脊髓压迫和半横断损伤小鼠模型的时空基因表达谱及修复机制研究

doi:10.12300/j.issn.1674-5817.2025.052

摘要/Abstract

摘要：

目的旨在深入探讨脊髓压迫损伤和半横断损伤小鼠模型在亚急性至慢性期（1~28 d），局部微环境中相关基因的表达次序及其分子机制，从而揭示其脊髓修复的分子特征，并为脊髓损伤治疗靶点的选择提供理论依据。方法选用36只8~9周龄SPF级ICR小鼠，随机分为假手术对照（CTR）组、脊髓半横断损伤（HSCI）组和脊髓压迫损伤（SCC）组，每组12只。CTR组小鼠经麻醉后，仅行椎板切除术暴露T₉~T₁₀节段的硬脊膜，并保持其完整，在空气中暴露10 min后缝合切口，不施加任何损伤干预；HSCI组小鼠在CTR组操作的基础上，通过显微器械横断70%的T₉节段脊髓组织构建脊髓半横断损伤模型；SCC组小鼠也在CTR组操作的基础上，采用自制压迫器（30 g实心小铁棒）持续压迫T₁₀节段脊髓10 min构建脊髓压迫损伤模型。在术后第1、3、7、14、21及28天，采用改良BBB（Basso-Beattie-Bresnahan）评分法评估各组小鼠运动功能的恢复情况；在术后第7、14天，将小鼠麻醉后，取损伤段脊髓组织，通过RNA测序（RNA-Seq）和富集分析来解析脊髓损伤小鼠模型中特异的分子网络演变，并利用实时荧光定量PCR法验证关键基因的表达情况。结果 BBB评分结果显示，SCC组小鼠的运动功能恢复情况显著优于HSCI组，其BBB评分在4周内呈持续上升趋势且高于HSCI组（P<0.001）。基于RNA-Seq差异表达基因的基因本体（gene ontology，GO）和京都基因与基因组数据库（Kyoto Encyclopedia of Genes and Genomes，KEGG）富集分析显示：与CTR组相比，SCC组在术后第7天，细胞外基质相关基因显著上调（P<0.05），而轴突引导相关基因显著下调（P<0.05）；在术后第21天，SCC组免疫调控和视黄醇信号通路相关基因被显著激活（P<0.05）。相比之下，HSCI组在术后第7天，炎症和免疫反应基因显著上调（P<0.001），神经元分化和突触形成相关基因则显著下调（P<0.001）；在术后第21天，细胞-基质连接和N-甲基-D-天门冬氨酸受体相关基因显著上调（P<0.001）。此外，与SCC组相比，HSCI组在损伤后第7天和第21天的GO和KEGG富集分析中表现出不同的通路富集特征。术后第7天，HSCI组中的NOD样受体信号通路以及补体和凝血级联反应相关基因显著上调（P<0.001）；而在术后第21天，细胞外基质-受体相互作用和神经活性配体-受体相互作用通路相关基因被显著激活（P<0.001）。最后，实时荧光定量PCR验证结果与RNA-Seq结果高度一致，进一步确认了SCC组和HSCI组关键基因表达趋势的差异。结论脊髓压迫损伤与脊髓半横断损伤模型可能驱动了不同的修复路径：SCC模型中部分轴突的保留使其更倾向于组织修复，而脊髓半横断损伤模型则需协调更复杂的分子网络以达成新的平衡。这一发现进一步深化了对脊髓损伤异质性调控机制的理解。

关键词: 脊髓损伤, ICR小鼠, 小鼠模型, 脊髓压迫损伤, 脊髓半横断损伤, RNA测序, 神经修复

Abstract:

Objective To investigate the gene expression sequence and molecular mechanisms in the local microenvironment during the subacute to chronic phases (1-28 days) in mouse models of spinal cord compression injury and hemisection spinal cord injury, thereby revealing the molecular characteristics of spinal cord repair and providing a theoretical basis for selecting therapeutic targets for spinal cord injury. Methods Thirty-six 8-9-week-old SPF-grade ICR mice were randomly divided into three groups (n=12 per group): sham-operated control (CTR) group, hemisection spinal cord injury (HSCI) group, and spinal cord compression injury (SCC) group. Mice in the CTR group underwent the same surgical preparation and anesthesia, followed by a dorsal midline incision at the T₉-T₁₀ segment. After layer-by-layer dissection and removal of the corresponding lamina, the spinal cord dura mater was fully exposed and kept intact. The cord was exposed to air for 10 minutes (matching the duration of the compression injury group), during which any instrument contact with the cord was avoided. The incision was then irrigated and sutured. The HSCI group underwent a 70% transection of the T₉ spinal cord segment using micro-instruments to establish a hemisection spinal cord injury model. The SCC group underwent sustained compression of the T₁₀ spinal cord segment for 10 minutes using a self-made compressor (a 30 g solid small iron bar) to establish a spinal cord compression injury model. Motor function recovery was assessed using the modified Basso-Beattie-Bresnahan (BBB) score on postoperative days 1, 3, 7, 14, 21, and 28. On days 7 and 14 post-operation, mice were anesthetized, and the injured spinal cord segments were harvested. The evolution of specific molecular networks in the spinal cord injury mouse models was analyzed via RNA sequencing (RNA-Seq) and enrichment analysis, and the expression of key genes was verified using real time fluorogenic quantitative PCR. Results BBB scores indicated that motor function recovery in the SCC group was significantly better than that in the HSCI group, with BBB scores showing a continuously increasing trend and remaining higher than those in the HSCI group over the 4-week period (P <0.001). Gene ontology （GO)and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analyses based on RNA-Seq differentially expressed genes revealed that, compared to the CTR group, genes related to the extracellular matrix were significantly up-regulated (P<0.05), while genes related to axon guidance were significantly down-regulated (P <0.05) in the SCC group on day 7 post-operation. On day 21, genes involved in immune regulation and the retinol signaling pathway were significantly activated in the SCC group (P<0.05). In contrast, in the HSCI group, genes associated with inflammation and immune response were significantly up-regulated (P<0.001), while genes related to neuronal differentiation and synapse formation were significantly down-regulated (P <0.001) on day 7. On day 21, genes related to cell-matrix junctions and N-methyl-D-aspartate receptors were significantly up-regulated (P<0.001) in the HSCI group. Furthermore, compared to the SCC group, the HSCI group exhibited different pathway enrichment characteristics in GO and KEGG analyses on days 7 and 21 post-injury. On day 7, genes involved in the NOD-like receptor signaling pathway and the complement and coagulation cascades were significantly up-regulated in the HSCI group (P<0.001). On day 21, genes related to the extracellular matrix-receptor interaction and the neuroactive ligand-receptor interaction pathways were significantly activated (P<0.001). Finally, real time fluorogenic quantitative PCR validation results were highly consistent with the RNA-Seq results, further confirming the differential expression trends of key genes between the SCC and HSCI groups. Conclusion The SCC and HSCI injury models may drive distinct repair pathways: the preservation of some axons in the SCC model predisposes it toward tissue repair, whereas the HSCI model requires the coordination of more complex molecular networks to achieve a new equilibrium. This finding further deepens the understanding of the heterogeneous regulatory mechanisms underlying spinal cord injury.

Key words: Spinal cord injury, ICR mice, Mouse model, Spinal cord compression injury, Hemisection spinal cord injury, RNA-Seq, Neural repair

中图分类号:

Q95-33

徐波,陈泰任,方乾,等. 脊髓压迫和半横断损伤小鼠模型的时空基因表达谱及修复机制研究[J]. 实验动物与比较医学, 2026, 46(1): 32-45. DOI: 10.12300/j.issn.1674-5817.2025.052.

XU Bo,CHEN Tairen,FANG Qian,et al. Research on Spatiotemporal Gene Expression Profiles and Repair Mechanisms of Spinal Cord Compression and Hemisection Spinal Cord Injury Mouse Models[J]. Laboratory Animal and Comparative Medicine, 2026, 46(1): 32-45. DOI: 10.12300/j.issn.1674-5817.2025.052.

图/表 7

注：在术后第1、3、7、21和28天采用改良BBB（Basso-Beattie-Bresnahan）评分评估假手术对照（CTR）组，脊髓半横断损伤（HSCI）组和脊髓压迫损伤（SCC）组小鼠运动功能的恢复情况，并在术后第7和21天对三组小鼠脊髓损伤段组织进行RNA测序（RNA-seq）。
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图1 模型构建和实验过程的技术路线图
注：在术后第1、3、7、21和28天采用改良BBB（Basso-Beattie-Bresnahan）评分评估假手术对照（CTR）组，脊髓半横断损伤（HSCI）组和脊髓压迫损伤（SCC）组小鼠运动功能的恢复情况，并在术后第7和21天对三组小鼠脊髓损伤段组织进行RNA测序（RNA-seq）。

Figure 1 Technical roadmap for model construction and experimental process
Note： On postoperative days 1， 3， 7， 21， and 28， the modified Basso-Beattie-Bresnahan scale was used to assess motor recovery in the sham-operated control （CTR） group， hemisection spinal cord injury （HSCI） group， and spinal cord compression injury （SCC） group. RNA-seq was performed on spinal cord tissue from the injured segments of mice in all three groups at days 7 and 21 post-surgery.

注：A，CTR组、SCC组和HSCI组小鼠的脊髓体视镜明场图（白色虚线框内为脊髓造模状态）；B，SCC组和HSCI组小鼠在术后第1、3、7、14、21和28天恢复情况；C，为术后第28天SCC组（标记为a）和HSCI组（标记为b）小鼠恢复情况的对比图；D，脊髓损伤术后不同时间点的BBB评分。CTR，假手术对照组；SCC，脊髓压迫损伤组；HSCI；脊髓半横断损伤组。^???与CTR组相比，P<0.001；^#与组内前一个时间点相比，P<0.05。
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图2 假手术对照组、脊髓半横断损伤组和脊髓压迫损伤组小鼠宏观形态、恢复过程及运动功能的比较
注：A，CTR组、SCC组和HSCI组小鼠的脊髓体视镜明场图（白色虚线框内为脊髓造模状态）；B，SCC组和HSCI组小鼠在术后第1、3、7、14、21和28天恢复情况；C，为术后第28天SCC组（标记为a）和HSCI组（标记为b）小鼠恢复情况的对比图；D，脊髓损伤术后不同时间点的BBB评分。CTR，假手术对照组；SCC，脊髓压迫损伤组；HSCI；脊髓半横断损伤组。^???与CTR组相比，P<0.001；^#与组内前一个时间点相比，P<0.05。

Figure 2 Comparison of macroscopic morphology， recovery process and motor function of mice in sham-operated control group， spinal cord compression and hemisection spinal cord injury group
Note： A， the spinal cord stereoscopy bright-field images of CTR group， SCC group and HSCI group， respectively（the white dashed box indicates the site of spinal cord modeling）； B， recovery of mice in SCC and HSCI groups at 1， 3， 7， 14， 21 and 28 days after modeling； C， the comparison chart of the recovery of mice in the SCC group （marked as a） and the HSCI group （marked as b） on the 28th day after surgery； D， BBB scores at different time points after SCI modeling. CTR， sham-operated control group； SCC， spinal cord compression injury group； HSCI， hemisection spinal cord injury group. ^???P<0.001， compared with CTR group； ^#P<0.05， compared with the previous time point within the group.

注：A~B，与CTR组相比，SCC组小鼠在术后第7天和第21天基因的表达情况；C~D，与CTR组相比，HSCI组小鼠在第7天和第21天基因的表达情况。红色圆点代表上调的差异表达基因（DEGs），蓝色圆点代表下调的DEGs，灰色点代表表达无显著差异的基因。CTR，假手术对照组；HSCI，脊髓半横断损伤组；SCC，脊髓压迫损伤组。
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图3 脊髓损伤组小鼠与假手术对照组小鼠在第7天和第21天基因表达差异的火山图
注：A~B，与CTR组相比，SCC组小鼠在术后第7天和第21天基因的表达情况；C~D，与CTR组相比，HSCI组小鼠在第7天和第21天基因的表达情况。红色圆点代表上调的差异表达基因（DEGs），蓝色圆点代表下调的DEGs，灰色点代表表达无显著差异的基因。CTR，假手术对照组；HSCI，脊髓半横断损伤组；SCC，脊髓压迫损伤组。

Figure 3 Volcano plots of differentially expressed genes in spinal cord injury models compared with the sham-operated control group at day 7 and day 21 post-injury
Note： A-B， gene expression in the SCC group compared with the CTR group at postoperative day 7 and day 21； C-D， gene expression in the HSCI group compared with the CTR group at day 7 and day 21. Red dots represent up-regulated differentially expressed genes （DEGs）， blue dots represent down-regulated DEGs， and gray dots represent genes with no significant difference in expression. CTR， sham-operated control group； HSCI， hemisection spinal cord injury group； SCC， spinal cord compression injury group.

注：A~D，与CTR组相比，SCC组在第7和21天上调和下调关键基因的实时荧光定量PCR结果；E~H，与CTR组相比，HSCI组在第7和21天上调和下调关键基因的实时荧光定量PCR结果。CTR，假手术对照组；SCC，脊髓压迫损伤组；HSCI，脊髓半横断损伤组。图中数据均以CTR组基因表达水平为1进行归一化展示。实时荧光定量PCR实验使用RNA测序后剩余的同一批小鼠组织样本进行检测（n=3）。^?P<0.05，^??P<0.01，^???P<0.001。
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图4 实时荧光定量PCR验证表达差异基因的mRNA相对表达水平
注：A~D，与CTR组相比，SCC组在第7和21天上调和下调关键基因的实时荧光定量PCR结果；E~H，与CTR组相比，HSCI组在第7和21天上调和下调关键基因的实时荧光定量PCR结果。CTR，假手术对照组；SCC，脊髓压迫损伤组；HSCI，脊髓半横断损伤组。图中数据均以CTR组基因表达水平为1进行归一化展示。实时荧光定量PCR实验使用RNA测序后剩余的同一批小鼠组织样本进行检测（n=3）。^?P<0.05，^??P<0.01，^???P<0.001。

Figure 4 Validation of mRNA expression levels of differentially expressed genes by real time fluorogenic quantitative PCR
Note： A-D， real time fluorogenic quantitative PCR results of the key genes that up-regulated and down-regulated in the SCC group compared with the CTR group at days 7 and 21； E-H， real time fluorogenic quantitative PCR results of the key genes that up-regulated and down-regulated at days 7 and 21 in the HSCI group compared with the CTR group. CTR， sham-operated control group； SCC， spinal cord contusion injury group； HSCI， hemisection spinal cord injury group. All data in the figure are normalized to a baseline of 1 for the CTR group. Real time fluorogenic quantitative PCR experiments were performed using the same batch of mouse tissue samples remaining after RNA-seq（n=3）. ^?P<0.05， ^??P<0.01， ^???P<0.001.

注：A~B，术后第7天，SCC组和CTR组之间上调和下调差异表达基因的基因本体（GO）富集分析结果；C~D，术后第7天，SCC组和CTR组之间上调和下调差异表达基因的京都基因与基因组数据库（KEGG）富集分析结果；E~F，术后第21天，SCC组和CTR组之间上调和下调差异表达基因的GO富集分析结果；G~H，术后第21天，SCC组和CTR组之间上调和下调差异表达基因的KEGG富集分析结果。图中条形颜色代表对应功能类别的P值，反映富集显著性。SCC，脊髓压迫损伤组；CTR，假手术对照组。
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图5 脊髓压迫损伤组与假手术对照组小鼠在术后第7天和第21天差异表达基因的富集分析结果
注：A~B，术后第7天，SCC组和CTR组之间上调和下调差异表达基因的基因本体（GO）富集分析结果；C~D，术后第7天，SCC组和CTR组之间上调和下调差异表达基因的京都基因与基因组数据库（KEGG）富集分析结果；E~F，术后第21天，SCC组和CTR组之间上调和下调差异表达基因的GO富集分析结果；G~H，术后第21天，SCC组和CTR组之间上调和下调差异表达基因的KEGG富集分析结果。图中条形颜色代表对应功能类别的P值，反映富集显著性。SCC，脊髓压迫损伤组；CTR，假手术对照组。

Figure 5 Enrichment analysis of differentially expressed genes in the spinal cord compression group compared to the sham-operated control group at postoperative days 7 and 21
Note： A-B， gene ontology （GO） enrichment analysis results of up-regulated and down-regulated differentially expressed genes between the SCC and CTR groups on postoperative day 7； C-D， Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis results of up-regulated and down-regulated differentially expressed genes between the SCC and CTR groups on postoperative day 7； E-F， GO enrichment analysis results of up-regulated and down-regulated differentially expressed genes between the SCC and CTR groups on postoperative day 21； G-H， KEGG enrichment analysis results of up-regulated and down-regulated differentially expressed genes between the SCC and CTR groups on postoperative day 21. The color of the bars represents the P value of the corresponding functional category， reflecting the significance of enrichment. SCC， spinal cord compression group； CTR， sham-operated control group.

注：A~B，术后第7天，HSCI组和CTR组之间上调和下调差异表达基因的基因本体（GO）富集分析结果；C~D，术后第7天，HSCI组和CTR组之间上调和下调差异表达基因的京都基因与基因组数据库（KEGG）富集分析结果；E~F，术后第21天，HSCI组和CTR组之间上调和下调差异表达基因的GO富集分析结果；G~H，术后第21天，HSCI组和CTR组之间上调和下调差异表达基因的KEGG富集分析结果。图中条形颜色代表对应功能类别的P值，反映富集显著性。HSCI组，脊髓半横断损伤组；CTR组，假手术对照组。
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图6 脊髓半横断损伤组与假手术对照组小鼠在第7天与第21天的差异表达基因的功能富集分析结果
注：A~B，术后第7天，HSCI组和CTR组之间上调和下调差异表达基因的基因本体（GO）富集分析结果；C~D，术后第7天，HSCI组和CTR组之间上调和下调差异表达基因的京都基因与基因组数据库（KEGG）富集分析结果；E~F，术后第21天，HSCI组和CTR组之间上调和下调差异表达基因的GO富集分析结果；G~H，术后第21天，HSCI组和CTR组之间上调和下调差异表达基因的KEGG富集分析结果。图中条形颜色代表对应功能类别的P值，反映富集显著性。HSCI组，脊髓半横断损伤组；CTR组，假手术对照组。

Figure 6 Functional enrichment analysis of differentially expressed genes between the hemisection spinal cord injury group and the sham-operated control group at postoperative days 7 and 21
Note： A-B， gene ontology （GO） enrichment analysis results of up-regulated and down-regulated differentially expressed genes between the HSCI and CTR groups on postoperative day 7； C-D， Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis results of up-regulated and down-regulated differentially expressed genes between the HSCI and CTR groups on postoperative day 7； E-F， GO enrichment analysis results of up-regulated and down-regulated differentially expressed genes between the HSCI and CTR groups on postoperative day 21； G-H， KEGG enrichment analysis results of up-regulated and down-regulated differentially expressed genes between the HSCI and CTR groups on postoperative day 21. The color of the bars represents the P value of the corresponding functional category， reflecting the significance of enrichment. HSCI， hemisection spinal cord injury group； CTR， sham-operated control group.

注：A~B，术后第7天，HSCI组和SCC组之间上调和下调差异表达基因的基因本体（GO）富集分析结果；C~D，术后第7天，HSCI组和SCC组之间上调和下调差异表达基因的京都基因与基因组数据库（KEGG）富集分析结果；E~F，术后第21天，HSCI组和SCC组之间上调和下调差异表达基因的GO富集分析结果；G~H，术后第21天，HSCI组和SCC组之间上调和下调差异表达基因的KEGG通路富集分析结果。条形颜色表示相应功能条目的P值，用于反映富集的显著性水平。HSCI组，脊髓半横断损伤组；SCC组，脊髓压迫损伤组。
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图7 脊髓半横断损伤组与脊髓压迫损伤组在术后第7天与第21天表达差异基因的功能富集分析结果
注：A~B，术后第7天，HSCI组和SCC组之间上调和下调差异表达基因的基因本体（GO）富集分析结果；C~D，术后第7天，HSCI组和SCC组之间上调和下调差异表达基因的京都基因与基因组数据库（KEGG）富集分析结果；E~F，术后第21天，HSCI组和SCC组之间上调和下调差异表达基因的GO富集分析结果；G~H，术后第21天，HSCI组和SCC组之间上调和下调差异表达基因的KEGG通路富集分析结果。条形颜色表示相应功能条目的P值，用于反映富集的显著性水平。HSCI组，脊髓半横断损伤组；SCC组，脊髓压迫损伤组。

Figure 7 Functional enrichment analysis of differentially expressed genes between the hemisection spinal cord injury group and the spinal cord compression injury group at postoperative days 7 and 21
Note： A-B， gene ontology （GO） enrichment analysis results of up?regulated and down?regulated differentially expressed genes between the HSCI and SCC groups on postoperative day 7； C-D， Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis results of up?regulated and down?regulated differentially expressed genes between the HSCI and SCC groups on postoperative day 7； E-F， GO enrichment analysis results of up?regulated and down?regulated differentially expressed genes between the HSCI and SCC groups on postoperative day 21； G-H， KEGG pathway enrichment analysis results of up?regulated and down?regulated differentially expressed genes between the HSCI and SCC groups on postoperative day 21. The color of the bars represents the P value of the corresponding functional term， indicating the significance level of enrichment. HSCI， hemisection spinal cord injury group； SCC， spinal cord compression group.

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