脚桥被盖核神经元激活可改善PD大鼠模型的步态行为——基于光遗传学技术研究

Activation of pedunculopontine tegmental nucleus neurons improves gait behavior of rat models of Parkinson's disease:a study based on optogenetics method

目的应用光遗传学技术探讨脚桥被盖核(PPTN)低频电刺激改善PD大鼠模型步态行为的作用机制。方法(1)24只健康成年雄性SD大鼠按随机数字表法分为假手术1组、毁损1组和光激活组,每组8只,其中假手术1组大鼠于右侧内侧前脑束(MFB)注射生理盐水,毁损1组和光激活组大鼠注射6-羟基多巴胺(6-OHDA)制备成PD模型。造模手术后第2周在各组大鼠右侧PPTN处注射腺相关病毒hSynapsin-ChR2-mcherry后,光激活组同时在PPTN处植入光纤并给予蓝光刺激。(2)24只健康成年雄性SD大鼠按随机数字表法分为假手术2组、毁损2组和光抑制组,每组8只,其中假手术2组大鼠于右侧MFB注射生理盐水,毁损2组和光抑制组大鼠注射6-OHDA制备成PD模型。造模手术后第2周在各组大鼠右侧PPTN处注射腺相关病毒hSynapsin-NpHR-mcherry后,光抑制组同时在PPTN处植入光纤并给予黄光刺激。(3)腺相关病毒注射后第3周,采用Catwalk步态分析评估各组大鼠的行为学能力。结果(1)光激活实验中,与假手术1组比较,毁损1组大鼠的前爪间距和后爪间距均明显增加,毁损侧肢体和对侧肢体的步幅长度均明显下降,毁损侧肢体和对侧肢体的压强均明显下降,对侧肢体的摆动速度明显下降,差异均有统计学意义(P<0.05);与毁损1组比较,光激活组大鼠的前爪间距和后爪间距均明显下降,毁损侧肢体和对侧肢体的步幅长度均明显增加,毁损侧肢体和对侧肢体的压强均明显增加,差异均有统计学意义(P<0.05)。(2)光抑制实验中,与假手术2组比较,毁损2组大鼠的前爪间距和后爪间距均明显增加,毁损侧肢体的步幅长度明显下降,毁损侧肢体和对侧肢体的压强均明显下降,毁损侧肢体和对侧肢体的摆动速度均明显下降,差异均有统计学意义(P<0.05);与毁损2组比较,光抑制组大鼠的前爪间距和后爪间距均无明显变化,毁损侧肢体和对侧肢体的步幅长度均无明显变化,毁损侧肢体和对侧肢体的压强均无明显变化,毁损侧肢体和对侧肢体的摆动速度均无明显变化,差异均无统计学意义(P>0.05)。结论PPTN低频电刺激改善PD模型大鼠步态行为的作用机制与PPTN神经元激活有关。

Objective To investigate the mechanism of improvement of gait behavior in PD rat models by low frequency electrical stimulation of pedunculopontine tegmental nucleus(PPTN)by optogenetics method.Methods(1)Twenty-four healthy adult male SD rats were randomly divided into a sham-operated group 1,a lesion group 1 and a photoactivation group(n=8);normal saline was injected into the right medial frontal tract(MFB)of the sham-operated group 1;6-hydroxydopamine(6-OHDA)was injected into the lesion group 1 and photoactivation group to induce PD models;two weeks after modeling,injection of adeno-associated virus hsynapsin-ChR2-mcherry into the right PPTN of the three groups was performed,and the photoactivation group received blue-ray stimulation by implanting optical fibers into the PPTN at the same time.(2)Twenty-four healthy adult male SD rats were randomly divided into a sham-operated group 2,a lesion group 2 and a photoinhibition group(n=8);normal saline was injected into right MFB of the sham-operated group 2;6-OHDA was injected into the lesion group 2 and photoinhibition group to induce PD models;two weeks after modeling,injection of adeno-associated virus hsynapsin-NpHR-mcherry into the right PPTN of the three groups was performed,and the photoinhibition group received yellow-ray stimulation by implanting optical fibers into the PPTN at the same time.(3)Three weeks after injection of adeno-associated virus,Catwalk gait analysis was used to assess the behavioral ability of rats in each group.Results(1)As compared with the sham-operated group 1,lesion group 1 had significantly increased front claw spacing and back front claw spacing,and significantly decreased stride length and pressure of damaged lateral and contralateral limbs,and significantly decreased swing speed of contralateral limb(P<0.05);as compared with those in the lesion group 1,the front claw spacing and back claw spacing were significantly shortened,and stride length and pressure of damaged lateral and contralateral limbs were statistically increased in the photoactivation group(P<0.05).(2)As compared with the sham-operated group 2,lesion group 2 had significantly increased front claw spacing and back front claw spacing,significantly decreased stride length of damaged lateral limb,and significantly decreased pressure and swing speed of damaged lateral and contralateral limbs(P<0.05);no significant differences were noted on the front claw spacing and back front claw spacing,pressure and swing speed of damaged lateral and contralateral limbs between lesion group 2 and photoinhibition group(P>0.05).Conclusion The mechanism of low frequency electrical stimulation of PPTN improving gait behavior of PD rat models is related to activation of PPTN neurons.