Research on Spatiotemporal Gene Expression Profiles and Repair Mechanisms of Spinal Cord Compression and Hemisection Spinal Cord Injury Mouse Models

doi:10.12300/j.issn.1674-5817.2025.052

Abstract

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

CLC Number:

Q95-33

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.

Figures/Tables 7

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.
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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.

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.

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.
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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.

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.

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.
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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.

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.
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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.

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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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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