍(major depressive disorder, MDD)和双相情感障碍(bipolar disorder, BD)。抑郁症的主要发病机制包括过度激活的应激反应调节系统,即下丘脑-垂体-肾上腺(hypothalamo-pituitary-adrenal, HPA)轴。抑郁症的另一主要病理特征是生物钟也即下丘脑视交叉上核(suprachiasmatic nucleus, SCN)功能紊乱,导致睡眠一觉醒节律紊乱以及进食障碍等症状。血管加压素(arginine vasopressin, AVP)是SCN合成的主要神经肽之一。在人类和动物下丘脑都观察到SCN神经元有向下丘脑室旁核(paraventricular nucleus, PVN)的直接或间接神经投射,提示SCN与PVN之间具有相互调节作用。此外,这两个核团均受到来自下丘脑其他部位例如下丘脑食欲素(orexin)系统的神经支配,提示下丘脑内由神经递质和神经调质所介导的神经环路功能障碍。值得注意的是,尽管SCN神经元几乎均为γ-氨基丁酸(Gamma amino butyric acid, GABA)能神经元,迄今为止抑郁症患者SCN内GABA变化尚未获得研究,它们与下丘脑orexin及其和PVN活性改变之间的关联也尚未被研究。因此,本研究旨在阐明抑郁症患者下丘脑SCN中GABA能变化及其和SCN内神经肽即AVP、下丘脑orexin变化之间的关联。方法：在13位抑郁症患者(6位MDD,7位BD)和13位按照性别、年龄、死亡时间、组织固定和贮存时间等参数良好匹配的对照组的下丘脑SCN中,采用免疫细胞化学法(immunocytochemistry, ICC)测定SCN内GABA能合成关键酶,即谷氨酸脱羧酶(glutamic acid decarboxylase, GAD)65/67-免疫反应性(immunoreactivity, ir)和AVP-ir;采用原位杂交技术(in situ hybridization, ISH)测定SCN内GAD67-mRNA水平,采用图像分析方法(image analysis)定量研究上述参数。对SCN数据和我们先前研究的相同个体的下丘脑orexin-ir表达水平做相关分析。结果：AVP-ir神经元和神经纤维广泛分布于下丘脑SCN, PVN、视上核(supraoptic nucleus, SON)以及位于PVN和SON之间的附加核(accessory nuclei). GAD65/67-ir神经纤维密集分布于丘脑、下丘各核团包括SCN、PVN、SON等。GAD67-mRNA-ISH信号也分布于丘脑以及下丘脑的SCN, PVN、SON等核团。抑郁症患者SCN内GAD65/67-ir较对照组显著上升约54%(P= 0.044), GAD67-mRNA较对照组显著上升165%(P=0.029)。此外,抑郁症女性患者SCN内AVP-ir较女性对照显著相升高155%(P=0.008),而抑郁症男性SCN内中AVP-ir和男性对照相比没有显著差异。在对照组,SCN-AVP-ir和年龄呈显著负相关(Rho=-0.745, P=0.003),男性抑郁症组也观察到SCN-AVP-ir和年龄之间的显著负相关(Rho=-0.766, P= 0.027),而女性抑郁症组内这种相关性消失。此外,在男性对照组下丘脑orexin-ir和SCN内GAD65/67-ir呈显著正相关(Rho= 0.790, P= 0.020),女性对照组内则无此相关性。上述所有参数在MDD和BD患者之间相比无显著差异。结论：抑郁症患者SCN内GABA显著升高(表现为GAD65/67-ir和GAD67-mRNA均显著升高)可能在SCN功能紊乱中扮演重要角色；SCN内AVP-ir以及下丘脑orexin-ir的性别相关性异常表达也和抑郁症中的生物学节律紊乱及HPA轴活性改变的性别差异密切相关。第二部分Sprague-Dawley大鼠应激反应中的性别差异：下丘脑和外周激素改变背景：已有研究表明,性别差异在抑郁症中扮演着重要角色,过度激活的应激反应是抑郁症的重要发病机制。应激反应调节系统下丘脑—垂体-肾上腺(hypothalamo-pituitary-adrenal, HPA)轴在抑郁症中活性异常,下丘脑促肾上腺皮质激素释放素(corticotropin-releasing hormone, CRH)过度激活。而个体的过度应激也可以导致抑郁症发生和/或加剧。我们组和其他研究组的先前研究发现,性激素例如雌二醇(estradiol, E2),睾酮(testosterone, T)可以通过其相应受体即雌激素受体α或者β(estrogen receptor α, β; ERa, ERβ)、雄激素受体(androgen receptor, AR)而作用于CRH基因启动子调节CRH基因表达,进而调节HPA轴活性。雌激素促进CRH基因表达而雄激素抑制该过程,这种机制可能是应激反应中的性别差异以及抑郁症发病性别差异的基础。为了进一步利用应激动物模型进行抑郁症相关研究,我们探索了大鼠急、慢性应激反应中的性别差异,测定血浆皮质酮(corticosterone, CORT)以及性激素(T和E2)水平；测定下丘脑应激相关分子包括ERα、ERβ、AR、芳香化酶(aromatase, ARO)—将雄激素转化为雌激素的关键酶、CORT受体包括盐皮质激素受体(mineralcorticoid receptor,MR)、糖皮质激素受体(glucocorticoid receptor, GR),以及神经肽CRH、血管加压素(arginine vasopressin, AVP)和催产素(oxytocin, OXT)的mRNA表达改变。方法：雌性Sprague-Dawley (SD)大鼠随机分组：将慢性不可预知性温和应激(chronic unpredicted mild stress, CUMS)组、急性足底电击(foot shock, FS)组以及对照组三组再细分为动情前期、动情间期牺牲组(共6组)。雄性SD大鼠随机分为组：CUMS组、FS组以及对照组(共3组)。CUMS组大鼠在三周应激以后与对照组进行行为学测定,包括旷场实验、糖水偏好实验。牺牲各组大鼠留取躯干血、下丘脑等样本。采用酶联免疫吸附法测定血浆CORT,放射性免疫法测定血浆T和E2,采用实时定量PCR (Quantitative reverse transcription PCR, Q-PCR)法测定下丘脑应激相关分子ERα、ERβ、AR、ARO、MR、GR、CRH、AVP和OXT的mRNA表达水平。结果：CUMS导致雌鼠的性周期紊乱,表现为动情间期延长。相比于雄鼠,雌鼠在行为学测试中显示更多的焦虑、抑郁样行为；雌性CUMS鼠比雄性CUMS鼠具有更多的下丘脑应激相关分子显著改变包括CRH-mRNA显著升高(P=0.05),MR-mRNA和GR-mRNA显著减低(P=0.003,P=0.001)等；而雄鼠CUMS组AVP-mRNA显著高于雄性对照组(P0.001)。CUMS、FS雌鼠血浆CORT相对于基础水升高的幅度显著大于雄鼠(雌鼠,CUMS组为对照组水平的18.7倍,FS组34.3倍；雄鼠,CUMS组1.8倍,FS组3.6倍)。CUMS和FS应激后,雌鼠血浆E2和T水平显著降低(P ≤0.039),而FS雄性大鼠的E2水平有升高趋势(P=0.056), CUMS雄性大鼠的血浆T水平显著降低(P=0.0047)。结论：急性或慢性应激导致SD大鼠在行为学、血浆激素以及下丘脑应激相关分子表达等方面表现出明显性别差异性变化,和人类应激反应、抑郁症中性别差异改变之间存在许多符合处。第三部分戊巴比妥钠或异氟烷麻醉对大鼠应激反应研究结果的影响背景：应激反应紊乱与抑郁症发病之间的关联促进了人们对应激动物模型的研究。调节应激反应的关键系统下丘脑—垂体—肾上腺(hypothalamo-pituitary-adrenal, HPA)轴在抑郁症中过度激活。研究发现,性激素例如睾酮(testosterone,T)和雌二醇(estradiol, E2)以及神经肽例如促肾上腺皮质激素释放素(corticotropin-releasing hormone, CRH),精氨酸血管加压素(arginine vasopressin, AVP)和催产素(oxytocin, OXT)等都在应激反应调节中起重要作用。值得注意的是,在应激反应的研究中,牺牲动物前使用麻醉剂是常用的试验方法,然而麻醉剂对应激反应研究结果的影响尚未获得明确结论。有报道麻醉剂可以通过抑制脑内兴奋性受体例如天冬氨酸(N-Methyl-D-Aspartate, NMDA)受体亚型NR2B、神经元型烟碱样乙酰胆碱受体(neuronal-type nicotinic acetylcholine receptor, nnAChR),以及激活脑内抑制性受体例如γ-氨基丁酸(Gamma amino butyric acid, GABA) A受体(GABAA receptor, GABAAR)而影响脑功能。迄今为止,常用的牺牲实验动物前所采用的麻醉方法,例如腹腔注射戊巴比妥钠麻醉、异氟烷气体吸入麻醉等是否影响急性应激性反应研究中所获得的参数结果尚属未知。本文旨在研究不同的牺牲动物的方式,即直接断头牺牲、腹腔注射戊巴比妥钠后断头牺牲、异氟烷吸入麻醉后断头牺牲对急性应激反应研究结果的影响,包括对血浆应激激素皮质酮(corticosterone, CORT)、血浆性激素T和E2激素水平的影响,以及对下丘脑应激相关分子如CRH、AVP、OXT的mRNA表达,以及额叶应激相关分子如NR2B、nnAChR、GABAAR的mRNA表达的影响。方法：雄性Sprague-Dawley (SD)大鼠随机分为足底电击(foot shock, FS)组和对照组,并根据牺牲大鼠的方法进一步分亚组：直接断头牺牲组、腹腔注射戊巴比妥钠后断头牺牲组、异氟烷吸入麻醉后断头牺牲组(共6组)。此外还设立非应激对照的腹腔注射生理盐水后断头牺牲组。留取躯干血、下丘脑和额叶标本。采用酶联免疫吸附法测定血浆CORT,放射性免疫法测定血浆T,酶免疫法测定血浆E2,实时定量PCR (Quantitative reverse transcription PCR, Q-PCR)法测定下丘脑应激相关分子CRH、AVP和OXT以及前额叶应激相关分子NR2B,GABAAR和nnAChR-mRNA表达的改变。结果：在直接断头牺牲组和异氟烷吸入后牺牲组,FS显著升高了血浆CORT水平(P0.001),而在戊巴比妥钠腹腔注射牺牲组,未观察到FS对血浆CORT水平的显著影响(P=0.132)。在对照组中,腹腔注射生理盐水或者腹腔注射戊巴比妥钠都显著升高了血浆CORT的水平(P=0.008,P=0.004)。性激素和脑内应激相关分子的mRNA水平在各组之间均无无显著性差异(P≥0.132)。结论：腹腔注射,而非麻醉药物本身,导致了动物的额外应激。这种额外应激导致血浆CORT水平升高,使得原本由急性FS应激所导致的血浆CORT水平在应激组和对照组之间的显著差异“消失”,因而干扰了研究结果。这类麻醉方法并未对血浆性激素或者脑中应激相关分子mRNA的表达造成显著影响。异氟烷气体吸入麻醉对急性应激反应无显著影响。从动物伦理学角度考虑,异氟烷气体吸入麻醉后牺牲是一项较优的实验方法。
[Abstract]:The study background of GABA changes in the hypothalamic suprachiasmatic nucleus in the first part of depression: depression is also called affective disorder, mainly including heavy depressive disorder (major depressive disorder, MDD) and bipolar affective disorder (bipolar disorder, BD). The main pathogenesis of depression includes the overly activated stress response regulation system, that is, the hypothalamus. The brain - pituitary - adrenal (hypothalamo-pituitary-adrenal, HPA) axis. Another major pathological feature of depression is the dysfunction of the suprachiasmatic nucleus (SCN) in the hypothalamus, which leads to the symptoms of sleep disorder and eating disorders. The vasopressin (arginine vasopressin, AVP) is a SCN synthesis. One of the major neuropeptides. In both human and animal hypothalamus, SCN neurons have direct or indirect nerve projections to the paraventricular paraventricular nucleus (paraventricular nucleus, PVN), suggesting the interaction between SCN and PVN. In addition, these two nuclei are derived from other parts of the hypothalamus, such as the hypothalamus orexin (orexin) system. It is worth noting that although SCN neurons are almost all Gamma amino butyric acid (GABA) neurons, the changes in SCN GABA in the patients with depression so far have not been studied, and they are with the hypothalamus orexin. The association between PVN activity and its activity has not been studied. Therefore, this study aims to elucidate the correlation between the changes in the GABA energy in the hypothalamus SCN and the changes in the SCN neuropeptide, AVP, and the hypothalamus orexin. Methods: in 13 depressive patients (6 MDD, 7 BD) and 13 according to sex, age, death time, and tissue fixation In the hypothalamic SCN of the control group with good matching of storage time and other parameters, immunocytochemistry (ICC) was used to determine the key enzyme of GABA in SCN, that is, glutamic acid decarboxylase, GAD 65/67- immunoreactivity (immunoreactivity, IR). Ridization, ISH) measured the level of GAD67-mRNA within SCN, using the image analysis method (image analysis) to quantify the above parameters. The correlation analysis of the SCN data and the level of the hypothalamic orexin-ir expression in the same individuals we previously studied. Results: AVP-ir neurons and nerve fibers were widely distributed in the hypothalamus SCN, PVN, supra nuclear (supraoptic). Nucleus, SON) and the additional nucleus (accessory nuclei) between PVN and SON. The GAD65/67-ir nerve fibers are densely distributed in the thalamus, and the nuclei of the lower colliculus, including SCN, PVN, SON, etc., are also distributed in the thalamus and the hypothalamus. .044), GAD67-mRNA was significantly higher than that of the control group (P=0.029). In addition, the SCN AVP-ir in SCN was significantly higher in women with depression than in the female control (P=0.008), while there was no significant difference between AVP-ir and men in the SCN of depression men. In the control group, there was a significant negative correlation between SCN-AVP-ir and age (Rho=-0.745, P=0.003) and male depression in the control group. The group also observed a significant negative correlation between SCN-AVP-ir and age (Rho=-0.766, P= 0.027), but this correlation disappeared in the female depression group. In addition, there was a significant positive correlation between the orexin-ir and SCN in the hypothalamus orexin-ir and SCN in the male control group (Rho= 0.790, P= 0.020), and there was no such correlation in the female control group. All the parameters were in MDD and BD. There is no significant difference between patients. Conclusion: the significant increase in SCN GABA in the patients with depression (showing a significant increase in GAD65/67-ir and GAD67-mRNA) may play an important role in SCN dysfunction; the abnormal expression of AVP-ir in SCN and the sex correlation of orexin-ir in the hypothalamus is also associated with the biological rhythm disorder and HPA axis in the depression. Gender differences in sexual changes are closely related. Second the gender differences in the stress response in the second part of the rats: the changes in the hypothalamus and peripheral hormones: Previous studies have shown that gender differences play an important role in depression, and excessive activation of stress response is an important pathogenesis of depression. The hypothalamic - pituitary - adrenal (hypothalamo-pituitary-adrenal, HPA) axis is abnormally active in depression, and the hypothalamic corticotropin releasing hormone (corticotropin-releasing hormone, CRH) is excessively activated. And individual overstress can also lead to depression and / or aggravation. Our group and other research groups have previously studied Now, sex hormones such as estradiol, E2, and testosterone (testosterone, T) can be used to regulate the gene expression by the receptor (estrogen receptor alpha, beta, ERa, ER beta), androgen receptor (androgen receptor, AR) by its corresponding receptors, and then regulate the activity of the axis. This mechanism may be the basis of gender differences in stress responses and gender differences in depression. In order to further use stress animal models to study depression related studies, we explored the sexual differences in acute and chronic stress responses in rats, and the determination of plasma corticosterone (corticosterone, CORT). ) and the levels of sex hormones (T and E2); the determination of hypothalamic stress related molecules including ER alpha, ER beta, AR, and aromatase (aromatase, ARO) - the key enzymes that convert androgens into estrogen, CORT receptors including the corticosteroid receptor (mineralcorticoid receptor, MR), glucocorticoid receptors, and neuropeptides, MRNA expression changes of arginine vasopressin (AVP) and oxytocin (oxytocin, OXT). Methods: female Sprague-Dawley (SD) rats were randomly divided into three groups: chronic unpredictable mild stress (chronic unpredicted mild stress) group, acute foot shock group, and control group, and then divided into estrus. The male SD rats were randomly divided into groups: group CUMS, group FS and control group (3 groups), after three weeks of stress, the male rats were randomly divided into groups, including the open field experiment and the sugar water preference experiment. The rats left the trunk blood, the hypothalamus and other samples were sacrificed by the enzyme linked immunosorbent assay. The plasma T and E2 were determined by radioimmunoassay, and the expression level of the hypothalamic stress related molecules ER a was measured by real-time quantitative PCR (Quantitative reverse transcription PCR, Q-PCR). Results: the sexual cycle disorder of the female rats was caused by the prolongation of the interval of estrus. Compared with the male rats, it was compared with the male rat. The female mice showed more anxiety and depressive behavior during the behavioral test, and the significant changes in the hypothalamic stress related molecules in female CUMS mice were significantly higher than that of the male CUMS rats (P=0.05), MR-mRNA and GR-mRNA significantly decreased (P=0.003, P=0.001), while the AVP-mRNA of male rats in group CUMS was significantly higher than that of the male control group (P0.001).C. UMS, the increase of plasma CORT in FS female rats was significantly greater than that of the male rats (female rats, 18.7 times the level of the control group in the CUMS group, 34.3 times in the FS group, 1.8 times in the male, the CUMS group and 3.6 times in the FS group), and the level of E2 and T in the female rats decreased significantly after the.CUMS and FS stress (P < 0.039). The level of plasma T in rats was significantly decreased (P=0.0047). Conclusion: acute or chronic stress results in obvious gender differences in behavior, plasma hormone and hypothalamic stress related molecules expression in SD rats, and human stress response. There are many conforms between the changes of gender differences in depression. Third partial glutba is found. The influence of sodium or isoflurane on the results of stress response study in rats: the association between stress disorder and depression promotes the study of stress animal models. The hypothalamus pituitary adrenal (hypothalamo-pituitary-adrenal, HPA) axis, the key system regulating stress response, excesses depression The study found that sex hormones such as testosterone (T) and estradiol (estradiol, E2) as well as neuropeptides such as corticotropin-releasing hormone (CRH), arginine vasopressin (arginine vasopressin, AVP), and oxytocin (oxytocin, OXT) all play an important role in the regulation of stress response. It is worth noting that in the study of stress reactions, the use of anesthetics before animals is a common test method. However, the effects of the anesthetics on the results of the stress response have not been clearly concluded. It is reported that anesthetics can be used to inhibit the brain excitability, such as the N-Methyl-D-Aspartate, NMDA receptor subtype NR 2B, neuron type nicotinic acetylcholine receptor (neuronal-type nicotinic acetylcholine receptor, nnAChR), and activation of brain inhibitory receptors, such as gamma aminobutyric acid (Gamma amino butyric acid, GABA) A receptors, which affect brain power. So far, the common anesthetic was used before the sacrifice of experimental animals. Methods, such as whether intraperitoneal injection of pentobarbital sodium, or isoflurane inhalation anaesthesia, whether the results of the acute stress response study are unknown. This article aims to study the different ways of sacrificing animals, namely, direct end of the head sacrifice, intraperitoneal injection of pentobarbital, and disconnection after inhalation of isoflurane. The effects of head sacrifice on the results of acute stress response, including the effects on plasma stress hormone corticosterone (corticosterone, CORT), plasma sex hormone T and E2 levels, as well as the expression of mRNA in the hypothalamic stress related molecules such as CRH, AVP, OXT, as well as the effect of frontal stress related molecules such as NR2B, nnAChR, GABAAR mRNA. The male Sprague-Dawley (SD) rats were randomly divided into the sole electric shock (foot shock, FS) group and the control group. According to the method of sacrificing rats, the rats were further divided into subgroups: direct head sacrifice group, intraperitoneal injection of pentobarbital sodium after intraperitoneal injection of head sacrifice group and 6 groups after isoflurane inhalation anesthesia (a total of 6 groups). Plasma CORT was measured by enzyme linked immunosorbent assay, plasma T was measured by radioimmunoassay, plasma E2 was measured by enzyme immunoassay, and PCR (Quantitative reverse transcription PCR, Q-PCR) was used to determine hypothalamic stress related molecules CRH, AVP and OXT as well as OXT, as well as in the sample of blood, hypothalamus and frontal lobes. Changes in the expression of prefrontal stress related molecules NR2B, GABAAR and nnAChR-mRNA. Results: FS significantly increased plasma CORT level (P0.001) in the direct end of the head sacrifice group and after the inhalation of isoflurane, while in the intraperitoneal injection of pentobarbital sodium, the significant effect of FS on the plasma CORT level was not observed (P=0.132). In the control group, the abdominal cavity was in the control group. Intraperitoneal injection of saline or intraperitoneal injection of pentobarbital sodium significantly increased the level of plasma CORT (P=0.008, P=0.004). There was no significant difference in mRNA levels between sex hormones and brain stress related molecules (P > 0.132). Conclusion: intraperitoneal injection, not an anesthetic itself, leads to additional stress in animals. The plasma levels of plasma CORT, which was caused by acute FS stress, were "disappearing" between the stress group and the control group, thus disturbing the results. This type of anesthesia did not significantly affect the expression of plasma sex hormones or the expression of stress related molecules in the brain, mRNA, and isoflurane gas absorption. Anesthesia has no significant effect on acute stress response. From the perspective of animal ethics, sacrifice after isoflurane inhalation anesthesia is a better experimental method.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R749.4

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