Department of Rehabilitation, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, Guangdong, China.
Department of Rehabilitation, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, Guangdong, China.
Ann Anat. 2023 Oct;250:152136. doi: 10.1016/j.aanat.2023.152136. Epub 2023 Jul 26.
Our previous study has confirmed that constraint-induced movement therapy (CIMT) could promote neural remodeling in hemiplegic cerebral palsy (HCP) mice through Nogo-A/NgR/RhoA/ROCK signaling, however, the upstream mechanism was still unclear. Therefore, the present study aimed to further explore the mechanism of CIMT regulating the expression of Nogo-A in HCP mice.
HCP mice were well established through ligating the left common carotid artery of 7-day-old pups and being placed in a hypoxic box which was filled with a mixture of 8% oxygen and 92% nitrogen. CIMT intervention was conducted by taping to fix the entire arm of the contralateral side (left) to force the mice to use the affected limb (right). Bioinformatics prediction and luciferase experiment were performed to confirm that miR-182-5p was targeted with Nogo-A. The beam test and grip test were applied to examine the behavioral performance under the intervention of c-Jun and CIMT. Also, immunofluorescence, Golgi staining, and transmission electron microscopy were conducted to show that the lenti-expression of c-Jun could increases the expression of myelin, and downregulates the expression of Nogo-A under the CIMT on HCP mice.
(1) The beam walking test and grip test experiment results showed that compared with the control group, the HCP + nCIMT group's forelimb grasping ability and balance coordination ability were decreased (P < 0.05). (2) The results of Golgi staining, and transmission electron microscopy showed that the thickness of myelin sheath and the density of dendritic spines in the HCP + nCIMT group were lower than those in the control group (P < 0.05). Compared with the HCP + nCIMT group, the cerebral cortex myelin sheath thickness, dendrite spine density and nerve filament expression were increased in HCP + CIMT group (P < 0.05). (3) Immunofluorescence staining showed that the expression of Nogo-A in the cerebral cortex of the HCP + nCIMT group was higher than that of the HCP + CIMT group (P < 0.05). Compared with the HCP + CIMT group, the expression of Nogo-A in the HCP + LC + CIMT group was decreased and, in the HCP, + SC + CIMT group was significantly increased (P < 0.05). Compared with the HCP + nCIMT group, the expression of c-Jun in the control, HCP + CIMT, HCP + LC + nCIMT and HCP + LC + CIMT groups was significantly increased, and in the HCP + SC + CIMT was decreased (P < 0.05). (4) Real-time quantitative polymerase chain reaction (RT-qPCR) results showed that the expression level of miR-182-5p in the HCP + LC + CIMT group was more increased than that in the HCP + nCIMT group (P < 0.05). The expression level of miR-182-5p in the HCP + LC + CIMT group was higher than that in the HCP + LC + nCIMT group and the HCP + SC + CIMT group (P < 0.05).
These data identified that CIMT might stimulate the remodeling of neurons and myelin in the motor cortex by partially inhibiting the c-Jun/miR-182-5p/Nogo-A pathway, thereby facilitating the grasping performance and balance function of HCP mice.
我们之前的研究已经证实，通过抑制 Nogo-A/NgR/RhoA/ROCK 信号通路，强制性运动疗法（CIMT）可以促进偏瘫脑瘫（HCP）小鼠的神经重塑，然而，其上游机制仍不清楚。因此，本研究旨在进一步探讨 CIMT 调节 HCP 小鼠 Nogo-A 表达的机制。
通过结扎 7 日龄幼鼠的左颈总动脉并将其置于充满 8%氧气和 92%氮气的缺氧箱中，成功建立了 HCP 小鼠模型。通过将对侧（左侧）手臂固定在一起来进行 CIMT 干预，迫使小鼠使用受影响的肢体（右侧）。通过生物信息学预测和荧光素酶实验证实 miR-182-5p 是 Nogo-A 的靶标。在 c-Jun 和 CIMT 的干预下，进行了束状试验和握力试验，以检测行为表现。此外，还进行了免疫荧光、高尔基染色和透射电子显微镜检查，以显示 lenti-c-Jun 的表达可以增加髓鞘的表达，下调 HCP 小鼠 CIMT 下 Nogo-A 的表达。
（1）束状行走试验和握力试验结果显示，与对照组相比，HCP+nCIMT 组的前肢抓握能力和平衡协调能力下降（P<0.05）。（2）高尔基染色和透射电子显微镜结果显示，HCP+nCIMT 组的髓鞘厚度和树突棘密度低于对照组（P<0.05）。与 HCP+nCIMT 组相比，HCP+CIMT 组大脑皮质的髓鞘厚度、树突棘密度和神经丝表达增加（P<0.05）。（3）免疫荧光染色显示，HCP+nCIMT 组大脑皮质的 Nogo-A 表达高于 HCP+CIMT 组（P<0.05）。与 HCP+CIMT 组相比，HCP+LC+CIMT 组的 Nogo-A 表达减少，而 HCP+SC+CIMT 组的 Nogo-A 表达显著增加（P<0.05）。与 HCP+CIMT 组相比，对照组、HCP+CIMT 组、HCP+LC+nCIMT 组和 HCP+LC+CIMT 组的 c-Jun 表达显著增加，而 HCP+SC+CIMT 组的 c-Jun 表达减少（P<0.05）。（4）实时定量聚合酶链反应（RT-qPCR）结果显示，HCP+LC+CIMT 组 miR-182-5p 的表达水平比 HCP+nCIMT 组明显增加（P<0.05）。HCP+LC+CIMT 组的 miR-182-5p 表达水平高于 HCP+LC+nCIMT 组和 HCP+SC+CIMT 组（P<0.05）。
这些数据表明，CIMT 可能通过部分抑制 c-Jun/miR-182-5p/Nogo-A 通路来刺激运动皮层神经元和髓鞘的重塑，从而促进 HCP 小鼠的抓握性能和平衡功能。
