Integrated bioinformatics analysis identifies the effects of Sema3A/NRP1 signaling in oligodendrocytes after spinal cord injury in rats

Analysis method of the data spectrum

Animals

All healthy male SD rats (eight-week old, 220 ± 10 g) were provided by Shanghai Xipu Bikai Shanghai Xipu Laboratory Animal Co. (animal license No: SCXk (Shanghai) 2018-0006) and were housed in the Experimental Animal Center of Zhejiang Chinese Medical University (AAALAC, animal license No: SYXK (Zhejiang) 2018-0012). The animals were housed in environmentally-controlled standard conditions (illumination, 12/12 h light/dark cycle; humidity, 50–60%; room temperature, 21–23 °C) with ad libitum access to food and water. 3% pentobarbital sodium (0.15 ml/kg, i.p.) was intraperitoneally injected before the end of the experiment. Rats were divided into a sham group (sham, only underwent laminectomy does not damage the spinal cord), the SCI model group (SCI, underwent laminectomy and damage the T10 spinal cord, details in ‘SCI Rat Model Establishment’), the SCI+AAV Sema3A group (spinal cord stereotactic injection of AAV virus, details in 2.4), and the SCI+AAV NC group (spinal cord stereotactic injection of AAV control virus, details in ‘Virus Injection’). All experiments were performed in compliance with relevant ethical regulations and approved by the animal ethics committee of Zhejiang Chinese Medical University (IACUC-20190128-02). All the experimental protocols strictly followed the National Institutes of Health guidelines for the care and use of laboratory animals (NIH Publication No. 8023).

SCI rat model establishment

To establish a contusive SCI model at T₁₀, contusive injury was induced on the T₁₀ spinal cord using the MASCIS weight-drop device with a 5 × 10 g/cm gravitational potential energy after T₁₀ laminectomy. The severity and consistency of injury were verified by checking the bruise on the spinal cord or tail-flick of the rats after weight drop. All animals received intraperitoneal injections of penicillin (100 U/d) for three days to prevent infection.

Virus injection

To further verify the effect of Sema3A/NRP1 signaling after SCI, we designed an SNCA-shRNA sequence plasmid and integrated it into adeno-associated virus (AAV), as previously described (Hu et al., 2020), and injected it into rats to downregulate Sema3A expression. Briefly, at 21 days before modeling, 0.5 ul AAV2/9-U6-shRNA(Sema3A)-CAG-tdtomato or a negative control AAV2/9-U6-shRNA(luciferase)-CAG-td tomato virus (Shanghai Taitool Bioscience Co., Ltd.) were injected bilaterally between T9 and T10 spinal cord using a 10 ul Hamilton syringe after rats anesthetized with pentobarbital sodium (40 mg/kg, i.p.). Vessels and nerves were avoided while injecting the virus. The viral titer was 7.5 × 10¹² vg/ml. 7 × 10⁹ vg virus was given to every animal.

Nissl staining

Procedures for Nissl staining were as follows. Briefly, after dehydration in 30% sugar solution, the tissues were sliced with a freezing microtome. The slices were incubated with nissl staining solution in a 37 °C constant temperature water bath for 10 min, followed by alcohol gradient decolorization for 1 min each, and washed with distilled water for 30 s immediately. Finally, the slices were sealed with neutral resin. Images were taken with a light microscope.

Real-time quantitative PCR (qPCR) analysis

First, total RNA was extracted from the T10 spinal cord tissue and the quality of RNA tested (Invitrogen, Carlsbad, CA, USA), cDNA was synthesized using PrimeScript RT reagent Kit (TaKaRa). qRT-PCR was conducted by using the Fast Start Universal SYBR Green Master kit (TaKaRa Bio Inc, Beijing, China) with a 20 µl reaction system. The data for qRT-PCR were collected with CFX96 Real-Time System (BioRad, USA). All qRT-PCR results were analyzed using comparative Ct methods (2 − ΔΔCt). Primer sequences are listed in Table 1.

Western blotting

Rat spinal cord tissue was homogenized with radioimmunoprecipitation assay (RIPA) buffer. Protein concentrations were determined using the bicinchoninic acid (BCA) assay. Furthermore, equal quantities of protein were separated via 8% SDS-PAGE gel and transferred by electroblotting polyvinyl difluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk in TBST containing 0.1% Tween 20 at room temperature for 1.5 h and then incubated overnight at 4 °C with primary antibody: semaphorin3A (GTX37550, 1:500), neuropilin1 (ab81321, 1:1,000), Platelet-derived growth factor receptor α (PDGFR α) (ab203491, 1:1,000), and β-actin (ab20271, 1:5,000). The following day, the membrane was incubated at room temperature for 2 h with secondary antibodies (CST7074s, 1:2000). Finally, immunoreactive bands were detected by enhanced chemiluminescence and visualized with an Image Quant LAS 4000. For statistical comparisons, primary antibody values were normalized against β-actin for each run in the normal Western blots. The density of each band was measured by ImageJ analysis software.

Immunofluorescence staining

Frozen sections were used for immunofluorescence. Transverse spinal cord sections (25 µm) were cut on a frozen microtome and installed on gelatin-coated glass slides. Then, the tissues were incubated overnight with the following antibodies at 4 °C: semaphorin3A (GTX37550, 1:200), neuropilin1 (AF566, 1:50), PDGFR α (ab203491, 1:500). The signal was detected with the corresponding second antibodies conjugated to Goat anti-rabbit Alexa Fluor-488 (111-545-144, 1:600), Donkey anti-rabbit Alexa Fluor 647 (711-605-152, 1:600) or Donkey anti-goat Alexa Fluor 488 (ab150129, 1:600) and viewed by Nikon A1R laser scanning confocal microscope.

Behavioral testing

Hindlimb locomotor function was evaluated using Basso-Beattie-Bresnahan (BBB) score. As previously described (Wei et al., 2017), the BBB scale ranges from 0–21 and reflects the extent of hind limb movements , weight support, stepping ability, coordination, and trunk stability. Evaluation and analysis were conducted by a specialist with an independent assistant blinded to the group allocation. Then the motor performance of the rats in an open field was scored, including hind limb joint movement, weight support, plantar stepping, coordination, paw position, and trunk and tail control. Scoring was performed as illustrated in Table 2.

Statistical analyses

The statistical analyses between control and experimental groups were conducted by an unpaired t-test or one-way ANOVA followed by Tukey Kramer tests in GraphPad Prism7. Results are expressed as mean ± SEM. A p-value < 0.05 was statistically significant.

The expression of OLs and PDGFR α after SCI

The injured spinal cord tissues were harvested on days 1, 7, and 14 after SCI, and the expression of OLs was detected by qPCR. The results showed that compared with the sham group, olig2, the early specific marker of OLs was significantly reduced after SCI in the injured spinal cord tissue of rats (Fig. 1A). Sox10, the mature OLs specific marker, exhibited low levels after SCI (Fig. 1B). Furthermore, the specific marker of OPCs, Platelet-derived growth factor receptor α (PDGFR α), was significantly increased after SCI (Fig. 1C). We further detected the expression of PDGFR α in spinal cord tissue of SCI by western blotting, and the results showed that the expression was increased at 7 and 14 days (Fig. 1D). Consistent with previous studies (Assinck et al., 2017) that reported a large number of OL deaths after SCI, and the spontaneous differentiation of OPCs could be stimulated.

Increased expression of Sema3A and NRP1 after SCI

The dataset GSE450006 based on the GPL1355 platform contained four spinal cord samples from sham rats and 20 from SCI model rats. A total of 299 DEGs were obtained, including 176 upregulated and 123 downregulated genes (Fig. 2A), and the volcano plot showed upregulated and downregulated gene expression in each sample (Fig. 2B). We observed that OLs were significantly decreased (Fig. 1) while NRP1 and Sema3A were upregulated (Figs. 3A and 3B) in SCI rats. Based on the fact that NRP1 can only exert its signal transduction function after forming a complex with Sema3A extracellularly (Parker et al., 2012; Hu et al., 2020), we performed immunofluorescence staining on the spinal cord tissues of sham and SCI seven-day rats. We observed that Sema3A and NRP1 were co-expressed in the SCI model (Fig. 3C).

Analysis of the function and pathway of DEGs in SCI model rats

To further explore the mechanism of SCI, we performed GO and KEGG pathway enrichment analyses of DEGs with the cluster profiler package in R software. GO is one of the most widely used ontologies, which annotates genes in terms of their molecular functions, cellular constituent, and biological processes (Feunang et al., 2016). Moreover, the KEGG database is an important tool for identifying functional and metabolic pathways  (Sun et al., 2021). In this study, we used the R package “clusterProfiler” that automates the process of biological-term classification and the enrichment analysis of gene clusters. The screening criteria for statistically significant GO terms or pathways was a p-value less than 0.05. We found that the biological functions of DEGs were mainly related to wound healing, cell growth, chemotaxis and metabolism regulation, extracellulaar matrix organization in the SCI group (Fig. 4A). KEGG pathway analysis of DEGs between the SCI and the sham groups revealed significant enrichment in the interaction between cells and cytokines, cell proliferation, differentiation and maturation, ECM-receptor interaction (Fig. 4B). We further condensed all enriched pathways processed by KEGG and GO functional enrichment analysis, summarized all NRP1 involved in the results (Table S1), among which unexpected findings include the semaphorin-plexin signaling pathway involved in neuron projection guidance and axon guidance, mediating cell growth and migration (Table 3).

Sema3A regulates the expression of NRP1 after SCI

To further define the relationship between Sema3A and NRP1, we designed SNCA-shRNA sequence plasmids with rad fluorescent tags and integrated them into an adeno-associated virus (AAV). Subsequently, we injected AAV2/9-U6-shRNA (Sema3A)-CAG-tdtomato into the junction between T₉ and T₁₀ spinal cord rats 21 days before modeling to knock down the expression of Sema3A. Four weeks after injection, we took the thoracic spinal cord for sectioning and observed it under the microscope. It was found that the T10 segment had the best effect of AAV virus transfection (Fig. S1). Next, we observed the changes in NRP1 and Sema3A after SCI. The immunofluorescence analysis results showed that compared with the sham group, the expression of Sema3A and NRP1 in spinal cord tissue of SCI rats increased, and knockdown of Sema3A could significantly downregulate the expression of NRP1 (Figs. 5A–5C). The qPCR results were consistent with the results obtained by immunofluorescence analysis results(Figs. 5E–5F).

Sema3A/NRP1 signaling regulates the expression of PDGFR α and OLs

To investigate the role of Sema3A/NRP1 signaling in SCI, we constructed the PPI network using STRING database and visualized it with Cytoscape. The upregulated genes were highlighted in red, downregulated genes in green, key genes in blue, and gray lines represented the protein interaction relationships. The results imply a possible regulatory relationship between the Sema3A/NRP1 and PDGFR α (Fig. S2).

We measured PDGFR α mRNA and PDGFR α protein levels by real-time PCR, immunofluorescence, and western blotting. The results revealed that the overexpression of PDGFR α in SCI model rats was significantly reduced by inhibition of Sema3A/NRP1 signaling (Figs. 5A, 5D, 6C, 6D, and 6G). To better understand the cellular mechanism of Sema3A/NRP1signaling in mediating motor function recovery in SCI, we investigated whether Sema3A/NRP1 signaling was involved in the maturation of OLs after SCI, which is also a key link in neurological function recovery (Tetzlaff et al., 2011). The qPCR results indicated that the expression of Olig2 and Sox10 of SCI group rats (after Sema3A/NRP1 signaling inhibition) significantly increased on day seven (Figs. 6A–6B) but showed no significant change on day 14 (Figs. 6E–6F). The above data indicate that Sema3A/NRP1 signaling may regulate the expression of PDGFR α and OLs.

Inhibition of Sema3A/NRP1 signaling promotes function recovery after SCI

The recovery of hind limb function was assessed using the BBB rating scale to examine the effect of Sema3A/NRP1 signaling on locomotor recovery after SCI. The line graph shows the BBB score for each group within 1 week after SCI. We found that all rats had a BBB score of 21 before SCI and lost locomotor function after SCI. When Sema3A/NRP1 signaling was inhibited, the BBB score was higher than the SCI group after 7days (Fig. 7C).

Nissl staining showed that the color intensity and the number of nissl bodies in the SCI-7d group and the SCI-7d+AAV NC decreased significantly compared with the sham group. Compared with the SCI group, the SCI-7d+AAV Sema3A group showed deeper nissl bodies staining with significantly increased expression of tiger-spotted motor neurons that were smaller than in the sham group (Figs. 7A–7B). These results suggest that suppressing Sema3A/NRP1 signal transduction is beneficial to functional recovery after injury.