抗抑郁药物对实验性脑损伤大鼠的保护作用及机制研究

发布时间：2018-05-12 11:42

本文选题：创伤性脑损伤 + 脑外伤后抑郁　；参考：《第三军医大学》2015年博士论文

【摘要】：背景:创伤性颅脑损伤(Traumatic brain injury,TBI)于全球范围内都是45岁以下年轻人群首要致死和致残病因,TBI后最容易被临床医生所忽视的是精神情感障碍。外伤后抑郁(Post-trauma Depression)作为脑外伤后最常见的神经精神后遗症,目前发生机制尚不明确,也没有疗效明确的治疗手段。在奖励机制、认知、唤醒等方面发挥着重要作用的多巴胺(Dopamine,DA)被认为在抑郁症的发生过程中扮演着关键角色。TBI所造成的继发性脑损伤可能破坏黑质-纹状体通路(Nigrostriatial Pathway),阻碍多巴胺能神经元转运分泌的功能,从而影响中枢神经系统多巴胺系统的功能。我们推断TBI后抑郁的发生和脑内黑质-纹状体通路稳态的破坏密切相关。金刚烷胺,作为临床上应用于帕金森病(Parkinson’s disease,PD)的药物,具有增加多巴胺生成和抑制多巴胺重摄取的作用。近年来,金刚烷胺应用于TBI后意识、认知、运动等功能恢复中的作用引起了学术界的广泛关注。上述证据提示金刚烷胺可能成为TBI后有效的抗抑郁药物。脑出血(intracerebral hemorrhage,ICH)作为死残率极高的突发性疾病,迄今尚无有效的干预策略和治疗手段。ICH造成的局部血肿不仅可以对出血周围脑组织造成直接的应力压迫效应,而且在数小时到数天内引发继发性脑损伤(secondary brain injury)。大量的证据显示继发性脑损伤可能造成迟发性神经功能缺损(delayed neurological deficit),血脑屏障(Blood Brain Barrier,BBB)的破坏及白质纤维束的断裂,而针对继发性脑损伤的治疗是有一定的治疗时间窗。目前探索针对早期脑损伤(early brain injury,EBI)有保护作用的治疗手段显得格外的关键。亚低温治疗作为一种综合治疗手段,已经用于多项临床转化。但最近三项关于物理性亚低温治疗脑外伤的多中心随机临床试验结果都宣告失败,这可能和物理降温属于外源性体温调节导致的寒颤引发应激反应,激活交感神经,释放儿茶酚胺,减少局部脑供血量相关。8-OH-DPAT,药理作用和选择性五羟色胺再摄取抑制剂类抗抑郁药物(Selective Serotonin Reuptake Inhibitor,SSRI)所类似,神经保护作用逐渐被研究者们所认识。化学性降温作用于内源性体温调整系统,直接作用于下丘脑视前区-下丘脑前部(preopticanteriorhypothalamus,po/ah),较物理性降温的降温过程更符合生理过程。第一部分脑外伤后抑郁发生机制及干预目的:研究脑外伤后脑内多巴胺水平对外伤后抑郁发生的影响,并探究金刚烷胺对外伤后抑郁样行为的治疗效果材料和方法:1、健康成年雄性sd大鼠296只,随机分为4组:假手术组、tbi组、金刚烷胺低剂量治疗组和金刚烷胺高剂量治疗组。治疗组术后1小时第1次腹腔注射,之后每8小时给药1次,高剂量组每日注射金刚烷胺135mg/kg,低剂量组每日注射45mg/kg,sham组和tbi组动物予以等量生理盐水作为对照。2、tbi后7d,14d,28d完成头颅磁共振成像,观察动物脑挫伤程度。3、tbi后3d,7d,14d,28d进行行为学实验(蔗糖摄取,旷场试验,强迫游泳),评估动物tbi后抑郁样行为。4、4%多聚甲醛灌注后取脑组织,固定脱水后,完成he染色,fluoro-jadec染色观察tbi后神经元形态。5、westernblot法定量检测tbi后中脑黑质区tyrosinehydroxylase(th)活性。6、免疫荧光技术观察中脑黑质区多巴胺能神经元tbi后凋亡。7、高效液相色谱-电化学法检测tbi后3h,6h,12h,24h,3d,7d,14d,28d伤侧大脑纹状体多巴胺及其代谢产物(dopac和hva)含量。结果:1、tbi后7d,14d,28d,tbi组、tbi+a45组和tbi+a135组各组组间动物脑挫伤体积无明显差异。2、与sham组相比,tbi组于伤后3d,7d,14d,28d抑郁样行为显著增加,而金刚烷胺治疗有效地改善了各个时间点动物呈现出的抑郁样行为。3、he染色显示tbi7d各组纹状体病理形态无明显差异。fjc染色显示手术后7d,tbi组纹状体和中脑黑质神经元大量变性,而金刚烷胺治疗有效地减少了中脑黑质神经元变性但对纹状体神经元变性无显著性治疗效果。4、westernblot结果提示:tbi组较sham组动物中脑黑质区th表达量明显下调(p0.05),金刚烷胺治疗有效地逆转了th的下调趋势。5、中脑黑质区免疫荧光染色显示:tbi组大量的多巴胺能神经元发生凋亡,而金刚烷胺治疗组多巴胺神经元凋亡被有效的抑制了。6、tbi后各个时间点大脑纹状体经高效液相色谱-电化学法测定多巴胺及其代谢产物(dopac和hva)提示:tbi后3h,多巴胺能神经递质有一过性的升高,而在亚急性期及慢性期都呈现持续性下降趋势,只在14d有过反弹性的增高。慢性金刚烷胺的治疗可以有效地逆转这种下降趋势。结论:1、实验性tbi动物在伤后不同时间点呈现出不同程度的抑郁样行为,金刚烷胺治疗有效地改善了tbi所致的抑郁样行为。2、tbi后直接作用的纹状体区,he染色显示:大量的神经元坏死,细胞间水肿明显,fjc染色证明大范围地出现神经元变性。金刚烷胺治疗组纹状体病理形态和tbi组无显著性差异。3、tbi后,动物中脑黑质区多巴胺能神经元变性凋亡明显,同时中脑黑质th活性明显下调。予以金刚烷胺治疗后,中脑黑质变性及凋亡的多巴胺能神经元明显减少,th的活性显著上调。4、高效液相色谱-电化学法结果显示:tbi动物纹状体内多巴胺及其代谢产物含量呈下降趋势,而金刚烷胺治疗可以逆转tbi后纹状体多巴胺水平的降低。第二部分化学降温剂对脑出血早期脑保护研究目的:本课题旨在8-oh-dpat发挥的降温作用对ich后ebi及bbb的破坏是否具有保护作用,并比较化学性降温剂和物理性亚低温治疗对ich动物的脑保护效果。材料和方法:1、健康成年雄性sd大鼠207只,随机分为假手术组(sham)、脑出血组(ich)、8-oh-dpat治疗组(dpat)、物理性亚低温治疗组(hypo)。dpat组术后6小时完成第1次腹腔给药,之后每天腹腔注射8-oh-dpat(1mg/kg)。hypo组于术后6小时开始亚低温治疗,每日治疗12小时。sham组和ich组动物予以等量生理盐水作为对照。2、ich后24h,72h进行神经功能评分和转杆试验,观察运动功能缺损。3、伤后7d进行morris水迷宫试验,评估动物ich后学习记忆能力。4、ich后6h,12h,24h,72h分别测量核心体温和颅脑体表温度。5、术后72h电镜观察海马区神经元亚显微形态和结构。6、ICH后24h,72h分别测量动物脑水含量及BBB通透性。7、ICH后72h,应用Western Blot法检测出血灶周围IL-1β及TNF-α表达量。结果:1、大鼠ICH模型可在早期造成神经功能和学习记忆障碍,伴随脑温局部性升高。2、自体血注射模型引起显著的急性脑水肿和BBB破坏,伴随海马神经元大量变性坏死,出血周围炎症因子表达增加。3、物理性亚低温治疗一定程度上改善了ICH造成的神经功能和学习记忆能力缺损,保护海马区神经细胞免于变性坏死,但对ICH造成的早期BBB破坏的保护效果不明确。4、8-OH-DPAT治疗对ICH后脑温升高的调节过程平缓且持续,可以有效地减轻ICH后动物神经功能和学习记忆能力的损伤,保护了ICH造成的BBB通透性增加,减少了血肿周围炎症因子的表达。结论:1、大鼠ICH模型在损伤早期可造成脑温升高,同时引起神经功能缺损和学习记忆能力降低,伴随BBB的破坏和海马区神经元坏死,提示脑温异常可以是早期脑损伤的重要机制。2、化学性降温剂8-OH-DPAT在ICH早期的脑保护作用优于物理性亚低温治疗,可以有效地保护BBB的损伤,减少神经元的变性坏死,减轻脑水肿发生,改善动物ICH后神经功能和学习记忆能力的缺损,说明直接作用于体温调节中枢的温度干预可能成为中枢神经系统损伤的一个新的治疗靶点。
[Abstract]:Background: Traumatic brain injury (TBI) is the leading cause of death and disability of young people under 45 years of age. After TBI, the most likely to be neglected by clinicians is the mental and emotional disorder. Post traumatic depression (Post-trauma Depression) is the most common neuropsychiatric sequelae after brain trauma, and is currently occurring at present. The mechanism is not clear, and there is no effective treatment. Dopamine (DA), which plays an important role in the reward mechanism, cognition, and arousal, is considered to be a key role of.TBI in the process of depression. The secondary brain injury may break the substantia nigra striatum pathway (Nigrostriatial Pathway), and prevent it. The functions of dopaminergic neurons transport and secrete and affect the function of the dopamine system in the central nervous system. We infer that the occurrence of depression after TBI is closely related to the destruction of the homeostasis of the substantia nigra striatal pathway in the brain. Amantadine, a drug that is used clinically in Parkinson's disease (Parkinson 's disease, PD), has an increase in dopamine production. In recent years, the role of amantadine in TBI, cognitive, exercise and other functional recovery has attracted extensive attention in the academic world. The above evidence suggests that amantadine may become an effective antidepressant after TBI. Intracerebral hemorrhage (ICH) is a very high death rate. The local hematoma caused by.ICH, which has so far no effective intervention strategy and treatment, can not only cause direct stress stress on the brain tissue around the hemorrhage, but also cause secondary brain damage (secondary brain injury) within several hours to several days. A large number of evidence shows that secondary brain injury may cause late onset deity. After functional defect (delayed neurological deficit), the destruction of the blood brain barrier (Blood Brain Barrier, BBB) and the fracture of the white matter fiber bundle, there is a certain time window for the treatment of secondary brain injury. At present, the treatment of the early brain injury (early brain injury, EBI) is the key to the treatment of the secondary brain injury (early brain injury, EBI). As a comprehensive treatment, hypothermia therapy has been used for multiple clinical transformations, but the recent three multicenter randomized clinical trials of physical hypothermia treatment have failed, which may be associated with physical hypothermia due to exogenous thermoregulation induced stress response, activation of sympathetic nerve, and catechol release. Amines, reducing local brain blood supply related.8-OH-DPAT, pharmacological action and selective antidepressant (Selective Serotonin Reuptake Inhibitor, SSRI) of the selective five hydroxytryptamine reuptake inhibitor (Reuptake Inhibitor, SSRI), the neuroprotective effect is gradually recognized by the researchers. Chemical hypothermia acts on the endogenous hypothalamus, directly acting on the hypothalamus The preoptic region of the anterior hypothalamus (preopticanteriorhypothalamus, po/ah) is more suitable for physiological processes than physical cooling. Part 1: the mechanism and intervention of post traumatic depression: the study of dopamine levels in the brain after traumatic brain injury and the effect of amantadine on posttraumatic depression like behavior Treatment effect materials and methods: 1, 296 healthy adult male SD rats were randomly divided into 4 groups: sham operation group, TBI group, low dose amantadine treatment group and high dose amantadine treatment group. The treatment group was injected 1 hours and first times after operation, then 1 times every 8 hours, high dose group daily injection of amantadine 135mg/kg, low dose group daily injection. 45mg/kg, group sham and group TBI were given equal amount of normal saline as control.2, 7d, 14d, and 28d completed head magnetic resonance imaging after TBI, and observed the degree.3 of brain contusion in animals, 3D, 7d, and 7d. After dehydration, he staining was completed, fluoro-jadec staining was used to observe neuronal morphology after TBI, and Westernblot method was used to detect Tyrosinehydroxylase (th) active.6 in the mesencephalic substantia nigra after TBI, and the apoptosis of dopaminergic neurons in the mesencephalic substantia nigra region was observed by immunofluorescence technique. The content of dopamine and its metabolites (DOPAC and HVA) in the cerebral striatum of 28d. Results: 1, TBI after TBI, 7d, 14d, 28d, TBI, tbi+a45 group and tbi+a135 group, there was no significant difference in the volume of brain contusion between the groups of tbi+a45 and tbi+a135 groups. .3, he staining showed no significant difference in the pathomorphology of the striatum in tbi7d, and.Fjc staining showed that the striatum and the substantia nigra neurons in group TBI were significantly denatured after the operation, while amantadine therapy effectively reduced the degeneration of the substantia nigra neurons in the middle brain but had no significant treatment for the degeneration of the striatum neurons. The results of therapeutic effect.4, Westernblot results showed that the TH expression of th in group TBI was significantly lower than that in group sham (P0.05), and amantadine therapy effectively reversed the downward trend.5 of th. The immunofluorescence staining in the medium brain substantia nigra region showed that a large number of dopaminergic neurons in the group TBI were apoptotic, and the apoptosis of dopamine neurons in the amantadine group was also apoptotic. The.6 was effectively suppressed. After TBI, the brain striatum measured dopamine and its metabolites (DOPAC and HVA) by high performance liquid chromatography electrochemistry (DOPAC and HVA). After TBI 3h, the dopaminergic neurotransmitter had an excessive increase in the dopaminergic neurotransmitter, while both in the subacute and chronic stages showed a persistent downward trend, only in 14d with a higher anti elasticity. The treatment of chronic amantadine can effectively reverse this downward trend. Conclusion: 1, experimental TBI animals present different degrees of depressive behavior at different time points after injury. Amantadine therapy effectively improves the depressive behavior caused by TBI,.2, the striatum, which is directly affected by TBI, and he staining shows that a large number of neurons are necrotic. The intercellular edema was obvious, and FJC staining showed that the neuron degeneration appeared in a large range. There was no significant difference between the pathological morphology of the striatum and the TBI group in the treatment group of amantadine.3. After TBI, the degeneration of dopaminergic neurons in the substantia nigra area of the animal was obvious, and the activity of th in the middle cerebral substantia nigra was significantly lower. The apoptosis of dopaminergic neurons significantly decreased, the activity of th increased significantly by.4. The results of high performance liquid chromatography electrochemistry showed that the content of dopamine and its metabolites in the striatum of TBI animals decreased, while amantadine could reverse the decrease in the level of dopamine in the striatum after TBI. The second part of the chemical cooling agent was early on the brain hemorrhage. Objective: the purpose of this study is to investigate whether the cooling effect of 8-OH-DPAT plays a protective role on the destruction of EBI and BBB after ICH, and compares the protective effects of chemical cooling agents and physical hypothermia on ICH animals. Materials and methods: 1, 207 healthy adult male SD rats were randomly divided into sham operation group (sham) and cerebral hemorrhage. Group (ICH), 8-OH-DPAT treatment group (DPAT), physical hypothermia treatment group (hypo) group.Dpat after 6 hours to complete first times of intraperitoneal administration, then daily intraperitoneal injection of 8-OH-DPAT (1mg/kg).Hypo group at 6 hours to begin mild hypothermia treatment, 12 hours of daily treatment of.Sham group and ICH group of normal saline as a control.2, ICH after, proceed into the ICH .3 and 7d maze test after injury were performed to evaluate the learning and memory ability of animals after ICH and.4, 6h, 12h, 24h, 72h after ICH, respectively, to measure the temperature of the core and the temperature of the body surface in the brain after ICH, and the submicroscopic morphology and structure of the neurons in the hippocampus were observed after the operation. The brain water content, BBB permeability.7 and 72h after ICH were used to detect the expression of IL-1 beta and TNF- alpha around the hemorrhagic foci by Western Blot. Results: 1, the rat ICH model could cause neurological function and learning memory impairment in the early stage, with the brain temperature local increase.2, the autologous blood injection model led to significant acute cerebral edema and BBB destruction, accompanied by hippocampus God. After massive degeneration and necrosis, the expression of inflammatory factors around the hemorrhage increased.3. Physical hypothermia treatment improved the neural function and learning memory impairment caused by ICH to some extent and protected the hippocampal neurons from degeneration and necrosis, but the protective effect of the early BBB damage caused by ICH was not clear on the.4,8-OH-DPAT treatment of the ICH brain. The adjustment process of temperature rise is slow and continuous. It can effectively reduce the damage of nerve function and learning and memory ability after ICH, protect the increase of BBB permeability caused by ICH and reduce the expression of inflammatory factors around hematoma conclusion: 1, the rat ICH model can make the brain temperature rise at the early stage of injury and cause nerve function defect and learning at the same time. The decrease of memory ability, the destruction of BBB and the necrosis of neurons in the hippocampus suggest that abnormal brain temperature can be an important mechanism of early brain injury,.2. The protective effect of chemical cooling agent 8-OH-DPAT in early ICH is better than that of physical hypothermia. It can effectively protect the injury of BBB, reduce the degeneration and necrosis of neurons and reduce the occurrence of brain edema. To improve the impairment of neural function and learning and memory ability after ICH, it is suggested that the temperature intervention directly acting on the center of thermoregulation may be a new therapeutic target for the injury of central nervous system.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R651.15

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