【作者】 黄志强；

【导师】 刘景昌； 方以群； 蔺世龙； 姚健； 张理义； 李宁；

【作者基本信息】 第二军医大学 ， 航空、航天和航海医学， 2009， 博士

【摘要】 目的抑郁症是精神科常见病,在军事作业人员(包括航海人员)中也有发生。其治疗问题是精神医学界探索的一个焦点。目前,重症抑郁特别是内因性抑郁的治疗以药物为主,药物几乎都是通过单胺假说筛选出来的,主要有三类:①三环类(tricylic antidepressants, TCAs);②单胺氧化酶抑制剂(monoamine oxidase inhibitors, MAOIs);③选择性5-羟色胺再摄取抑制剂(selective serotonin reuptake inhibitors, SSRIs)。它们主要通过增加脑内神经突触间隙的5-羟色胺(5-hydroxytryptamine, 5-HT)或去甲肾上腺素(norepinephrine, NE)浓度,并使相应受体的敏感性下调,从而发挥抗抑郁作用。也有极个别的药物,如丁胺苯丙酮,在抑制NE再摄取的同时,还抑制多巴胺(dopamine, DA)这一种单胺的再摄取。无论使用何种药物,都遇到一个难题,即服药2周左右才起效。另外,部分患者由于其他疾病的原因而不适合采用药物治疗。此外,临床上还可见到部分“难治性抑郁症”,对常规的药物治疗无反应。可见,深入研究抑郁症的病因、机理,探讨安全有效的新疗法,具有重要的理论意义与应用价值。高压氧(hyperbaric oxygenation, HBO)可以增加氧在机体的溶解和弥散,改善微循环,并影响到细胞超微结构,对神经系统、内分泌、心血管等产生广泛的效应。合理应用HBO,可以治疗或者辅助治疗多种疾病。但是,将HBO用于抑郁症的治疗,目前报道很少。由于HBO对中枢神经系统有较大影响,而且增加正常机体血脑屏障的通透性,从而对精神药物发挥效用也有一定作用,所以我们考虑将HBO试用于治疗发病与中脑边缘系统改变有关的抑郁症。高压氧舱在我国已走向普及,若HBO能用于辅助治疗抑郁症,将有良好的应用前景,可以减少药物或其他疗法的副作用。本课题旨在通过建立抑郁症动物模型,进行HBO疗效的观察,并与经典抗抑郁药物比较,从而客观、科学地研究HBO治疗抑郁症的效用,并探讨其可能的作用机制。方法1.雄性SD大鼠120只,在体重筛选及旷场试验后选择行为学评分相近的80只,区组化随机分为正常对照组、抑郁症模型组、HBO治疗组、氟西汀药物治疗组、HBO与氟西汀联合治疗组。正常对照组按常规饲养,不接受刺激和治疗,只接受测试。其余4组大鼠均接受慢性温和不可预见应激(chronic unpredicted mild stress,CUMS)(CUMS在第30d模型建立后停止),并接受相应处置:HBO治疗组与联合治疗组均接收HBO治疗:纯氧加压至0.2MPa,停留60min,以5min匀速减压至常压,每天1次,7次为一疗程。模型组、氟西汀治疗组置于相同的容器,放进同型号的另一氧舱内,舱门打开不加压。氟西汀治疗组与联合治疗组用氟西汀按l.8 mg/kg灌胃,每日一次,每10d根据各组测得的平均体重调整一次剂量。正常对照组不灌胃,模型组、HBO治疗组用等量蒸馏水灌胃。2.每10d检测一次行为学指标(体重、摄食量、糖水偏爱度、旷场行为),监测抑郁症模型的建立成功与否,并监测大鼠抑郁症状的变化。3. 50d后麻醉处死动物,取材,用放射免疫法(radioimmunoassay, RIA)检测血浆中皮质醇(cortisol, CORT)、促肾上腺皮质激素(adrenocorticotrophic hormone, ACTH)的浓度,并用高效液相色谱法(high performance liquid chromatography, HPLC)检测脑组织中5-HT、NE、DA这三种单胺类神经递质的浓度,再用酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)检测脑组织中色氨酸羟化酶(tryptophan hydroxylase, TPH)、酪氨酸羟化酶(tyrosine hydroxylase, TH)浓度,还用分光光度计检测了单胺氧化酶(monoamine oxidase, MAO)活力,最后用显微镜观察了海马CA1、CA3和齿状回的病理形态学变化。结果1.第30d,各实验组大鼠的体重均显著低于对照组( P<0.001);抑郁症模型组的糖水偏爱度显著低于对照组(P<0.05),水平运动显著低于对照组(P<0.05),垂直运动显著低于对照组(P<0.01),理毛次数也显著低于对照组(P<0.001)。模型组的上述行为学指标显著劣于正常对照组,表明抑郁症大鼠模型已经建立。此后停止刺激,只进行治疗和检测。2.到第40d , 3个治疗组的糖水偏爱度均显著超过抑郁症模型组(P<0.01);HBO治疗组的理毛次数显著超过其他三个实验组(P<0.01),这种变化保持到D50。到第50d,HBO治疗组、氟西汀治疗组的水平运动均显著优于模型组(P<0.05),HBO治疗组的垂直运动显著优于抑郁症模型组(P<0.05),而此时氟西汀治疗组的垂直运动与模型组之间尚无显著性差异。上述结果提示HBO在改善抑郁症大鼠对“快感”、“奖赏”的反应方面同氟西汀疗效相当,而在改善抑郁症大鼠的“满足感”和探究行为方面先于氟西汀起效。至于HBO与氟西汀联合应用的疗效,在第20d,联合治疗组的垂直运动显著少于对照组(P<0.05),而此时,其它实验组与对照组无显著性差异;到第30d,联合治疗组的垂直运动不仅显著低于正常对照组(P<0.01),而且显著低于HBO治疗组(P<0.05)。然而在糖水偏爱度这一指标,联合治疗组没有出现类似变化,相反,到第50d,联合治疗组仍显著高于模型组,而由于模型组糖水偏爱度的提高,此时其它两个治疗组与模型组之间的差异不显著。联合治疗组在垂直运动与糖水偏爱度这两个指标方面的差异,提示HBO与氟西汀联用能提高抑郁症大鼠对“奖赏”和“快感”的反应,但可能降低抑郁症大鼠的兴奋性(即无助于抑郁症大鼠活动减少的症状)。不过,对待这一结论需持谨慎态度,因为联合治疗组垂直运动的初始值就较低(尽管与其它组相比没有显著性差异)。HBO与氟西汀联合应用还产生另一现象:联合治疗组“摄食比”较低,在第20d显著低于模型组(P<0.01)、HBO治疗组(P<0.01)和氟西汀治疗组(P<0.01),在第30d显著低于HBO治疗组(P<0.05),在第40d显著低于模型组(P<0.01),提示联用HBO增强了氟西汀的抑制食欲作用。2.各组大鼠在下丘脑、海马、颞叶和顶叶四个脑区的5-HT浓度均无显著性差异(P>0.05)。NE浓度的变化显示:在下丘脑,HBO治疗组显著高于抑郁症模型组(P<0.05);而在颞叶,模型组显著低于正常对照组(P<0.01),且显著低于联合治疗组和氟西汀治疗组(P<0.05);在顶叶,模型组显著低于HBO治疗组(P<0.05)。DA浓度的变化显示:在海马,HBO治疗组显著低于正常对照组(P<0.05);在颞叶,组间差异最显著,HBO治疗组显著低于正常对照组(P<0.05)和氟西汀治疗组(P<0.01),联合治疗组最低,与正常对照组(P<0.05)、抑郁模型组(P<0.05)、氟西汀治疗组(P<0.01)相比均有显著性差异。3.各组大鼠脑组织TH浓度均无显著性差异(P>0.05)。正常对照组与抑郁症模型组的TPH浓度均较低,与HBO治疗组、氟西汀药物治疗组或者联合治疗组比较,有显著性差异(P<0.05)。正常对照组、HBO治疗组与氟西汀药物治疗组的MAO活力均未升高,但是,抑郁症模型组同联合治疗组的MAO活力显著升高(P<0.001)。结论1.本研究通过慢性不可预见温和应激,成功制备了抑郁症大鼠动物模型。2. HBO暴露对改善慢性应激抑郁症大鼠的抑郁症状有良好效用:在促进食欲方面显著优于氟西汀,在改善快感丧失、动机不足、兴趣缺乏、活动减少等抑郁症状方面至少与氟西汀相当,此外在增进探究行为和修饰行为方面先于氟西汀起效。3.HBO改善大鼠抑郁症状可能是通过增加脑内NE神经递质浓度实现的,而与脑组织整体中5-HT浓度无关。HBO改善抑郁症状的机制与治疗抑郁症的经典药物氟西汀作用机制不同,表现为:氟西汀增加抑郁症大鼠血浆中的ACTH、CORT浓度,而HBO未显示此种作用;HBO组在广泛脑区(下丘脑、颞叶、顶叶)NE浓度均显著升高,但氟西汀治疗组NE仅在颞叶超过模型组;HBO降低海马和颞叶的DA浓度,而氟西汀增加颞叶的DA浓度。病理形态学结果还提示,HBO抗抑郁效用可能与其拮抗慢性应激所致海马神经元受损有关。4.HBO增加脑组织NE的浓度,可能系通过抑制其降解酶MAO(monoamine oxidase,单胺氧化酶)活力,而非增加其合成限速酶TH(tyrosine hydroxylase,酪氨酸羟化酶)浓度实现的。5.额外,本研究证实了经典抗抑郁药物氟西汀除了通常认为的选择性抑制5-HT再回收的药理作用之外,还能增加部分脑区NE含量以及脑组织中5-HT合成酶TPH(tryptophan hydroxylase,色氨酸羟化酶)的含量。更多还原

【Abstract】 ObjectiveDepression has a high incidence, and it has been burdening too much on the society as well as the patients. So far, drugs play a dominant role to cure Depression Major, especially to cure those with endogenous reasons. The drugs include:①tricylic antidepressants (TCAs);②monoamine oxidase inhibitors (MAOIs);③selective serotonin reuptake inhibitors (SSRIs). They mainly make influences by increasing the concentration of 5-hydroxytryptamine (5-HT) or norepinephrine (NE) in brain’s synaptic clefts, and downward regulating their receptors’sensitivity. A very small number of antidepressants, such as Bupropion, have a pharmacological mechanism related to the monomine dopamine (DA).Whatever drug administrated, a big problem baffles the therapy: it will cost the patient about 2 weeks for the drug to make a difference.Besides, we can see some patients have no reaction to normal anti-depressants. So, many efforts have been done to find new paths to cure depression.Hyperbaric oxygen (HBO) can magnify the solution and diffusion in organisms, improve the microcirculation, and influence the cell’s ultramicrostructure, making extensive effects on nervous, endocrine and cardiovascular systems. HBO has successfully cured some diseases, or played a supporting but important role to treat many diseases.HBO has a significant influence to central nervous system, and it can improve the permeability of blood-brain barrier (so, it can magnify the pharmacodynamic action of psychotropic drugs). So, we are considering using HBO to treat depression, whose etiology has much to do with the pathological changes of mesolimbic system. Due to the popularization of HBO chambers in China, HBO will have a bright future to benefit the patients, if it has a curative effect for depression.So, we decided to make a depressed rat model, and then treat it with HBO, and compare the effectivity with which of a classic anti-depressant, and look for the mechanism of the effectivity (if there is any).Methods1. Eighty male SD rats were selected by open-field test, etc., from 120 ones, to be randomized distributed into 5 groups: Control, Model, HBO treatment, Fluoxetine treatment, Combined treatment (both HBO and Fluoxetine). The Control group was normally bred, receiving only tests, not stimuli or therapies. The other 4 groups of rats received chronic unpredicted mild stress (CUMS) with corresponding therapies:HBO was taken by both HBO group and Combined group: Sodium lime was put at the bottom of the small animal HBO chamber to absorb CO2; pure oxygen was consumed to“wash”the chamber, and then was used to lift the inside pressure to 0.2MPa. 60min passed (during the span, pure oxygen was used to ventilate the chamber for 10min), 5min was taken to reduce the pressure to normal. One time a day, 7 days a course. When the rats were treated with HBO, the Model group or Fluoxetine group were put into a similar box, which was put into another chamber of the same making, but the chamber gate was open, and no pure oxygen was supplied.Fluoxetine, a classic anti-depressant, was given to Fluoxetine group and Combined group, with a dosage of 1.8 mg/kg for every morning (the dose was changed every 10d according to the body weight measured), by gavage. The Model group and the HBO group were given a gavage of distilled water of the same volume with the drug. The Control group didn’t receive gavage.2. Behavioral tests were administrated every 10 days, to monitor the emergence, changes or fadeaway of depression symptoms. 3. Thirty days later, behavioral changes showed that the animal model had been successfully established. Then, the stress was ceased. 50 days later, all of the animals were executed with mercy, and plasma or brain samples were taken, to detect plasma stress hormone such as cortisol (CORT) and adrenocorticotrophic hormone (ACTH) by radioimmunoassay (RIA), and to detect brain monoamine neurotransmitters such as 5-HT, NE and DA by high performance liquid chromatography (HPLC), and to detect the concentration of tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH) by enzyme linked immunosorbent assay (ELISA), and to detect the activity of monoamine oxidase (MAO) by spectrophotometer. Last but not least, a Nyssl’s staining was conducted to observe the morphological changes of the neurons in hippocampal CA1, CA3 and dentate gyrus area.Results1. Since D30, the bodyweight of Control group was very significantly heavier than those of another 4 groups ( P<0.001); Compared with Control group, Model group had a lower sucrose preference ( P<0.05) and fewer horizontal locomotion ( P<0.05), fewer vertical activities ( P<0.01), fewer grooming acts ( P<0.001) . These changes manifested the depression rat modeling was successful. Then, the stimuli were ceased, only therapies continued.2. Since D40, the 3 groups with therapies had their sucrose preference higher than Model group ( P<0.01) ; HBO group had their grooming acts much more than the other 3 experimental groups ( P<0.01) , and kept this advantage till the end. On D50, the horizontal locomotion of HBO group and Fluoxetine group was significantly frequent than Model group ( P<0.05) ; the vertical activities of HBO group was better than Model group ( P<0.05), while that of Fluoxetine had no this difference.The interaction between HBO and Fluoxetine were a little complicated. On D20, the vertical activities of Combined group were fewer than that of Control group ( P<0.05), while the other 2 experimental groups had no such a difference; On D30, the vertical activities of Combined group were not only fewer than Control group ( P<0.01) , but also than HBO group (P<0.05). As for sucrose preference, Combined group didn’t show such a change, on the contrary, it kept this indicator significantly higher than Model group, while the other 2 groups with therapies showed no significant difference with Model group (since Model group also lifted its sucrose sucrose preference after the stimuli ceased). These two opposite changes indicates that Fluoxetine with HBO can improve rats’responsiveness to pleasant sensation or“rewards”, but in the meantime, the combination maybe reduce depressed rats’activeness. We have to deal with this conclusion cautiously, because Combined group showed fewer intitial rearings (although the difference was not statistically significant). Another phenomenon also interested us, that the Combined group continuously had a lower grazing rate: on D20, lower than Model group ( P<0.01), HBO group (P<0.01) or Fluoxetine group (P<0.01); On D30, lower than HBO group (P<0.05); On D40, lower than Model group (P<0.01), indicating the combination with HBO perhaps synergized the anorectic effect of Fluoxetine.3. The 5-HT concentration of different brain areas of different groups (P>0.05), but the NE concentration showed some difference: in hypothalamus, HBO group was higher than Model group (P<0.05); in temporal lobe, Model group was lower than Control and Combined (P<0.05); in parietal lobe, Model group was lower than Combined and Fluoxetine (P<0.05). As for the concentration of DA, in hippocampus, HBO group was lower than Control group (P<0.05); in temporal lobe, HBO group was lower than Control group (P<0.05) and Fluoxetine group (P<0.01), Combined group was lower than Control group (P<0.05), Model group (P<0.05) and Fluoxetine group (P<0.01).4. The TH concentration had no significant difference among those groups in any brain areas (P>0.05). The TPH concentration of Control group and which of Model group were lower than HBO group, Fluoxetine group or Combined group (P<0.05). MAO activity was not activated in Control, HBO or Fluoxetine group, while it was highly lifted up in Model and Combined groups (P<0.001). Conclusions1. The CUMS-induced depression rats modeling was successful.2. HBO showed some therapeutic effect for chronic stress depression rats: compared with Fluoxetine, it was better to improve appetite, it had similar effects to cure anhedonia, motivelessness, inactiveness, and it had a faster respondence of depressed rats to improve self-satisfaction and exploratory activities.3. HBO maybe cure those depression symptoms by increasing the concentration of NE, the neurotransmitter, in brain. It suggests that HBO’s anti-depressant mechanisms are different from which of Fluoxetine, the prototypical anti-depressant classified as SSRIs. The evidence includes: HBO didn’t lifted up ACTH, CORT level in plasm, while Fluoxetine did; HBO increased NE concentration in hypothalamus, temporal lobe and parietal lobe, while Fluoxetine only did that in temporal lobe; HBO reduced DA concentration in hippocampus and temporal, while Fluoxetine increased the concentration in temporal lobe. Besides, HBO showed some protecting effects to the pathologically morphological changes caused by CUMS.4. HBO increased NE concentration in brain, perhaps by inhibiting MAO (which decomposes NE), other than by increasing TH (which synthesize NE).5. As a serendipity, we also confirmed Fluoxetine can increase TPH in rat brain, beside of its SSRI pharmacology.更多还原