【作者】 李娟；

【导师】 王红星；

【作者基本信息】 东南大学 ， 康复医学与理疗学， 2025， 博士

【摘要】 第一部分GRNs核团精准刺激在体稳定性、安全性及有效性研究[研究背景]脑干腹侧网状巨细胞核(Gigantocellular reticular nucleus,GRNs)是挫伤型脊髓损伤(spinal cord injury,SCI)后保存最为完好的网状脊髓束的主要来源之一。其下行纤维能够构建大脑皮层与脊髓之间的信号传输桥梁,从而实现皮层与脊髓损伤部位以下的神经环路的功能性连接。鉴于脑干独特的解剖学位置,长时间在此区域植入电极或光纤面临着巨大的挑战,同时重复经颅磁刺激(repetitive transcranial magnetic stimulation,rTMS)因其刺激深度有限与靶向性不佳,难以有效精确激活GRNs核团。为解决此难题,本研究利用超顺磁性氧化铁(superparamagnetic iron oxide,SPIO)纳米颗粒和外部磁场(magnetic field,MF),即联合磁刺激系统(combined magnetic stimulation system,c-MSS),精准靶向GRNs核团,初步探讨c-MSS的在体稳定性、安全性和有效性。[研究方法]本研究采用T10挫伤小鼠SCI模型,应用立体定位技术将SPIO纳米颗粒注射到SCI小鼠GRNs核团。通过小动物在体磁共振成像(magnetic resonance imaging,MRI)动态监测SPIO纳米颗粒在GRNs核团的分布与存留时间;利用动态体重检测与原位末端标记法(TdT-mediated dUTP Nick-End Labeling,TUNEL)在体评估SPIO纳米颗粒的安全性;通过BMS量表(Basso Mouse Scale)进行行为学评估,筛选出合适的刺激参数;利用早期时相蛋白c-Fos免疫荧光染色反映GRNs核团的激活情况。[研究结果](1)MRI显示,SPIO纳米颗粒在GRNs核团至少保留8周无明显降解,并且随着时间的推移缓慢降解(24周);(2)SPIO纳米颗粒不会影响SCI小鼠体重变化,TUNEL染色结果也显示SPIO纳米颗粒不会诱导GRNs核团细胞凋亡;(3)相比10 Hz,20 Hz的MF干预频率更有利于促进SCI小鼠的运动恢复(P<0.01);(4)c-MSS能精准激活GRNs核团神经元。[研究结论]SPIO纳米颗粒在GRNs核团中表现出良好的稳定性,并对局部细胞几乎不产生毒性影响。c-MSS能够激活GRNs核团,且20 Hz的MF干预频率更有利于促进SCI小鼠的运动恢复。第二部分c-MSS联合运动训练改善脊髓损伤小鼠运动功能及其机制的初步探索[研究背景]运动训练已被证实能改善脊髓损伤(spinal cord injury,SCI)患者的运动功能,但其改善效果存在平台期。为解决这一难题,研究发现神经调控技术能够进一步增强并维持运动治疗的效果。基于此,本研究设计了 c-MSS联合运动训练的方案,旨在探讨该方案能否进一步提升SCI小鼠的运动功能,并初步探讨其潜在的相关机制。[研究方法]本研究采用T10挫伤SCI模型,在造模后第5天,应用立体定位技术将超顺磁性氧化铁(superparamagnetic iron oxide,SPIO)纳米颗粒注射到SCI小鼠GRNs核团。实验分为六组:Sham组(仅椎板切除,无脊髓损伤),SCI组(仅脊髓损伤),SCI+SPIO组(GRNs核团注射SPIO纳米颗粒,无外加磁场干预),SCI+c-MSS组(脊髓损伤,c-MSS干预),SCI+Tr(脊髓损伤,运动平板训练),SCI+c-MSS+Tr(脊髓损伤,c-MSS联合运动平板训练)。造模后第7天开始进行为期8周的c-MSS干预,20 min/次,2次/天,每周5天,磁场频率为20 Hz。SCI+c-MSS+Tr组小鼠在c-MSS干预结束后,进行运动平板训练,30 min/次,2次/天,每周5天,速度从最小开始逐渐增加至8～10m/min。在造模后第5周进行顺行示踪病毒注射。通过BMS评分、网格步行实验和步态分析评估小鼠的运动功能;通过运动诱发电位(motor-evokedpotentials,MEPs)评估皮质运动通路的完整性;通过H反射评估腰髓前角运动神经元的兴奋性;通过顺行示踪分析GRNs核团下行投射纤维在T10损伤灶周围及腰髓前角的分布及相对定量分析;通过免疫荧光染色分析腰髓前角兴奋性与抑制性的变化情况。[研究结果](1)行为学评分:①BMS评分结果显示,各组在损伤后立即出现下肢瘫痪(BMS=0);损伤后1周,各组小鼠出现一些自发运动功能恢复(BMS≈1);与损伤后1周相比,各组小鼠在SCI后9周的运动功能均表现出不同程度的改善;SCI组和SCI+SPIO组的BMS评分在各时间节点上没有明显差异,两组评分均在约7周时趋于平稳;与SCI组相比,SCI+c-MSS组在损伤后6周时,该组小鼠的运动功能改善具有统计学意义(SCIvs SCI+c-MSS,P<0.05);与SCI组相比,SCI+Tr组在损伤后4周BMS评分达到4分左右,差异具有统计学意义(P<0.05);SCI+Tr组小鼠的BMS评分在各时间点高于SCI+c-MSS组,但两者没有统计性差异(P>0.05);除Sham组外,SCI+c-MSS+Tr组小鼠运动功能恢复最好,在损伤后9周,BMS≈7。②网格步行实验结果显示,SCI组和SCI+SPIO组在足落空(footdroops,FDs)次数之间没有显著差异(P>0.05);与SCI组相比,SCI+c-MSS组和SCI+Tr组的FDs次数减少,差异具有统计学意义(P<0.05);除了 Sham组,SCI+c-MSS+Tr组小鼠的FDs次数最少。③步态分析结果显示,与SCI组相比,SCI+c-MSS和SCI+Tr组小鼠的足印面积、足底压强和摆动速度均有显著改善;SCI+Tr和SCI+c-MSS组在足印面积、足底压强和摆动速度这些参数上没有观察到统计学上的显著差异(P>0.05);与SCI+c-MSS和SCI+Tr组相比,SCI+c-MSS+Tr组的小鼠在足印面积、足底压强和摆动速度等参数上表现更好(足印面积:SCI+c-MSS vs SCI+c-MSS+Tr,P<0.05)(2)MEPs结果显示,在损伤之前,各组均能一致地诱发出MEPs;受伤1周后,除Sham组外,其他各组的MEPs均消失;在损伤后9周,除Sham组与SCI+c-MSS+Tr组,其余4组均未诱发出MEPs;与Sham组相比(MEPs诱发率100%),SCI+c-MSS+Tr组中诱发的MEPs百分比为60%(10只小鼠中的6只成功诱导出MEPs);且SCI+c-MSS+Tr组的波幅较小(Shamvs SCI+c-MSS+Tr,9.55±1.0928 mV vs 0.70959±0.57379 mV)、潜伏期较长(Sham vs SCI+c-MSS+Tr,4.305±0.188 ms vs 23.085±1.60 ms)。(3)H反射结果显示,所有组在刺激频率为0.1、0.5、1和5 Hz时均表现出与频率相关的Hmax/Mmax 比值抑制和H反射潜伏期延长;与SCI组相比,SCI+Tr组的Hmax/Mmax 比值显著降低,尤其是在0.1 Hz的刺激频率下(P<0.0001);与SCI组相比,SCI+c-MSS组在各刺激频率下的Hmax/Mmax 比值降低不明显(P>0.05);c-MSS干预显著增加了各刺激频率下的H反射潜伏期(在0.1Hz时,SCI vs SCI+SCI+c-MSS,P<0.05;在 0.5Hz 时,SCI vs SCI+SCI+c-MSS,P<0.01;在 1Hz 时,SCIvs SCI+SCI+c-MSS,P<0.0001;在 5Hz 时,SCIvs SCI+SCI+c-MSS,P<0.0001);而Tr干预并未显著延长H反射的潜伏期(P>0.05);在SCI+c-MSS+Tr组中,H反射的Hmax/Mmax 比值降低、潜伏期延长,与Sham相比无显著差异(P>0.05)。(4)与Sham组相比,脊髓挫伤导致损伤部位下方GRNs下行投射纤维数量显著减少;与SCI组、SCI+SPIO组相比,c-MSS干预促使更多GRNs下行投射纤维数量跨越损伤部位(即0 μm)(P<0.05);运动训练组在0μm处的GRNs下行纤维与SCI组相比没有显著性差异(P>0.05);除了 Sham组,c-MSS联合运动训练干预促进了最多的GRNs核团纤维跨越损伤部位;SCI+c-MSS组与SCI+c-MSS+Tr组在中心下方700 μm和1400 μm处均显示出比SCI组更多的GRNs下行纤维,但差异无统计学意义(P>0.05);SCI+c-MSS组在腰髓冠状面的GRNs核团纤维分布明显高于SCI组和SCI+SPIO组(P<0.05);与SCI+c-MSS组和SCI+Tr组相比,SCI+c-MSS+Tr组在冠状面上拥有最大数量的GRNs下行投射纤维分布。(5)与SCI组相比,c-MSS和Tr干预均提高了 vglut2在腰髓前角的蛋白表达水平(SCIvs SCI+c-MSS,P<0.01;SCIvs SCI+Tr,P<0.0001);GAD67和vGAT在SCI后的脊髓前角中表达水平升高;与SCI组相比,c-MSS干预使腰髓前角GAD67和vGAT的蛋白表达显著降低(GAD67:SCIvs SCI+c-MSS,P<0.0001;vGAT:SCI vs SCI+c-MSS,P<0.05);与 SCI 组相比,Tr 组中 GAD67和vGAT的蛋白表达水平降低不明显(P>0.05);在SCI+c-MSS+Tr组中,vglut2、GAD67和vGAT的蛋白表达水平均下降,且接近Sham组的水平;超过50%的GRNs核团下行投射纤维与vGAT共标;与SCI组和SCI+Tr组相比,SCI+c-MSS组中运动神经元膜表面与vGAT共标的GRNs下行投射数量显著上调;但各组运动神经元区域vGAT的总表达没有统计学差异(P>0.05)。[研究结论]c-MSS联合运动训练显著提高SCI小鼠运动功能恢复,其机制可能与c-MSS联合运动训练通过促进GRNs核团下行纤维跨过损伤灶,降低腰髓运动神经元兴奋性,恢复运动神经元突触前抑制,并调节腰髓前角神经递质再平衡,从而重塑皮质—网状GRNs—脊髓环路有关。更多还原

【Abstract】 Part Ⅰ Study on the stability,safety,and efficacy of combined magnetic stimulation system applied to the GRNs in vivoBackground:The gigantocellular reticular nucleus(GRNs)in the ventral brainstem is the primary source of the reticulospinal tract that remains well-preserved following contusive spinal cord injury(SCI).Its descending fibers can establish a signaling bridge between the cerebral cortex and the spinal cord,enabling functional connections between the cortex and neural circuits below the injured spinal cord region.Given the unique anatomical location of the brainstem,long-term implantation of electrodes or optical fibers in this region poses significant challenges.Additionally,repetitive transcranial magnetic stimulation(rTMS)is insufficient in depth and poorly targeted,making it difficult to effectively activate the GRNs.To address this challenge,this study utilizes superparamagnetic iron oxide(SPIO)nanoparticles and extracorporeal magnetic field(MF),known as the combined magnetic stimulation system(c-MSS),to precisely target the GRNs.This study preliminarily investigates the in vivo stability,safety,and efficacy of this magnetic stimulation system applied to the GRNs.Methods:Using a Tio contusion mouse model of SCI,SPIO nanoparticles were injected into the GRNs of SCI mice via stereotactic apparatus.The distribution and retention time of SPIO nanoparticles in the GRNs were dynamically monitored using small animal in vivo magnetic resonance imaging(MRI).The safety of SPIO nanoparticles was evaluated in vivo through dynamic body weight monitoring and TdT-mediated dUTP Nick-End Labeling(TUNEL)assay on brain slices of the GRNs region.Appropriate stimulation parameters were screened using Basso Mouse Scale(BMS)behavioral scoring.The activation of the stimulated brain region was assessed by immunofluorescence staining of the immediate early protein c-Fos in brain slices of the GRNs region.Results:(1)MRI showed that SPIO nanoparticles remained in the GRNs nucleus for at least 8 weeks without significant degradation and slowly degraded over time(up to 24 weeks).(2)SPIO nanoparticles did not affect the body weight changes of SCI mice,and TUNEL staining results also showed that SPIO nanoparticles did not induce apoptosis of cells in the GRNs nucleus.(3)Compared to 10 Hz,a stimulation frequency of 20 Hz was more effective in promoting motor recovery in SCI mice(P<0.01).(4)The c-MSS could precisely activate neurons in the GRNs region.Conclusion:The SPIO nanoparticles in the GRNs region were relatively stable and had minimal toxic effects on local cells.The c-MSS system was able to activate GRNs neurons,with an intervention frequency of 20 Hz being more effective in promoting motor recovery in SCI mice.Part Ⅱ Magnetic selected stimulation of gigantocellular reticular nucleus combined treadmill training improving locomotion recovery in mice after spinal cord contusion and exploring the mechanismBackground:Treadmill training has been proven to elevate locomotion in patients with spinal cord injury(SCI),but the improvement tends to plateau.To overcome this bottleneck,research has shown that neuromodulation techniques can further enhance and sustain the effects of locomotor therapy.Based on the research background,this study designed a protocol combining c-MSS stimulation with treadmill training,aiming to explore whether this combined therapy can further enhance locomotion function in SCI mice and to investigate the potential underlying mechanisms.Methods:On day 5 post-injury(T10 contusion),superparamagnetic iron oxide(SPIO)nanoparticles were injected into the GRNs using stereotaxic apparatus.Starting from day 7 post-injury,the mice underwent 8 weeks of c-MSS intervention,with a twice daily for 20 minutes each session,stimulation frequency of 20 Hz,five days a week.Mice in the c-MSS combined with treadmill training group received treadmill training after c-MSS intervention,consisting of two sessions per day for 30 minutes each,five days a week,starting at minimal speed and gradually increasing to 8-10 m/min.Anterograde tracing viral injections were performed in week 5 post-injury.Locomotor function was assessed using Basso Mouse Scale(BMS),grid-walking test,and gait analysis.The integrity of the corticospinal tract was evaluated using motor-evoked potentials(MEPs),and the excitability of lumbar spinal anterior horn motoneurons was assessed using H-reflex.The distribution of GRNs’ descending projections was analyzed in the T10 lesion and lumbar spinal cord anterior horn using anterograde tracing viruses.Changes in the expression of excitatory and inhibitory neurotransmitters in the lumbar spinal anterior horn were analyzed using immunofluorescence staining.Results:(1)Behavioral scoring:①BMS scores showed immediate lower limb paralysis(BMS=0)in all groups post-injury;by week 1 post-injury,all groups exhibited some spontaneous functional recovery(BMS≈1);from week 1 to week 9 post-SCI,all groups showed varying degrees of improvement in motor function;there was no significant difference in BMS scores between the SCI and SCI+SPIO groups at all time points,with scores stabilizing around week 7;compared to the SCI group,the SCI+c-MSS group showed statistically significant improvement in motor function at week 6 post-injury(P<0.05);compared to the SCI group,the SCI+Tr group reached a BMS score of around 4 by week 4,with statistically significant differences(P<0.05);the BMS scores of the SCI+Tr group were higher than those of the c-MSS group at all time points,but there was no statistical significance between them(P>0.05);except for the Sham group,the SCI+c-MSS+Tr group had the best motor function recovery with BMS≈7 by week 9 post-injury.②Grid walking test results showed no significant difference in foot droops(FDs)counts between the SCI and SCI+SPIO groups(P>0.05);compared to the SCI group,both the SCI+c-MSS and SCI+Tr groups had a reduction in FDs counts with statistically significant differences(P<0.05);apart from the Sham groups,the SCI+c-MSS+Tr group had the fewest FDs counts.③Gait analysis revealed significant improvements in footprint area,foot strength,and swing speed in the SCI+c-MSS and SCI+Tr groups compared to the SCI group;no statistically significant differences were observed between the SCI+Tr and SCI+cMSS groups in these parameters(P>0.05);the SCI+c-MSS+Tr group showed better performance in footprint area,foot pressure,and swing speed compared to the SCI+cMSS and SCI+Tr groups(SCI+c-MSS vs SCI+c-MSS+Tr in footprint area,P<0.05).(2)Before the injury,MEPs could be consistently elicited in all groups;one week after the injury,MEPs disappeared in all groups except the Sham group;by week 9 postinjury,MEPs were not induced in the remaining four groups except the Sham group and the SCI+c-MSS+Tr group;compared to the Sham group(MEPs induction rate of 100%),the percentage of MEPs induced in the SCI+c-MSS+Tr group was 60%(6 out of 10 mice);the amplitude in the SCI+c-MSS+Tr group was smaller(Sham vs SCI+c-MSS+Tr,9.55±1.0928 mV vs 0.70959±0.57379 mV),and the latency was longer(Sham vs SCI+c-MSS+Tr,4.305±0.188 ms vs 23.085±1.60 ms).(3)H-reflex results showed that all groups exhibited frequency-dependent inhibition of the Hmax/Mmax ratio and prolongation of the H-reflex latency at stimulation frequencies of 0.1,0.5,1,and 5 Hz.Compared to the SCI group,the SCI+Tr group had a significantly lower Hmax/Mmax ratio,especially at 0.1 Hz(P<0.0001).In contrast,the SCI+c-MSS group showed no significant reduction in the Hmax/Mmax ratio at any stimulation frequency compared to the SCI group(P>0.05).c-MSS intervention significantly increased the H-reflex latency at all stimulation frequencies(at 0.1 Hz,SCI vs SCI+c-MSS,P<0.05;at 0.5 Hz,SCI vs SCI+c-MSS,P<0.01;at 1 Hz,SCI vs SCI+c-MSS,P<0.0001;at 5 Hz,SCI vs SCI+c-MSS,P<0.0001).However,Tr intervention did not significantly prolong the H-reflex latency(P>0.05).In the SCI+c-MSS+Tr group,the Hmax/Mmax ratio was reduced and the latency prolonged,with no significant difference compared to the Sham group(P>0.05).(4)Compared to the Sham group,spinal cord contusion resulted in a significant reduction in GRNs projections below the injury site;compared to the SCI and SCI+SPIO groups,c-MSS intervention promoted more GRNs’ fibers crossing the injury site(0 μm)(P<0.05);SCI+Tr did not show a significant difference in the number of GRNs’descending fibers at 0 μm compared to the SCI group(P>0.05);apart from the Sham group,the combination of c-MSS and Tr intervention promoted the most GRNs’ fibers crossing the injury site;both the SCI+c-MSS group and the SCI+c-MSS+Tr group showed more GRNs’ descending fibers below 700 μm and 1400 μm compared to the SCI group,but not to a statistically significant level(P>0.05);in the coronal sections of the lumbar spinal cord,the distribution of GRNs’ descending fibers was significantly higher in the SCI+c-MSS group compared to the SCI and SCI+SPIO groups;compared to the SCI+c-MSS and SCI+Tr groups,the SCI+c-MSS+Tr group had the largest GRNs’ projection distribution in the coronal plane.(5)Compared to the SCI group,both c-MSS and Tr interventions increased the protein expression levels of vglut2 in the lumbar spinal cord anterior horn(SCI vs SCI+cMSS,P<0.01;SCI vs SCI+Tr,P<0.0001).The expression levels of GAD67 and vGAT were elevated in the anterior horn of the spinal cord after SCI.Compared to the SCI group,c-MSS intervention significantly reduced the protein expression of GAD67 and vGAT in the lumbar spinal cord anterior horn(GAD67:SCI vs SCI+c-MSS,P<0.0001;vGAT:SCI vs SCI+c-MSS,P<0.05).In the Tr group,the protein expression levels of GAD67 and vGAT were not significantly reduced compared to the SCI group(P>0.05).In the SCI+c-MSS+Tr group,the protein expression levels of vglut2,GAD67,and vGAT all decreased and approached the levels seen in the Sham group.More than 50%of the descending projection fibers from the GRNs nucleus co-localized with vGAT.Compared to the SCI group and the SCI+Tr group,the number of GRNs descending projections co-localized with vGAT on the motor neuron membrane surface was significantly upregulated in the SCI+c-MSS group.However,there was no statistically significant difference in the total expression of vGAT around the motor neuron area among the groups(P>0.05).Conclusion:c-MSS combined with treadmill training significantly enhanced locomotion function recovery in SCI mice.The mechanism may involve c-MSS and treadmill training promoting the crossing of descending fibers from the GRNs over the injury site,reducing the excitability of lumbar motor neurons,restoring presynaptic inhibition of motor neurons,regulating the neurotransmitters rebalance in the anterior horn of the lumbar spinal cord,and thereby reshaping the cortico-reticulo/GRNs-spinal circuit.更多还原