本文選題：帕金森病 + 腦橋被蓋核　； 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究目的:當(dāng)帕金森病進(jìn)展到晚期階段,有大約一半的患者會(huì)出現(xiàn)軸性癥狀,比如:嚴(yán)重的步態(tài)和姿勢(shì)障礙(更具體的說(shuō),凍僵步態(tài)和跌倒),并且不被左旋多巴與STN-DBS治療改善。近年來(lái),有越來(lái)越多的證據(jù)表明,電刺激腦橋核(PPN-DBS)在緩解軸性癥狀中發(fā)生起著至關(guān)重要的作用。通過(guò)研究PD中的PPN和它的功能將有助于理解軸性癥狀的病理基礎(chǔ)和PPN-DBS治療的機(jī)制。因此,我們可以合理推測(cè)難治性軸性癥狀的發(fā)生與膽堿能神經(jīng)元變性更相關(guān),而不是多巴胺能神經(jīng)元變性。在本研究中,我們進(jìn)一步探討PPN膽堿能神經(jīng)元在大鼠發(fā)生運(yùn)動(dòng)障礙中的作用。特別是軸性運(yùn)動(dòng)障礙。研究方法本實(shí)驗(yàn)選用42只健康成年雄性SD大鼠隨機(jī)分四組:(1)生理鹽水注射假手術(shù)組(2)6-羥基多巴胺(6-OHDA)毀損單側(cè)黑質(zhì)致密部(SNc)組(3)anti-ChAT-SAP毀損單側(cè)腳橋被蓋核(PPTg)組(4)6-羥基多巴胺(6-OHDA)毀損單側(cè)黑質(zhì)致密部(SNc)合并anti-ChAT-SAP毀損同側(cè)腳橋被蓋核(PPTg)組(聯(lián)合毀損組)。anti-ChAT-SAP是一種帶皂素(SAP)(皂類植物種子中提取的可使核糖體失活蛋白)的特異性抑制膽堿乙酰轉(zhuǎn)移酶(ChAT)的兔源性抗體毒藥。右側(cè)定位內(nèi)側(cè)前腦束(MFB)注射4ul6-OHDA。靶向抗膽堿乙酰轉(zhuǎn)移酶IgG免疫毒素(anti-ChAT-SAP)注入右側(cè)PPTg毀損膽堿能神經(jīng)元,通過(guò)注射相同劑量的生理鹽水到右側(cè)MFB和PPTg造成假毀損。我們使用Catwalk XT系統(tǒng)(Noldus信息技術(shù),瓦赫寧恩,荷蘭)分析自由活動(dòng)的大鼠的步態(tài)。在毀損術(shù)后重新進(jìn)行行為學(xué)評(píng)估的過(guò)程中,除了假手術(shù)組,其他大鼠在測(cè)試中均表現(xiàn)出輕到中度的凍僵步態(tài)(起步延遲)。由于凍結(jié)步態(tài)(起步延遲)而不能完成整個(gè)階段的(包括少于三次通過(guò)通道的)計(jì)入凍結(jié)步態(tài)的數(shù)量,計(jì)算每一組中凍僵步態(tài)數(shù)量占測(cè)試階段的全部數(shù)量的百分比。最后完成行為學(xué)測(cè)試后,SNc通過(guò)酪氨酸羥化酶(TH)免疫組織化學(xué)染色法如前所述。此程序適用于PPTg的ChAT免疫組織化學(xué)染色方法,染色后,使用Leica顯微攝像程序組獲取圖片。根據(jù)光學(xué)分餾器原理對(duì)SNc中TH+的神經(jīng)元或者PPTg中ChAT+的神經(jīng)元數(shù)量進(jìn)行定量分析評(píng)估。以每6張覆蓋了完整SNc或者PPTg的切片取得的均值為計(jì)數(shù)值。數(shù)據(jù)以完整一側(cè)相應(yīng)區(qū)域的百分比記錄。研究結(jié)果在SNc毀損組DA神經(jīng)元的平均損失90.19%,單純SNc毀損組與聯(lián)合毀損組之間沒有組間差異。在PPTg毀損組的膽堿能神經(jīng)細(xì)胞的損失達(dá)到了 77.62%,在聯(lián)合毀損組達(dá)到81.75%,兩者與假手術(shù)組相比明顯降低。單純PPTg毀損組與聯(lián)合毀損組膽堿能細(xì)胞的損失沒有顯著差異。Catwalk行為學(xué)分析系統(tǒng)捕捉動(dòng)態(tài)和靜態(tài)的步態(tài)參數(shù)。損傷后,與假手術(shù)組和PPTg毀損組相比SNc毀損組和聯(lián)合毀損組的跑動(dòng)時(shí)長(zhǎng)、站立時(shí)間和步替循環(huán)明顯增加步幅長(zhǎng)度和擺動(dòng)速度明顯下降。SNc毀損組與聯(lián)合毀損組無(wú)顯著差異。最大接觸面積和平均強(qiáng)度的相互作用只出現(xiàn)在左側(cè)爪印(對(duì)側(cè)腦損傷)。與假手術(shù)組和PPTg毀損組相比,左前爪和左后爪在SNc毀損組和聯(lián)合毀損組中這兩個(gè)步態(tài)參數(shù)顯著下降。而SNc毀損組和聯(lián)合毀損組之間無(wú)差異。與假手術(shù)組和PPTg毀損組比較,SNc毀損組和聯(lián)合毀損組的爪(前或后)間寬距參數(shù)明顯增加,而在前肢只有微弱增加。在SNc毀損組和聯(lián)合毀損組中所有足印的終末雙足著地時(shí)間都有顯著增加。并且聯(lián)合毀損組高于SNc毀損組。在四組中SNc毀損組和聯(lián)合毀損組出現(xiàn)了最嚴(yán)重的凍結(jié)步態(tài)癥狀(57.58%,p0.05)。從某種程度上說(shuō),與假手術(shù)組相比,單純毀損SNc也會(huì)誘發(fā)大鼠出現(xiàn)FOG(26.66%,p0.05,)。假手術(shù)和PPTg毀損組大鼠無(wú)顯著差異(0%vs.13.88%)。在PPTg毀損組中沒有發(fā)現(xiàn)膽堿能神經(jīng)元與步態(tài)參數(shù)變化存在相關(guān)性。在聯(lián)合毀損組,只有左側(cè)肢體的終末雙足著地時(shí)間與膽堿能神經(jīng)元存在相關(guān)(偏相關(guān)系數(shù):左前肢:-0.77,P = 0.04;左后肢:-0.79,P = 0.02),其他參數(shù)無(wú)顯著相關(guān)性。研究結(jié)論結(jié)果表明,單純毀損PPTg大鼠不會(huì)引起步態(tài)異常。但是6-OHDA毀損多巴胺神經(jīng)元合并PPTg膽堿能神經(jīng)元?dú)p可加重PD大鼠的凍僵步態(tài)癥狀。我們的數(shù)據(jù)表明,PPN膽堿能神經(jīng)元在PD患者FOG的發(fā)生過(guò)程中起著至關(guān)重要的作用。后續(xù)研究可以更進(jìn)一步探究PPN膽堿能神經(jīng)元與參與PD相關(guān)的結(jié)構(gòu)或神經(jīng)元在PPN-DBS治療機(jī)制的關(guān)系。
[Abstract]:Research purposes: when Parkinson's disease progresses to a late stage, about half of the patients have axial symptoms, such as severe gait and postural disorders (more specifically, frozen gait and fall), and not treated by levodopa and STN-DBS. In recent years, there is more evidence that the electrical stimulation of the pontine nucleus (PPN-DBS) is relieved. The study of PPN and its function in PD will help to understand the pathological basis of axial symptoms and the mechanism of PPN-DBS therapy. Therefore, we can reasonably speculate that the occurrence of intractable axial symptoms is more related to the degeneration of cholinergic neurons, rather than the degeneration of dopaminergic neurons. In the study, we further explored the role of PPN cholinergic neurons in dyskinesia in rats, especially axonal dyskinesia. The study method selected 42 healthy adult male SD rats randomly divided into four groups: (1) physiological saline injection sham group (2) 6- hydroxyl dopamine (6-OHDA) damaged unilateral substantia nigra dense part (3) anti-C HAT-SAP destruction of unilateral foot bridge tegmentum (PPTg) group (4) 6- hydroxyl dopamine (6-OHDA) damage unilateral substantia nigra dense part (SNc) combined with anti-ChAT-SAP damage in the same lateral foot bridge tegmental nucleus (PPTg) group (joint damage group).Anti-ChAT-SAP is a kind of specific inhibitory choline B with saponin (SAP) (the ribosome inactivation protein extracted from the seed of soap plant) The right localization medial forebrain bundle (MFB) injected 4ul6-OHDA. target to the cholinergic acetyltransferase IgG immuno toxin (anti-ChAT-SAP) injecting the right PPTg damage to the cholinergic neurons by injecting the right side of the medial forebrain bundle (ChAT) into the right side. We used the Catwalk XT system (Nold) by injecting the same dose of the saline to the right MFB and PPTg. Us information technology, wakningen, Holland) analysis of the gait of free active rats. In the process of reassessment of behavior after the damage, other rats showed mild to moderate frozen gait in the test, except for the sham operation group (starting delay). The whole stage was not completed due to the freezing step (starting delay). The number of frozen gait in the three passage was counted, and the percentage of the total number of frozen gait in each group was calculated. After the final behavioral test, the SNc immunohistochemical staining of tyrosine hydroxylase (TH) was described as before. This program is suitable for the ChAT immunohistochemical staining method of PPTg and dyeing. Then, the images are obtained using the Leica microcamera group. According to the optical fractionator principle, the number of neurons in the neurons of TH+ in the SNc or the number of ChAT+ in the PPTg is quantitatively evaluated. The mean value obtained with every 6 slices covered with complete SNc or PPTg is calculated. The data is recorded with the percentage of the corresponding region on the complete side. The average loss of DA neurons in the SNc damage group was 90.19%. There was no difference between the simple SNc damage group and the combined damage group. The loss of cholinergic neurons in the PPTg damage group reached 77.62%, and 81.75% in the combined damage group, which was significantly lower than that in the sham group. The cholinergic activity in the simple PPTg damage group and the combined damage group was fine. The loss of cell was not significantly different from the.Catwalk behavioral analysis system to capture dynamic and static gait parameters. After injury, the running time of the SNc damage group and the combined damage group was longer than the sham operation group and the PPTg damage group. The standing time and the step cycle significantly increased the length and the swinging speed of the.SNc damage group and the joint damage group. There was no difference. The interaction between the maximum contact area and the average strength only appeared in the left claw print (contralateral brain damage). Compared with the sham operation group and the PPTg damage group, the two gait parameters of the left front and left posterior claw in the SNc damage group and the combined damage group were significantly decreased. There was no difference between the SNc damage group and the combined group. Compared with the PPTg damage group, the width parameters of the claw (before and after) in the SNc damage group and the joint damage group increased significantly, but only a faint increase in the forelimb. All the end of foot prints in the SNc damage group and the combined damage group increased significantly. And the joint destruction group was higher than the SNc damage group. In the four groups, the SNc damage group and the joint destruction were destroyed. The most serious frozen gait symptoms (57.58%, P0.05) were found in the loss group. To some extent, compared with the sham group, the simple destruction of SNc could induce FOG (26.66%, P0.05,) in rats. There was no significant difference between the sham operation and the PPTg damage group (0%vs.13.88%). The changes of the cholinergic neurons and gait parameters were not found in the PPTg damage group. Correlation. In the combined damage group, only the left limb terminal bipedal time was associated with cholinergic neurons (partial correlation coefficient: left forelimb: -0.77, P = 0.04; left hind limbs: -0.79, P = 0.02), and no significant correlation was found in other parameters. The results of the study showed that the simple destruction of PPTg rats would not cause abnormal gait, but 6-OHDA was much damaged. Our data suggest that PPN cholinergic neurons play a vital role in the occurrence of FOG in PD patients. Further studies can further explore the PPN cholinergic neurons associated with PD related structures or neurons in the treatment of PD in the PPN-DBS treatment. The relationship of the therapeutic mechanism.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R742.5

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