丙泊酚对新生小鼠大脑梨状皮层中间神经元的影响及相关作用研究

发布时间：2018-01-15 20:07

本文关键词：丙泊酚对新生小鼠大脑梨状皮层中间神经元的影响及相关作用研究　出处：《第三军医大学》2016年硕士论文　论文类型：学位论文

【摘要】：研究背景:丙泊酚(Propofol)是一种短效的、新型快速的全身麻醉药物,因其起效迅速、作用时间短、苏醒快、术后恶心呕吐发生率低而被广泛应用于临床麻醉及ICU镇静。近年来,麻醉药物对发育大脑认知、学习记忆的影响是大家关注的热点问题。临床研究结果表明,两岁以下婴幼儿全麻药物暴露可致较长时间的人格及行为改变。然而,全麻药物丙泊酚所致神经毒性的机制仍不明确。大脑梨状皮层(Piriform cortex)位于前脑腹外侧,源于古皮质,属边缘系统,是哺乳动物最大的嗅觉皮层,接受嗅球的单突触输入,且与相关皮层及海马间存在复杂的纤维投射。梨状皮层由三层皮质组成,第一层称为网织层,主要接受外侧嗅束的投射纤维,第二、三层称为细胞层,主要接收外侧嗅束、相邻皮层的兴奋输入及局部中间神经元的抑制传入。发育期大脑梨状皮层神经前体细胞具有增殖及分化发育为成熟神经元的能力,该皮层同海马及室管膜下区一样存在神经发生的潜能。此外,梨状皮层的细胞层主要由数量较多的释放谷氨酸的椎体神经元及数量较少释放γ-氨基丁酸的中间神经元组成。梨状皮层的中间神经元按其细胞表面标记物主要分为钙结合蛋白(PV—Parvalbumin、CB—Calbindin、CR—Calretinin)中间神经元和神经肽(SOM—Somatostatin、NPY—Neuropeptide Y、CCK—Cholecystokinin)中间神经元两大类。该皮层内上述中间神经元的功能颇为复杂,涉及树突的传入、轴突的输出以及各中间神经元的远程联接。正是因为对信息传入、输出的调控,该区域内的中间神经元在嗅觉的学习、记忆过程中起重要作用。既往研究发现,对阿尔茨海默病(Alzheimer’s disease,AD)患者而言,其嗅觉损害症状早于认知障碍的发生,并且其大脑梨状皮层SOM及CR中间神经元与AD病理性标记物β-淀粉样蛋白(Amyloidβ,Aβ)及Tau蛋白之间存在共同标记,这提示梨状皮层中间神经元在AD的发生及发展中的易损性及其与认知障碍的密切相关性。此外,多项研究证实,尿烷(Urethane)麻醉可导致大脑梨状皮层选择性的中间神经元损伤,这可能与麻醉所致的认知功能损害相关。换而言之,梨状皮层可能是麻醉药物发挥其神经毒性作用新的靶区。神经行为障碍(neurobehavioraldisorders)是由多种与大脑疾病或损伤相关联的行为功能损害组成。神经行为学(neuroethology)则是对动物的行为及神经系统机制研究的方法,目的是通过动物的特殊行为学改变了解中枢神经系统(centralnervoussystem,cns)病变。既往研究表明,新生期吸入麻醉药七氟烷暴露不仅引发学习认知功能受损,还可导致类似自闭症样的社交行为学异常。结合本课题组前期研究成果:丙泊酚抑制发育大脑海马齿状回神经干细胞的增殖,进一步探究丙泊酚对认知相关行为的影响及其可能的机制。研究方法:本课题首先采用出生后7天(postnatal7,p7)的新生小鼠进行丙泊酚(30mg/kg组及60mg/kg组)或脂肪乳腹腔注射以建立丙泊酚模型,应用c-fos免疫组织化学染色方法筛选丙泊酚对发育大脑的激活靶区及定性该靶区内易损神经元的类别;同样地,采用免疫组织化学方法检测丙泊酚激活区域(梨状皮层)神经前体细胞增殖以明确丙泊酚对发育大脑梨状皮层神经发生的影响。此外,建立丙泊酚多次注射模型,即p7-p9连续注射丙泊酚并饲养至成年以观察发育期丙泊酚暴露对远期神经行为的影响。采用食物埋藏实验(buriedfoodtest)检测丙泊酚模型小鼠与对照组小鼠的嗅觉识别及嗅觉学习记忆功能;采用社交实验(sociabilityandpreferencefornoveltytest)检测丙泊酚模型小鼠与对照组小鼠的社会行为及社会偏好;采用新物体识别实验(novelobjectrecognitiontest)检测丙泊酚模型小鼠与对照组小鼠的认知记忆功能。最后,应用免疫组织化学方法检测p21小鼠大脑梨状皮层易损神经元的改变以探讨丙泊酚影响发育大脑神经行为可能的机制。研究结果:1、丙泊酚激活发育大脑梨状皮层中间神经元(1)c-fos免疫组织化学结果表明:与脂肪乳对照组小鼠相比,丙泊酚30mg/kg组(p0.05)及丙泊酚60mg/kg组(p0.05)小鼠大脑梨状皮层c-fos+细胞均显著增多。(2)c-fos与neun及gfap双重标记免疫组织化学结果表明:三组间,neun标记的成熟神经元数量无差异;丙泊酚30mg/kg组及丙泊酚60mg/kg组与脂肪乳对照组相比c-fos与neun双重标记阳性细胞数量均显著增多(p0.05);然而,c-fos与gfap无共标阳性细胞。(3)c-fos与cb、cr、som、npy双重标记免疫组织化学结果表明:丙泊酚30mg/kg组及丙泊酚60mg/kg组与脂肪乳对照组相比c-fos与cb双重标记阳性细胞数量均显著增多(p0.01);然而,c-fos与cr中神经元及神经肽类中间神经元标记物som、npy之间几乎没有共标阳性细胞。2、丙泊酚对发育大脑梨状皮层神经前体细胞增殖的影响(1)brdu免疫组织化学结果表明:与脂肪乳对照组小鼠相比,丙泊酚30mg/kg组(p0.05)及丙泊酚60mg/kg组(p0.01)小鼠大脑梨状皮层brdu+细胞均显著减少;且与丙泊酚30mg/kg组相比,丙泊酚60mg/kg组小鼠大脑梨状皮层brdu+细胞减少更为显著(p0.05)。(2)sox2与brdu双重标记免疫组织化学结果表明:与脂肪乳对照组小鼠相比,丙泊酚30mg/kg组小鼠大脑梨状皮层sox2+细胞无显著差异,丙泊酚60mg/kg组小鼠大脑梨状皮层sox2+细胞则减少显著(p0.01)。同样地,brdu-sox2双标阳性细胞数量丙泊酚30mg/kg组与脂肪乳对照组相比,改变无显著差异,而丙泊酚60mg/kg组则显著减少(p0.01)。(3)nestin免疫组织化学结果表明:与脂肪乳对照组小鼠相比,丙泊酚30mg/kg组及丙泊酚60mg/kg组小鼠大脑梨状皮层nestin标记神经干细胞纤维突起分布稀疏且排列紊乱(p0.01)。3、发育期丙泊酚麻醉暴露对成年期神经行为学的影响及可能机制(1)食物埋藏实验结果表明:丙泊酚30mg/kg组小鼠与对照组小鼠相比探索埋藏食物的潜伏期无明显改变,然而丙泊酚60mg/kg组小鼠与对照组及丙泊酚30mg/kg组小鼠相比,探索埋藏食物的潜伏期则明显增加(p0.01,p0.05)。(2)社交实验结果表明:对照组小鼠较喜欢呆在有小鼠的一侧箱体(p0.05),而丙泊酚30mg/kg组及丙泊酚60mg/kg组小鼠在两侧箱体的时间则无差异;在该实验的第二阶段即社会新奇偏好性测试中,对照组小鼠较偏好与陌生小鼠交流(p0.01),而丙泊酚30mg/kg组及丙泊酚60mg/kg组小鼠则未有如此表现。(3)新物体识别实验结果表明:对照组小鼠新物体的探索时间较旧物体探索时间明显增多(p0.001),而丙泊酚30mg/kg组及丙泊酚60mg/kg组小鼠则无明显偏好性。换言之,丙泊酚30mg/kg组及丙泊酚60mg/kg组小鼠较对照组小鼠新物体分辨率明显下降(p0.001)。(4)p21小鼠大脑切片免疫组织化学染色结果显示,丙泊酚30mg/kg组及丙泊酚60mg/kg组小鼠大脑梨状皮层cb中间神经数量均显著减少,提示梨状皮层cb中间神经元在发育期丙泊酚麻醉作用中表现出特异性地易损性。研究结论:上述研究结果提示:丙泊酚通过抑制发育大脑梨状皮层神经前体细胞增殖及激活该区域内CB中间神经元,进而影响远期认知、学习、记忆功能。
[Abstract]:Background: Propofol (Propofol) is a short acting, all new fast narcotic drugs, because of its rapid onset, short duration, fast recovery, low incidence of postoperative nausea and vomiting have been widely used in clinical anesthesia and sedation in ICU. In recent years, anesthetics on brain development of cognition, learning and memory effect is a hot issue of concern. The clinical research results show that the change of infants under two general anesthesia drug exposure can cause a long time personality and behavior. However, the mechanism of general anesthesia of propofol induced neurotoxicity is not clear. The brain in piriform cortex (Piriform cortex) is located in the ventrolateral forebrain, originated from the ancient cortex, belongs to the limbic system, is the largest mammalian olfactory cortex, single synaptic input accept of the olfactory bulb, and there are projections associated with complex cortex and hippocampus. The piriform cortex consists of three layers of cortex, the first layer is called the net The main layer, accept projection fibers in the lateral olfactory tract, the second, third layer is a layer of cells, mainly for receiving the lateral olfactory tract, inhibition of excitatory inputs and local interneurons adjacent cortical afferent. During the development of brain in piriform cortex neural precursor cells proliferation and differentiation ability of mature neurons, in the hippocampus and cortex of the same the subventricular zone exists neurogenesis potential. In addition, interneurons in piriform cortex cell layer is mainly composed of a large number of glutamate release vertebral neurons and fewer release GABA interneurons. Pear shaped skin layer according to its cell surface markers consists of calcium binding protein (PV - Parvalbumin, CB - Calbindin, CR - Calretinin) and neuropeptide interneurons (SOM - Somatostatin, NPY - Neuropeptide Y, CCK Cholecystokinin). The two types of interneurons in the cortex. The intermediate neuron function is quite complex, involving afferent dendrites, axons and neurons of the intermediate output remote join. It is precisely because of the incoming information, output control, the area of the interneurons in the olfactory learning, plays an important role in the process of memory. Not only to study found in Alzheimer's disease (Alzheimer 's disease, AD) patients, the olfactory impairment symptoms of early cognitive dysfunction in the brain, and piriform cortex SOM and CR neurons and AD pathological markers of beta amyloid (Amyloid beta, beta A) are common markers between Tau and protein, suggesting that the close relationship between the vulnerability of piriformis cortical interneurons in the occurrence and development of AD in and cognitive disorders. In addition, a number of studies have confirmed that urethane (Urethane) anesthesia can cause brain damage among neurons in piriform cortex selectively, which may be related to Related to cognitive impairment caused by anaesthesia. In other words, the piriform cortex may be the anesthetic drug exerts its neurotoxic effects of new target areas. Neurobehavioral disorders (neurobehavioraldisorders) is composed of a variety of disease or injury related behavior and brain function damage. Combined neural behavior (neuroethology) is a method of behavior and neural mechanism Research on animal, animal behavior through special purpose is to observe the histological change of central nervous system (centralnervoussystem, CNS) disease. Previous studies showed that neonatal exposure not only caused seven halothane inhalation anesthetics learning impaired cognitive function, can also lead to similar social behavior in autism kind of abnormal. Combined with the previous research results: propofol inhibits development of hippocampus neural stem cell proliferation, to further explore the effects of propofol on cognitive behavior and The possible mechanism. Methods: firstly, using 7 days after birth (postnatal7, P7) of newborn mice were propofol (group 30mg/kg and group 60mg/kg) model was established by propofol or Intralipid intraperitoneal injection, c-fos immunohistochemistry screening of propofol on activation of target area and the qualitative target area of vulnerable neurons the categories of brain development; similarly, the detection of propofol activation region by immunohistochemical method (piriform cortex) affect the proliferation of neural precursor cells to determine propofol on brain development in piriform cortex neurogenesis. In addition, the establishment of multiple propofol injection model, namely p7-p9 continuous injection of propofol and reared to adulthood in order to observe the development of propofol effects of exposure on neurobehavioral food. The burial experiment (buriedfoodtest) detection of propofol and the control group of mice model mice olfactory recognition and olfactory Feel the learning and memory function; the social experiment (sociabilityandpreferencefornoveltytest) detection model mice and control mice with social behavior and social preference; the new object recognition test (novelobjectrecognitiontest) detection of propofol mice and control mice cognitive memory function. Finally, the mechanism of immunohistochemical detection of p21 mouse brain piriform cortex in vulnerable neurons in order to investigate the effect of propofol may change the development of brain behavior. Results: 1, the development of brain activation of propofol in piriform cortex neurons (1) c-fos immunohistochemistry results showed that compared with fat emulsion group mice, propofol 30mg/kg group (P0.05) and propofol group (60mg/kg P0.05) in brain of mouse piriform cortex c-fos+ the cells were significantly increased. (2) c-fos and NeuN and GFAP double labeling immunohistochemistry results showed that Between three groups, no difference in the number of mature neurons labeled with NeuN; group 30mg/kg propofol and propofol 60mg/kg group and control group compared to the number of fat emulsion c-fos and NeuN double labeled positive cells were significantly increased (P0.05); however, c-fos and GFAP were labeled positive cells. (3) c-fos and CB, Cr, SOM. NPY double labeling immunohistochemistry results showed that propofol 30mg/kg group and propofol group 60mg/kg with fat emulsion group compared to the number of c-fos and CB double labeled positive cells were significantly increased (P0.01); however, c-fos and Cr in neurons and neuropeptides in neuronal markers SOM, NPY almost no labeled.2 positive cells. Effect of propofol on brain development in piriform cortex the proliferation of neural precursor cells (1) BrdU immunohistochemistry results showed that compared with fat emulsion group mice, propofol 30mg/kg group (P0.05) and propofol group (60mg/kg P0.01) in mouse brain The piriform cortex brdu+ cells were significantly reduced; compared with propofol 30mg/kg group, propofol group 60mg/kg mice brain piriform cortex brdu+ cells reduced significantly (P0.05). (2) Sox2 and BrdU double labeling immunohistochemistry results showed that compared with the fat milk control mice, there was no significant difference between the propofol group 30mg/kg mice brain the piriform cortex sox2+ cells, propofol group 60mg/kg mice brain piriform cortex of sox2+ cells decreased significantly (P0.01). Similarly, the number of brdu-sox2 positive cells in group 30mg/kg propofol and Intralipid were compared with the control group, no significant difference in change, and propofol 60mg/kg group decreased significantly (P0.01). (3) nestin immunohistochemistry the results showed that: compared with chemical fat emulsion group mice, propofol 30mg/kg group and propofol group 60mg/kg mice brain piriform cortex nestin neural stem cells labeled fibers projecting sparse and arranged orderly Chaos (P0.01).3, during the development of propofol exposure on the neurobehavioral effects of adult learning and the possible mechanism of buried food (1) the experimental results show that the propofol 30mg/kg group mice compared with control mice had no obvious change in the exploration of buried food latency, however compared with propofol 60mg/kg group with the control group and propofol group 30mg/kg mice. Exploration of buried food latency increased significantly (P0.01, P0.05). (2) social experiment results showed that the control group of mice than love stay in the side box with the mouse (P0.05), and the time on both sides of the box of propofol 30mg/kg group and propofol group 60mg/kg mice had no significant difference; in the second phase of the experiment that the social novelty preference test, the mice of control group were unfamiliar with the preference of mice (P0.01), and the exchange of propofol 30mg/kg group and propofol group 60mg/kg mice were not so. (3) a new object recognition experiment The results showed that the mice of control group new object of exploration time than the old object exploration time increased significantly (p0.001), and propofol 30mg/kg group and propofol group 60mg/kg mice had no obvious preference. In other words, propofol group 30mg/kg and propofol in 60mg/kg group decreased significantly compared with the control group of mice (p0.001). The new resolution (4 p21 mouse brain slices) immunohistochemical staining showed that the number of propofol 30mg/kg group and propofol group 60mg/kg mice brain piriform cortex CB intermediate nerve were significantly reduced, suggesting that showing vulnerability specific piriform cortex CB interneurons in the development period of anesthetic action of propofol. Conclusions: the results suggest that propofol by inhibiting the development of brain nerve anterior piriform cortex and the region of CB interneurons activated cell proliferation, thereby affecting the long-term cognitive, learning, and memory.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R614

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