在體光遺傳學—電生理相結合研究—側半球控制雙側上肢運動的腦功能重塑機制
發(fā)布時間：2018-08-22 14:05
【摘要】：背景：作為中樞神經損傷后較為嚴重的后遺癥之一,上肢痙攣性癱瘓的治療方法及效果均有限。課題組前期研究發(fā)現(xiàn)：通過改變周圍神經通路增強同側神經纖維對肢體的支配可以實現(xiàn)偏癱患者的健存大腦半球同時司管雙側上肢。但具體涉及到的神經通路以及患肢運動功能恢復的動態(tài)中樞機制尚不明確。本課題使用Thy1-ChR2-EYFP轉基因小鼠建立腦外傷和健側頸七神經根移位至患側頸七神經根的模型,通過光遺傳學-電生理相結合的技術、行為學以及逆行跨多突觸的神經示蹤技術,研究癱瘓肢體運動功能恢復的中樞機制。該技術具有皮層定位及刺激精確可控、可重復性佳以及高效無創(chuàng)的不可替代優(yōu)勢,研究成果將有助于揭示一側皮層司管雙側上肢的動態(tài)重塑規(guī)律,為后續(xù)積極干預腦重塑,促進卒中、腦癱、腦外傷后遺癥的上肢功能恢復研究提供依據(jù)。方法：我們建立了小鼠左側控制性皮層撞擊腦外傷(CCI)及健側頸七-患側頸七神經根切斷及移位模型,術后通過滾軸實驗和階梯步行實驗檢測上肢運動功能的損傷及恢復情況,在體光遺傳學-電生理技術相結合用于繪制正常小鼠初級運動皮層M1、前肢各肌肉代表區(qū)圖譜以及動態(tài)記錄術后不同時間點健側皮層刺激后雙側上肢各靶肌肉代表區(qū)位置及運動誘發(fā)電位(MEP)參數(shù)的變化。采用偽狂犬病毒PRV-Bartha株dsred自患肢頸七神經根注射,連續(xù)冰凍切片及免疫組化觀察其在健側皮層的神經元標記情況。結果：左側CCI后,小鼠對側肢體在滾軸實驗和階梯步行實驗的評分均出現(xiàn)顯著的降低,對照組(CCI+雙側頸七切斷組以及單純CCI組)的評分在CCI后1月內均出現(xiàn)了一定程度的上升,但之后直至術后10個月對照組的患肢行為學評分未進一步恢復,而實驗組(CCI+健側頸七-患側頸七移位組)的患肢在術后5月時滾軸實驗的姿態(tài)、頭部姿勢以及前屈項目行為學評分開始好于對照組,至術后6個月時更為顯著且一直持續(xù)至術后10月。術后7月時患肢在滾軸實驗的提攜項目評分以及階梯步行實驗評分開始好于對照組,至術后8個月時更為顯著且一直持續(xù)至術后10月。而健側肢體功能僅在術后1月內評分下降,術后1月恢復至術前并維持至術后10月。我們通過光遺傳學方法繪制了轉基因小鼠M1圖譜,并發(fā)現(xiàn)前肢代表區(qū)由位于偏前方較小面積的代表區(qū)RFA(6±1個刺激位點,主要誘發(fā)出腕及趾活動)以及偏后方較大面積的代表區(qū)CFA(44±4個刺激位點,主要誘發(fā)出肩肘及部分腕活動)構成。術后4月以內,右(健)側皮層刺激僅能記錄到左(健)側肢體靶肌肉的MEP；術后5月時,右(健)側皮層刺激可以同時記錄到雙側肱三頭肌的MEP,且術后右(患)側肱三頭肌代表區(qū)逐漸縮小并向左(健)側肱三頭肌代表區(qū)匯聚；術后7月時,右(健)側皮層刺激可以同時記錄到雙側前臂伸肌群的MEP,且術后右(患)側前臂伸肌群代表區(qū)呈現(xiàn)出逐漸縮小并向左(健)側前臂伸肌群代表區(qū)匯聚的趨勢,而右(健)側皮層內刺激始終無法誘發(fā)出右(患)側肱二頭肌的MEP。右(健)側皮層刺激誘發(fā)出左(健)側肢體靶肌肉的代表區(qū)及波幅未見明顯變化。單純CCI組小鼠及CCI+雙側頸七神經根切斷的小鼠的右(健)側皮層在各時間點均未發(fā)現(xiàn)被標記的神經元,而CCI+健側頸七移位的小鼠在術后5月,健側皮層中己能找到少量被標記的神經元,術后7月直至10月健側皮層中被標記的神經元密度進一步增高。結論：對于重度CCI小鼠,健側頸七神經根移位術可以促進患肢粗大(伸肘)及部分精細(伸腕指及協(xié)調)功能的恢復,前者恢復更為顯著。術后健側皮層參與了對雙側上肢運動的支配,患肢代表區(qū)在術后早期覆蓋了健肢代表區(qū),且前者呈現(xiàn)出向后者逐漸縮小匯聚、精確有序的趨勢。
[Abstract]:BACKGROUND: As one of the more serious sequelae after central nerve injury, the therapeutic methods and effects of spastic paralysis of the upper extremity are limited. Previous studies have found that hemiplegic patients can survive cerebral hemisphere and manage bilateral upper extremities simultaneously by changing peripheral nerve pathways to enhance the innervation of ipsilateral nerve fibers to the extremities. The specific neural pathways involved and the dynamic central mechanism underlying the recovery of motor function in the affected limbs are still unclear. In this study, we used Thy1-ChR2-EYFP transgenic mice to establish models of brain injury and translocation of contralateral cervical seven nerve roots to the affected cervical seven nerve roots. Neural tracing technique is an irreplaceable technique with precise and controllable cortical location and stimulation, good repeatability and high efficiency. The results of this study will help to reveal the dynamic remodeling regularity of bilateral upper limbs of unilateral cortical canal, and actively intervene in brain remodeling and promote the follow-up. Methods: The models of CCI and CCI were established in mice with stroke, cerebral palsy and sequelae of traumatic brain injury. The combination of somatogenetics and electrophysiology was used to map the primary motor cortex M1, the representative areas of forelimb muscles, and to dynamically record the position of target muscles and the changes of motor evoked potential (MEP) parameters in bilateral upper limbs after stimulation of contralateral cortex at different time points. Results: After CCI, the scores of contralateral limbs in rolling test and stepped walking test were significantly lower than those in control group (CCI + bilateral cervical 7 amputation group and CCI group) within 1 month after CCI. However, the behavioral scores of the affected limbs in the control group did not recover further until 10 months after operation. The posture, head posture and bending item behavioral scores of the affected limbs in the experimental group (CCI + CCI + CCI + CCI + CCI) were better than those in the control group at 5 months after operation. 7 months after operation, the scores of lifting items and step walking test of the affected limbs were better than those of the control group, and were more significant at 8 months after operation and lasted until 10 months after operation. We mapped the M1 map of transgenic mice by photogenetics. We found that the forelimb representative region was composed of RFA (6 + 1 stimulus loci, mainly inducing wrist and toe activity) located in a small area in the front and CFA (44 + 4 stimulus loci, mainly inducing shoulder, elbow and part of wrist) in a large area in the rear. Within 4 months after surgery, the right (healthy) cortical stimulation could only record the MEP of the target muscle of the left (healthy) limb; 5 months after surgery, the right (healthy) cortical stimulation could simultaneously record the MEP of the bilateral triceps brachii, and the representative area of the right (affected) triceps brachii decreased gradually and converged to the left (healthy) triceps brachii. MEP of bilateral forearm extensors could be recorded simultaneously by right (healthy) cortical stimulation, and the representative area of right (affected) forearm extensors decreased gradually and converged to the representative area of left (healthy) forearm extensors, while the right (healthy) cortical stimulation could not induce MEP of right (affected) biceps brachii. No marked neurons were found in the right (healthy) cortex of CCI group mice and CCI + bilateral rhizotomy mice at all time points, while a small number of marked neurons were found in the contralateral cortex of CCI + transposition mice 5 months after operation. The density of the labeled neurons in the contralateral cortex was further increased from July to October. Conclusion: For severe CCI mice, transposition of the contralateral seven nerve roots can promote the recovery of the thick (elbow extension) and some fine (wrist extension and coordination) functions of the affected limbs, and the former is more significant. During the early postoperative period, the representative area of the affected limb covered the representative area of the healthy limb, and the former gradually reduced to the latter, accurately and orderly.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R741
本文編號：2197326
[Abstract]:BACKGROUND: As one of the more serious sequelae after central nerve injury, the therapeutic methods and effects of spastic paralysis of the upper extremity are limited. Previous studies have found that hemiplegic patients can survive cerebral hemisphere and manage bilateral upper extremities simultaneously by changing peripheral nerve pathways to enhance the innervation of ipsilateral nerve fibers to the extremities. The specific neural pathways involved and the dynamic central mechanism underlying the recovery of motor function in the affected limbs are still unclear. In this study, we used Thy1-ChR2-EYFP transgenic mice to establish models of brain injury and translocation of contralateral cervical seven nerve roots to the affected cervical seven nerve roots. Neural tracing technique is an irreplaceable technique with precise and controllable cortical location and stimulation, good repeatability and high efficiency. The results of this study will help to reveal the dynamic remodeling regularity of bilateral upper limbs of unilateral cortical canal, and actively intervene in brain remodeling and promote the follow-up. Methods: The models of CCI and CCI were established in mice with stroke, cerebral palsy and sequelae of traumatic brain injury. The combination of somatogenetics and electrophysiology was used to map the primary motor cortex M1, the representative areas of forelimb muscles, and to dynamically record the position of target muscles and the changes of motor evoked potential (MEP) parameters in bilateral upper limbs after stimulation of contralateral cortex at different time points. Results: After CCI, the scores of contralateral limbs in rolling test and stepped walking test were significantly lower than those in control group (CCI + bilateral cervical 7 amputation group and CCI group) within 1 month after CCI. However, the behavioral scores of the affected limbs in the control group did not recover further until 10 months after operation. The posture, head posture and bending item behavioral scores of the affected limbs in the experimental group (CCI + CCI + CCI + CCI + CCI) were better than those in the control group at 5 months after operation. 7 months after operation, the scores of lifting items and step walking test of the affected limbs were better than those of the control group, and were more significant at 8 months after operation and lasted until 10 months after operation. We mapped the M1 map of transgenic mice by photogenetics. We found that the forelimb representative region was composed of RFA (6 + 1 stimulus loci, mainly inducing wrist and toe activity) located in a small area in the front and CFA (44 + 4 stimulus loci, mainly inducing shoulder, elbow and part of wrist) in a large area in the rear. Within 4 months after surgery, the right (healthy) cortical stimulation could only record the MEP of the target muscle of the left (healthy) limb; 5 months after surgery, the right (healthy) cortical stimulation could simultaneously record the MEP of the bilateral triceps brachii, and the representative area of the right (affected) triceps brachii decreased gradually and converged to the left (healthy) triceps brachii. MEP of bilateral forearm extensors could be recorded simultaneously by right (healthy) cortical stimulation, and the representative area of right (affected) forearm extensors decreased gradually and converged to the representative area of left (healthy) forearm extensors, while the right (healthy) cortical stimulation could not induce MEP of right (affected) biceps brachii. No marked neurons were found in the right (healthy) cortex of CCI group mice and CCI + bilateral rhizotomy mice at all time points, while a small number of marked neurons were found in the contralateral cortex of CCI + transposition mice 5 months after operation. The density of the labeled neurons in the contralateral cortex was further increased from July to October. Conclusion: For severe CCI mice, transposition of the contralateral seven nerve roots can promote the recovery of the thick (elbow extension) and some fine (wrist extension and coordination) functions of the affected limbs, and the former is more significant. During the early postoperative period, the representative area of the affected limb covered the representative area of the healthy limb, and the former gradually reduced to the latter, accurately and orderly.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R741
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