本文選題：外傷性腦損傷 + 經(jīng)顱磁刺激��； 參考：《北京協(xié)和醫(yī)學(xué)院》2014年博士論文

【摘要】：研究背景和目的 外傷性腦損傷(traumatic brain injury, TBI)在所有人群中都很常見,不分年齡、背景或健康狀況。時(shí)代發(fā)展使得交通事故愈加頻繁,TBI必然會(huì)成為我國一個(gè)沉重的公共衛(wèi)生問題。影響TBI患者功能恢復(fù)的最大障礙是腦實(shí)質(zhì)缺失。雖然腦損傷時(shí)機(jī)體內(nèi)源性神經(jīng)干細(xì)胞會(huì)增殖,但增殖水平有限且增殖后的存活率很低。促進(jìn)內(nèi)源性神經(jīng)干細(xì)胞增殖和存活或許是治療TBI的一個(gè)可行策略。 經(jīng)顱磁刺激(transcranial magnetic stimulation, TMS)是一種無創(chuàng)腦刺激(noninvasive brain stimulation, NBS)技術(shù),通過激活神經(jīng)元或改變神經(jīng)元興奮性而發(fā)揮神經(jīng)調(diào)控作用。在恰當(dāng)?shù)膮?shù)下,TMS能影響大腦局部血流,改變局部氧化應(yīng)激水平,調(diào)節(jié)神經(jīng)再塑(neuroplasticity)。另外,有研究提示TMS具有促進(jìn)神經(jīng)再生的能力。基于TMS已知的多種作用,結(jié)合TBI的病理生理過程,可得出TMS具有治療TBI潛能的推論。但至今沒有用TMS治療TBI的研究發(fā)表,也沒有專門的研究探討TMS是否能影響TBI后內(nèi)源性干細(xì)胞增殖。 本研究嘗試用重復(fù)經(jīng)顱磁刺激(rTMS)治療中度TBI大鼠,重點(diǎn)觀察大腦內(nèi)源性神經(jīng)干細(xì)胞增殖情況。 方法 成年雄性SD大鼠,體重210-260g,用Feeney自由落體打擊法造成中度TBI。用改良神經(jīng)功能缺失評(píng)分(mNSS)評(píng)價(jià)大鼠PO1、PO7、PO14和P028的行為學(xué)表現(xiàn)。根據(jù)PO1行為評(píng)分,將大鼠分層隨機(jī)分為對(duì)照組和經(jīng)顱磁刺激組(TMS組)。從PO2開始行TMS,參數(shù)如下：電壓700V,頻率5Hz,TMS組每只大鼠每日接受900個(gè)脈沖刺激。在PO7、PO14、PO28三個(gè)時(shí)間點(diǎn)將大鼠分批處死,生理鹽水和4%多聚甲醛心臟灌流后取腦,石蠟包埋切片,進(jìn)行HE和免疫組化染色。利用HE切片測定冠狀面腦組織面積,以此計(jì)算損傷側(cè)腦組織相對(duì)健康側(cè)的減少率。免疫組化染色指標(biāo)包括細(xì)胞增殖標(biāo)志物BrdU,神經(jīng)干細(xì)胞標(biāo)志物musashil,成熟神經(jīng)元標(biāo)志物NeuN,細(xì)胞凋亡標(biāo)記物caspase-3。少數(shù)在PO14處死的大鼠(n=5)取腦后單獨(dú)剝離SVZ行免疫熒光染色,染色指標(biāo)包括BrdU、DAPI、DCX(神經(jīng)元前體細(xì)胞標(biāo)志物)和laminin(血管標(biāo)志物)。BrdU、NeuN、caspase-3和DCX染色情況用視野內(nèi)的陽性細(xì)胞數(shù)評(píng)價(jià)(陽性細(xì)胞密度),musashil用視野內(nèi)陽性面積占全部腦組織面積的比例評(píng)價(jià)(陽性面積比)。部分大鼠在P02和P013行18F-FDG micro-PET。 結(jié)果 (1)TMS組意外死亡率有明顯低于對(duì)照組的傾向(15.79%vs37.14%,p=0.09)。 (2)大鼠的mNSS評(píng)分在損傷后PO1-PO14逐漸改善,TMS組vs對(duì)照組,P07的評(píng)分近似(7.00±1.27vs6.20±1.92),而PO14評(píng)分有TMS組明顯更低的傾向(3.38±1.47vs5.40±1.14,p=0.085)。 (3)外傷性腦損傷后大鼠損傷側(cè)大腦半球的腦組織相對(duì)減少率逐漸提高,其中P028時(shí)TMS組有明顯低于對(duì)照組的傾向(32.80±3.0%vs38.59±3.2%,p=0.083)。 (4)免疫組化染色中,兩組損傷側(cè)腦室背外角的BrdU陽性細(xì)胞密度在三個(gè)時(shí)間點(diǎn)均無顯著差別；損傷側(cè)腦室背外角musashil陽性面積比從P07到P028逐漸減少,兩組間比較,對(duì)照組P07時(shí)高于TMS組,而P014和P028時(shí)低于TMS組,差別都不具有統(tǒng)計(jì)學(xué)意義；兩組損傷灶周邊區(qū)NeuN陽性細(xì)胞密度在三個(gè)時(shí)間點(diǎn)均無顯著差別；損傷灶周邊Caspase-3陽性細(xì)胞密度,三個(gè)時(shí)間點(diǎn)對(duì)照組的平均值都低于TMS組,其中P014時(shí)有對(duì)照組明顯低于TMS組的傾向(p=0.062)。 (5)P014取SVZ進(jìn)行的免疫熒光染色中,對(duì)照組BrdU和DCX陽性細(xì)胞密度平均值都低于TMS組,差別沒有統(tǒng)計(jì)學(xué)意義。 (6)P02時(shí),對(duì)照組和TMS組損傷側(cè)大腦皮層和紋狀體代謝水平都較正常側(cè)減低；P013時(shí),兩組損傷側(cè)皮層代謝水平都接近于正常側(cè),而紋狀體代謝水平都較正常側(cè)升高。對(duì)照組和TMS組之間比較無明顯差別。 結(jié)論 對(duì)于遭受中度TBI的大鼠,TMS表現(xiàn)出了以下有利趨勢：(1)明顯降低大鼠TBI后死亡率的趨勢,(2)明顯改善TBI后行為恢復(fù)的趨勢,(3)明顯減少TBI后損傷側(cè)腦實(shí)質(zhì)丟失的趨勢,這些結(jié)果與基于已有研究結(jié)果和TBI病理生理作出的推論相符,但對(duì)于另一個(gè)推論,即促進(jìn)TBI后內(nèi)源性神經(jīng)干細(xì)胞增殖,本研究結(jié)果不能作為支持。TMS對(duì)刺激停止后24h的腦局部代謝無明顯影響。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R651.15;R-332

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