外伤性脑损伤模型大鼠中经颅磁刺激对内源性神经干细胞作用的研究

发布时间：2018-05-30 05:26

本文选题：外伤性脑损伤 + 经颅磁刺激　；参考：《北京协和医学院》2014年博士论文

【摘要】：研究背景和目的外伤性脑损伤(traumatic brain injury, TBI)在所有人群中都很常见,不分年龄、背景或健康状况。时代发展使得交通事故愈加频繁,TBI必然会成为我国一个沉重的公共卫生问题。影响TBI患者功能恢复的最大障碍是脑实质缺失。虽然脑损伤时机体内源性神经干细胞会增殖,但增殖水平有限且增殖后的存活率很低。促进内源性神经干细胞增殖和存活或许是治疗TBI的一个可行策略。经颅磁刺激(transcranial magnetic stimulation, TMS)是一种无创脑刺激(noninvasive brain stimulation, NBS)技术,通过激活神经元或改变神经元兴奋性而发挥神经调控作用。在恰当的参数下,TMS能影响大脑局部血流,改变局部氧化应激水平,调节神经再塑(neuroplasticity)。另外,有研究提示TMS具有促进神经再生的能力。基于TMS已知的多种作用,结合TBI的病理生理过程,可得出TMS具有治疗TBI潜能的推论。但至今没有用TMS治疗TBI的研究发表,也没有专门的研究探讨TMS是否能影响TBI后内源性干细胞增殖。本研究尝试用重复经颅磁刺激(rTMS)治疗中度TBI大鼠,重点观察大脑内源性神经干细胞增殖情况。方法成年雄性SD大鼠,体重210-260g,用Feeney自由落体打击法造成中度TBI。用改良神经功能缺失评分(mNSS)评价大鼠PO1、PO7、PO14和P028的行为学表现。根据PO1行为评分,将大鼠分层随机分为对照组和经颅磁刺激组(TMS组)。从PO2开始行TMS,参数如下：电压700V,频率5Hz,TMS组每只大鼠每日接受900个脉冲刺激。在PO7、PO14、PO28三个时间点将大鼠分批处死,生理盐水和4%多聚甲醛心脏灌流后取脑,石蜡包埋切片,进行HE和免疫组化染色。利用HE切片测定冠状面脑组织面积,以此计算损伤侧脑组织相对健康侧的减少率。免疫组化染色指标包括细胞增殖标志物BrdU,神经干细胞标志物musashil,成熟神经元标志物NeuN,细胞凋亡标记物caspase-3。少数在PO14处死的大鼠(n=5)取脑后单独剥离SVZ行免疫荧光染色,染色指标包括BrdU、DAPI、DCX(神经元前体细胞标志物)和laminin(血管标志物)。BrdU、NeuN、caspase-3和DCX染色情况用视野内的阳性细胞数评价(阳性细胞密度),musashil用视野内阳性面积占全部脑组织面积的比例评价(阳性面积比)。部分大鼠在P02和P013行18F-FDG micro-PET。结果 (1)TMS组意外死亡率有明显低于对照组的倾向(15.79%vs37.14%,p=0.09)。 (2)大鼠的mNSS评分在损伤后PO1-PO14逐渐改善,TMS组vs对照组,P07的评分近似(7.00±1.27vs6.20±1.92),而PO14评分有TMS组明显更低的倾向(3.38±1.47vs5.40±1.14,p=0.085)。 (3)外伤性脑损伤后大鼠损伤侧大脑半球的脑组织相对减少率逐渐提高,其中P028时TMS组有明显低于对照组的倾向(32.80±3.0%vs38.59±3.2%,p=0.083)。 (4)免疫组化染色中,两组损伤侧脑室背外角的BrdU阳性细胞密度在三个时间点均无显著差别；损伤侧脑室背外角musashil阳性面积比从P07到P028逐渐减少,两组间比较,对照组P07时高于TMS组,而P014和P028时低于TMS组,差别都不具有统计学意义；两组损伤灶周边区NeuN阳性细胞密度在三个时间点均无显著差别；损伤灶周边Caspase-3阳性细胞密度,三个时间点对照组的平均值都低于TMS组,其中P014时有对照组明显低于TMS组的倾向(p=0.062)。 (5)P014取SVZ进行的免疫荧光染色中,对照组BrdU和DCX阳性细胞密度平均值都低于TMS组,差别没有统计学意义。 (6)P02时,对照组和TMS组损伤侧大脑皮层和纹状体代谢水平都较正常侧减低；P013时,两组损伤侧皮层代谢水平都接近于正常侧,而纹状体代谢水平都较正常侧升高。对照组和TMS组之间比较无明显差别。结论对于遭受中度TBI的大鼠,TMS表现出了以下有利趋势：(1)明显降低大鼠TBI后死亡率的趋势,(2)明显改善TBI后行为恢复的趋势,(3)明显减少TBI后损伤侧脑实质丢失的趋势,这些结果与基于已有研究结果和TBI病理生理作出的推论相符,但对于另一个推论,即促进TBI后内源性神经干细胞增殖,本研究结果不能作为支持。TMS对刺激停止后24h的脑局部代谢无明显影响。
[Abstract]:Background and purpose of research
Traumatic brain injury (TBI) is common in all people, without age, background or health. Time development makes traffic accidents more frequent. TBI will inevitably become a heavy public health problem in China. The biggest obstacle to the function recovery of TBI patients is the loss of brain parenchyma. Although brain damage is in the body, the body is damaged. Endogenous neural stem cells proliferate, but the proliferation level is limited and the survival rate after proliferation is very low. Promoting the proliferation and survival of endogenous neural stem cells may be a feasible strategy for the treatment of TBI.
Transcranial magnetic stimulation (TMS) is a non-invasive brain stimulation (noninvasive brain stimulation, NBS) technology that plays neural regulation by activating neurons or changing neuronal excitability. Under appropriate parameters, TMS can affect the local blood flow of the brain, change the level of local oxidative stress, and regulate the nerve again. Plastic (neuroplasticity). Furthermore, some studies suggest that TMS has the ability to promote nerve regeneration. Based on a variety of known effects of TMS, combined with the pathophysiological process of TBI, the inference of TMS for the treatment of TBI potential is obtained. However, there has been no research published by TMS in the treatment of TBI, and there is no study on whether TMS can affect the endogenous dry after TBI. Cell proliferation.
In this study, repetitive transcranial magnetic stimulation (rTMS) was used to treat moderate TBI rats. The proliferation of endogenous neural stem cells in the brain was observed.
Method
Adult male SD rats, weight 210-260g, use Feeney free falling body strike method to cause moderately TBI. modified nerve function loss score (mNSS) to evaluate the behavioral performance of PO1, PO7, PO14 and P028 in rats. According to the PO1 behavior score, the rat stratification was randomly divided into the control group and the transcranial magnetic stimulation group (TMS group). The parameters were as follows: voltage: voltage 700V, frequency 5Hz, group TMS received 900 pulses per day. At the three time points of PO7, PO14, and PO28, the rats were executed in batches, the brain was taken after the perfusion of physiological saline and 4% polyformaldehyde, and the paraffin embedded sections were embedded into HE and immunohistochemical staining. The relative brain tissue area of the injured side was measured by HE section to calculate the relative brain tissue in the injured side. The reduction rate of the health side. The immunohistochemical staining indexes include cell proliferation marker BrdU, neural stem cell marker musashil, mature neuron marker NeuN, and apoptotic marker caspase-3. in PO14 executed rats (n=5) separately stripped to SVZ and immunofluorescence staining, and the staining indexes include BrdU, DAPI, DCX (neuron precursor). Cell markers) and laminin (vascular markers).BrdU, NeuN, Caspase-3, and DCX staining were evaluated by positive cells in the field of vision (positive cell density), and musashil used the positive area of the visual field to evaluate the proportion of the total area of the brain (positive area ratio). Some rats were in P02 and P013 18F-FDG micro-PET..
Result
(1) the accidental death rate of group TMS was significantly lower than that of the control group (15.79%vs37.14%, p=0.09).
(2) the mNSS score of rats was gradually improved after the injury, and the vs control group in group TMS was similar to (7 + 1.27vs6.20 + 1.92) in the vs control group, while the PO14 score was significantly lower in TMS group (3.38 + 1.47vs5.40 + 1.14, p=0.085).
(3) after traumatic brain injury, the relative decrease rate of brain tissue in the injured cerebral hemisphere was increased gradually, and the tendency of TMS group was significantly lower than that of the control group at P028 (32.80 + 3.0%vs38.59 + 3.2%, p=0.083).
(4) in immunohistochemical staining, there was no significant difference in the density of BrdU positive cells in the two groups of the lateral ventricle of the lateral ventricle at three time points, and the musashil positive area of the lateral ventricle of the injured lateral ventricle decreased gradually from P07 to P028, compared with the group TMS in the control group, while the P014 and P028 were lower than the TMS group, and the difference was not statistically significant. There was no significant difference in the density of NeuN positive cells in the peripheral area of the two groups at three time points, the density of Caspase-3 positive cells around the lesion and the average of the control group at three time points were lower than that of the TMS group, in which the control group was significantly lower than the TMS group (p=0.062).
(5) in P014 immunofluorescence staining with SVZ, the average density of BrdU and DCX positive cells in the control group were lower than those in the TMS group, and the difference was not statistically significant.
(6) the metabolic levels of the cerebral cortex and striatum in the control group and the TMS group were all lower than those of the normal side in the control group and the TMS group. At P013, the metabolic level of the injured side of the lateral cortex was close to the normal side, while the metabolism level of the striatum was higher than that in the normal side. There was no significant difference between the control group and the TMS group.
conclusion
For the rats suffering from moderate TBI, TMS showed the following favorable trends: (1) the tendency to reduce the mortality after TBI was obviously reduced, and (2) the tendency to improve the recovery after TBI was obviously improved. (3) the trend of the loss of brain parenchyma after TBI was obviously reduced. These results were consistent with the inference based on the results and the pathophysiology of TBI. Another inference is to promote the proliferation of endogenous neural stem cells after TBI, and the results of this study do not have a significant effect on the local metabolism of the brain in 24h after the withdrawal of.TMS.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R651.15;R-332

【参考文献】