PDF(Pubmed)
Abstract：
This study aimed to investigate the effect of perineuronal net (PNN) and neurocan (NCAN) on spinal inhibitory parvalbumin interneuron (PV-IN), and the mechanism of electroacupuncture (EA) in promoting spinal cord injury (SCI) repair through neurocan in PNN.
A mouse model of SCI was established. Sham-operated mice or SCI model mice were treated with chondroitin sulfate ABC (ChABC) enzyme or control vehicle for 2 weeks (i.e., sham+veh group, sham+ChABC group, SCI+veh group, and SCI+ChABC group, respectively), and then spinal cord tissues were taken from the T10 lesion epicenter for RNA sequencing (RNA-seq). MSigDB Hallmark and C5 databases for functional analysis, analysis strategies such as differential expression gene analysis (DEG), Kyoto Encyclopedia of Genes and Genomes (KEGG), gene set enrichment analysis (GSEA), and protein-protein interaction (PPI). According to the results of RNA-seq analysis, the expression of NCAN was knocked down or overexpressed by virus intervention, or/and EA intervention. Polymerase chain reaction (PCR), immunofluorescence, western blot, electrophysiological, and behavioral tests were performed.
After the successful establishment of SCI model, the motor dysfunction of lower limbs, and the expression of PNN core glycan protein at the epicenter of SCI were reduced. RNA-seq and PCR showed that PNN core proteoglycans except NCAN showed the same expression trend in normal and injured spinal cord treated with ChABC. KEGG and GSEA showed that PNN is mainly associated with inhibitory GABA neuronal function in injured spinal cord tissue, and PPI showed that NCAN in PNN can be associated with inhibitory neuronal function through parvalbumin (PV). Calcium imaging showed that local parvalbumin interneuron (PV-IN) activity decreased after PNN destruction, whether due to ChABC treatment or surgical bruising of the spinal cord. Overexpression of neurocan in injured spinal cord can enhance local PV-IN activity. PCR and western blot suggested that overexpression or knockdown of neurocan could up-regulate or down-regulate the expression of GAD. At the same time, the activity of PV-IN in the primary motor cortex (M1) and the primary sensory cortex of lower (S1HL) extremity changed synchronously. In addition, overexpression of neurocan improved the electrical activity of the lower limb and promoted functional repair of the paralyzed hind limb. EA intervention reversed the down-regulation of neurocan, enhanced the expression of PNN in the lesioned area, M1 and S1HL.
Neurocan in PNN can regulate the activity of PV-IN, and EA can promote functional recovery of mice with SCI by upregulating neurocan expression in PNN.
A mouse model of SCI was established. Sham-operated mice or SCI model mice were treated with chondroitin sulfate ABC (ChABC) enzyme or control vehicle for 2 weeks (i.e., sham+veh group, sham+ChABC group, SCI+veh group, and SCI+ChABC group, respectively), and then spinal cord tissues were taken from the T10 lesion epicenter for RNA sequencing (RNA-seq). MSigDB Hallmark and C5 databases for functional analysis, analysis strategies such as differential expression gene analysis (DEG), Kyoto Encyclopedia of Genes and Genomes (KEGG), gene set enrichment analysis (GSEA), and protein-protein interaction (PPI). According to the results of RNA-seq analysis, the expression of NCAN was knocked down or overexpressed by virus intervention, or/and EA intervention. Polymerase chain reaction (PCR), immunofluorescence, western blot, electrophysiological, and behavioral tests were performed.
After the successful establishment of SCI model, the motor dysfunction of lower limbs, and the expression of PNN core glycan protein at the epicenter of SCI were reduced. RNA-seq and PCR showed that PNN core proteoglycans except NCAN showed the same expression trend in normal and injured spinal cord treated with ChABC. KEGG and GSEA showed that PNN is mainly associated with inhibitory GABA neuronal function in injured spinal cord tissue, and PPI showed that NCAN in PNN can be associated with inhibitory neuronal function through parvalbumin (PV). Calcium imaging showed that local parvalbumin interneuron (PV-IN) activity decreased after PNN destruction, whether due to ChABC treatment or surgical bruising of the spinal cord. Overexpression of neurocan in injured spinal cord can enhance local PV-IN activity. PCR and western blot suggested that overexpression or knockdown of neurocan could up-regulate or down-regulate the expression of GAD. At the same time, the activity of PV-IN in the primary motor cortex (M1) and the primary sensory cortex of lower (S1HL) extremity changed synchronously. In addition, overexpression of neurocan improved the electrical activity of the lower limb and promoted functional repair of the paralyzed hind limb. EA intervention reversed the down-regulation of neurocan, enhanced the expression of PNN in the lesioned area, M1 and S1HL.
Neurocan in PNN can regulate the activity of PV-IN, and EA can promote functional recovery of mice with SCI by upregulating neurocan expression in PNN.
摘要：
目的：本研究旨在研究神经周网状物（PNN）和神经蛋白聚糖（NCAN）对脊髓抑制性小白蛋白中间神经元（PV-IN）的影响，以及电针（EA）通过PNN中的Neurocan促进脊髓损伤（SCI）修复的机制。
方法：建立SCI小鼠模型。假手术小鼠或SCI模型小鼠用硫酸软骨素ABC（ChABC）酶或对照载体治疗2周（即，sham+veh组，sham+ChABC组,SCI+veh集团，SCI+ChABC组，分别)，然后从T10病变中心取脊髓组织进行RNA测序(RNA-seq)。用于功能分析的MSigDBHallmark和C5数据库，差异表达基因分析(DEG)等分析策略，京都基因和基因组百科全书（KEGG），基因集富集分析(GSEA)，和蛋白质-蛋白质相互作用（PPI）。根据RNA-seq分析的结果,NCAN的表达被病毒干预敲低或过表达，或/和EA干预。聚合酶链反应（PCR），免疫荧光，westernblot,电生理学,并进行行为测试。
结果：成功建立SCI模型后，下肢运动功能障碍，SCI中心PNN核心聚糖蛋白表达降低。RNA-seq和PCR显示，除NCAN外，PNN核心蛋白聚糖在ChABC处理的正常和损伤脊髓中表现出相同的表达趋势。KEGG和GSEA显示PNN主要与损伤脊髓组织中抑制性GABA神经元功能有关,和PPI显示PNN中的NCAN可以通过小清蛋白（PV）与抑制性神经元功能有关。钙成像显示PNN破坏后局部小白蛋白中间神经元（PV-IN）活性降低，无论是由于ChABC治疗还是脊髓的手术擦伤。神经蛋白聚糖在受损脊髓中的过表达可增强局部PV-IN活性。PCR和westernblot提示过表达或敲低的Neurocan可以上调或下调GAD的表达。同时,初级运动皮层（M1）和下肢初级感觉皮层（S1HL）的PV-IN活性同步变化。此外,神经的过度表达可以改善下肢的电活动，促进瘫痪后肢的功能修复。EA干预逆转了神经聚糖的下调，增强了病变区PNN的表达，M1和S1HL。
结论：PNN中的Neurocan可以调节PV-IN的活性，EA可以通过上调PNN中Neurocan的表达来促进SCI小鼠的功能恢复。
方法：建立SCI小鼠模型。假手术小鼠或SCI模型小鼠用硫酸软骨素ABC（ChABC）酶或对照载体治疗2周（即，sham+veh组，sham+ChABC组,SCI+veh集团，SCI+ChABC组，分别)，然后从T10病变中心取脊髓组织进行RNA测序(RNA-seq)。用于功能分析的MSigDBHallmark和C5数据库，差异表达基因分析(DEG)等分析策略，京都基因和基因组百科全书（KEGG），基因集富集分析(GSEA)，和蛋白质-蛋白质相互作用（PPI）。根据RNA-seq分析的结果,NCAN的表达被病毒干预敲低或过表达，或/和EA干预。聚合酶链反应（PCR），免疫荧光，westernblot,电生理学,并进行行为测试。
结果：成功建立SCI模型后，下肢运动功能障碍，SCI中心PNN核心聚糖蛋白表达降低。RNA-seq和PCR显示，除NCAN外，PNN核心蛋白聚糖在ChABC处理的正常和损伤脊髓中表现出相同的表达趋势。KEGG和GSEA显示PNN主要与损伤脊髓组织中抑制性GABA神经元功能有关,和PPI显示PNN中的NCAN可以通过小清蛋白（PV）与抑制性神经元功能有关。钙成像显示PNN破坏后局部小白蛋白中间神经元（PV-IN）活性降低，无论是由于ChABC治疗还是脊髓的手术擦伤。神经蛋白聚糖在受损脊髓中的过表达可增强局部PV-IN活性。PCR和westernblot提示过表达或敲低的Neurocan可以上调或下调GAD的表达。同时,初级运动皮层（M1）和下肢初级感觉皮层（S1HL）的PV-IN活性同步变化。此外,神经的过度表达可以改善下肢的电活动，促进瘫痪后肢的功能修复。EA干预逆转了神经聚糖的下调，增强了病变区PNN的表达，M1和S1HL。
结论：PNN中的Neurocan可以调节PV-IN的活性，EA可以通过上调PNN中Neurocan的表达来促进SCI小鼠的功能恢复。
