Resection of scar tissue in rats with spinal cord injury can promote the expression of βⅢ-tubulin in the injured area

doi:10.21203/rs.3.rs-1539960/v1

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Resection of scar tissue in rats with spinal cord injury can promote the expression of βⅢ-tubulin in the injured area

https://doi.org/10.21203/rs.3.rs-1539960/v1

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Background The scar tissue formed in the injured area after spinal cord injury is one of the reasons that hinder nerve regeneration and tissue repair. In previous studies, the researchers tried to prevent the formation of scar tissue to promote nerve regeneration and tissue repair, but it ran counter to the their desire, which also reflected the favorable side of scar tissue in the formation process. Further studies confirmed that scar formation of tissue is a dynamic process of hindering and promoting, nerve regeneration and tissue repair. By grasping the dual action of glial scar, understanding the underlying mechanism of its diversity is the key to break through the regeneration of nerve function after spinal cord injury.

Results Based on the rat models of the transection injury of the T8-T9 spinal cord, we selected several different key time points to resect the scar tissue formed after spinal cord injury and we found that the resection of scar tissue promoted the recovery of motor function and the expression of βⅢ-tubulin in the injured area, especially significant after scar excision in the second week after spinal cord injury. Further studies have shown that the mechanism of recovery might be a combination of promoting nerve axons regeneration, promoting nerve signal transmission, forming functional blood vessels, protecting neurons survival, and reducing scarring, etc. Meanwhile, we found that Tubb3 and Tubb6 genes were significantly different in the tissue before and after early scar resection.

Conclusions The motor function of the rats with spinal cord injury after scar resection was improved, and it could be seen that the expression of βⅢ-tubulin increased in the re-formed scars. The Tubb3, Tubb6 genes expression and other neural regeneration pathways were significantly different in the tissue before and after early resection. It was proved that excision of scar tissue promoted nerve regeneration.

Spinal cord injury

Scar tissue

Neuron

Motor function

neural regeneration

Spinal cord injury (SCI) can lead to severe limb dysfunction below the injured segment. The main reasons for this are the lack of regeneration ability of spinal cord nerve tissue, disorders of the microenvironment after injury, and the formation of a large number of glial scars in the injured area to hinder the signal transmission between neurons and axons, thus affecting the functional repair of the nervous system [1, 2]. After SCI, a large number of astrocytes are activated, proliferate rapidly, and aggregate around the lesion to form a dense cell barrier, that is, early glial scars [3, 4], which block further damage to the tissue in the injured area to reduce the scope of the injury. Previous studies showed that early glial scars had an important protective effect, limiting the spread of inflammatory responses to protect surrounding undamaged nerve tissue [5]. However, studies also show that glial scar hyperplasia is not conducive to the regeneration of neuronal axons, whose physical barrier effect prevents axons from functioning across the injured area [6, 7].

In recent years, studies have found that astrocytes can secrete molecules such as chondroitin sulfate proteoglycan, proteoglycan-NG2 and tyrosine kinase, which inhibits the activation of neurotrophic factors, cell adhesion molecules, and extracellular matrix to hinder the growth and regeneration of axons [8, 9], thus causing scar tissue to exert physical and chemical dual inhibitory effects on axons regeneration and myelination during the process [10]. However, some studies have found that inhibiting scar tissue formation did not promote tissue regeneration and functional repair, but aggravated the tissue damage [11, 12]. Therefore, scarring has both advantages and disadvantages for the recovery of nerve function after SCI. The roles of scar formation in functional recovery after injury need further research.

In order to further explore the role of scar tissue after SCI, we established a rat model of the complete transection of T8-T9 spinal cord. Based on the model, we analyzed the formation process of scar tissue after SCI and the post-injury functional repair. In this experiment, different time points were selected to completely resect the scar tissue formed after SCI. We found that the motor function of the SCI model rats had a significant recovery after resecting the scar tissue in the second week after SCI modeling, which was positively correlated with the expression of βⅢ-tubulin. High-throughput sequencing analysis showed that significantly differential genes related to functional recovery were mainly enriched in pathways related to promoting nerve axon regeneration, strengthening nerve signal transmission, forming functional blood vessels, protecting neuronal survival and reducing scar formation, etc.. Tubb3 and Tubb6 genes were significantly different in tissue before and after early scar resection. Our research explored the mechanisms related to the improvement of microtubule stability in the area of injured tissue, which is expected to become a new target for SCI therapy.

Ethological and histopathological Characteristics of SCI model rats

We selected different critical time points to remove and collect scar tissue from SCI model rats (Fig. 1). We first checked the pathology characteristics after SCI by HE, Nissl, Tunel and immunofluorescence methods until Day 180 (Fig. 2). Tissue staining showed that a large number of cell apoptosis occurred within 24 hours after SCI, the cell bodies of neurons shrunk, and the number of Nissl bodies decreased rapidly. Nestin (GFAP confined the border of lesion) cells began to appear in the surrounding tissue of the injured area on the 3rd day after injury. On the 5th day after injury, a transparent scar tissue visible to the naked eye appeared in the tissue absent area. At this time, Tunel staining showed that the number of apoptotic cells reached a peak, and the number of Nestin⁺ cells increased significantly. On the 7th day after injury, some Nestin positive stained cells began to increase at the center of the injured area, and the maximum number was on the 10th day after injury. On the 15th day after injury, transparent scar formation with high toughness was visible to the naked eye. At this time, cell apoptosis stopped, and no Nestin and Tuj1 positive stained cells were found in the injured area. After 30 days, there was no significant changes in histopathological staining until the 180th day after the injury. During this period, the scar tissue began to shrink gradually, and the spinal cord tissue at the end of the injured area also began to shrink. HE staining showed that at the early stage of injury, the tissue was wrapped and entangled by fiber strips and isolated from the external environment. The fiber strips gradually present a grid-like structure, and later forms a cavity.

Resected the scar tissue of the SCI model rats can promotes functional recovery

According to the pathology characteristics of different stages after SCI. We selected the 7 time points – the 5th, 7th, 10th, 15th, 30th, 45th, 60th days – as the research time points for the scar resection. We grouped the SCI rats according to time points: Day 5 group, Day 7 group, Day 10 group, Day 15 group, Day 30 group, Day 45 group, Day 60 group, and the model rats without scar resection were the control group.

After the scar tissue was resected, the motor function of the rats began to show rapid recovery. The BBB results showed that significantly-obvious motor improvements in rats, whose scar tissue was resected within 30 days after SCI, especially those in Day 7, Day 10, and Day 15 groups (Fig. 3a).

The CMEP waveforms were detected in rats with SCI after excised scar tissue (Fig. 3b). The CSEP test results of rats in each group showed that N33 waves and P40 waves were tested in rats with scar tissue resected after 30 days, and the latent period of the main wave was delayed and smaller than normal (Fig. 3c).

The CMEP test results of the rats whose scar tissue was resected in each group showed that the peak-to-peak amplitude of P-N waves of the single excitation decreased by more than 80% of the normal value (Fig. 3d), but there were differences among the groups. On the 120th day after modeling, the latent period of the main wave of the left limbs of the rats in Day 7 group and Day 10 group was significantly shortened compared with the control group, and the latent period of the main wave of right limbs of the rats in Day 10 and Day 15 was significantly shortened (Fig. 3e). The peak- to-peak amplitude of P-N waves of the single excitation of the bilateral limbs showed the Day 10 group was higher than that of other groups (Fig. 3f).

Neuronal and axonal regeneration in the re-formed scar

In order to further verify and find out the reasons for the recovery of motor function of rats, we detected the neural regeneration after injury. The results showed that after the first scar tissue resection, Tuj1-positive neurons and axonal regeneration were found in the reformed scar tissue of each group. In contrast, no Tuj1-positive cells and no axons survival were found in the injured areas of the rats without scar tissue resection (Fig. 4, 5). In the re-formed scar tissues after scar resection, the number of Tuj1-positive cells (Fig. 3g) and regenerated axons also increased significantly, especially the total amount of axons in Day 7, Day 10 and Day 15 groups increased (Fig. 3h).

High-throughput sequencing analysis suggest that the recovery mechanism is related to changes in the scar tissue environment

To investigate the mechanisms involved in functional recovery, we selected scar tissues at 7th, 10th and 15th when motor function recovery was most significant and performed RNA-seq analysis. S27-Day 7 group, S210-Day 10 group and S215-Day 15 group is that after the scar tissue was resected according to the corresponding resection time point, and starting from the resection time point, the reformed scar tissue was collected after 15 days of continuous feeding; S230-Day 7 group, S220-Day 10 group and S210-Day 15 group is that the scar tissue of the model rats was resected on the 10th day after the establishment of the rat model, and starting from the resection operation time, the re-formed scar tissue was respectively collected after 7,10,15 days of continuous feeding (Fig. 1c).

Comparison is S17-Day 7 group, S110-Day 10 group, S115-Day 15 group, starting from the 1st day after modeling, the scar tissue at the corresponding time was collected on the 7th day, 10th day and 15th day respectively and then used as a comparison (Fig. 1c).

We compared the differential genes in the following groups: S27-S17; S210-S110; S215-S115; S230-S17; S220-S110; S210-S115 (Fig. 6a, b). The differential genes and significant degree in scar tissues before and after scars resection were the most obvious on the 7th day after SCI, and the number of the significant difference genes were the greatest and the difference was the most obvious. As time went by, the significant differential genes showed a rapidly reduction. GO enrichment analysis showed that the significantly differential genes were distributed in the pathways related to promoting recovery of nerve function such as synaptic function, axonal myelin, reducing irregular scars, reducing tissue damage, promoting angiogenesis, and promoting neuron regeneration, etc. (Fig. 7a). KEGG enrichment analysis showed that significantly differential genes were related to nervous system, immune system, synaptic function and PI3k-Akt signaling pathway (Fig. 7b, c). After family distribution of differential genes, functional genes were most significantly distributed in zf-C2H2, Homeobox, and bHLH families, and were significantly correlated with the expression of βⅢ-tubulin (Fig. 7d). These key genes jointly promoted the expression of βⅢ-tubulin by playing different roles. At the same time, it was found that Tubb3 and Tubb6 genes were significantly different in tissues before and after early scar resection, and maintaining the stability of microtubules played a key role in promoting axons regeneration. We used Venn diagram to analyze the shared differential genes having entered the stable phase and found that the immune system was involved throughout the process of promoting regeneration. The genes related to immunity and neutrophil regulation were significantly different in the group of promoting βⅢ-tubulin expression and these differential genes were significantly associated with the release of neutrophil extracellular traps.

The formed scars and irreversible loss of neurons after SCI caused the paralysis of controlled muscle tissue, and the rupture of long spinal tracts caused the loss of sensory and motor regulation function. Although the experimental treatment of regenerative medicine has made progress in enhancing tissue repair and neural plasticity, the spinal cord lacks the ability of nerve regeneration, and moreover, the scar tissue formed after injury hinders nerve regeneration to some extent. Early research suggested that scar tissue formed in the acute phase of trauma formed a physical barrier to hinder the extension of neuronal axons while protecting the damaged tissue from external adverse factors. This view lasted for decades, and people gradually began to try to inhibit the formation of scars through various averages, only to find that it could not help axon regeneration [23, 24]. The studies in the later phase showed that scar tissue could continuously produce a variety of inhibitory molecules during formation and after stabilization, participated in the construction of a microenvironment that inhibits axonal regeneration, and meanwhile, it could also secrete a variety of neurotrophic factors to promote neuron regeneration. Therefore, the specific role of scar formation on functional repair after injury is still a research hotspot in this field.

In this paper, we used a model of complete transection of the spinal cord. The complete transection of the spinal cord is more suitable for the study of scar repair and axonal regeneration after SCI of lower mammals [25]. In the rat model of complete transection of the above T10 spinal cord, the lower nerve center on which the recovery of spontaneous motor function of hind limbs depends is located in the T11-L4 segment of spinal cord [26]. Some researchers believe that there is a central pattern generator (CPG) in the spinal cord of lower mammals such as rats, and CPG can generate alternating rhythmic movements of hind limbs without afferent sensory impulses [27], which belong to the spontaneous rhythmic impulses of lower neurons. After 30 days of spinal cord transection of rats, the formed scar tissue entered a stable phase, and no significant changes were found in HE staining. Surprisingly, the rats possessed the same ability of the hind limb movement after completely resecting the scar tissue. We could tell that the key site for the recovery of hind limb motor function of the SCI model rats was not in the area of the damaged scar tissue, which we speculated was in the lower motor neurons of the T11-L4 spinal cord guiding the recovery of the autonomic motor function. High-throughput sequencing analysis showed that significant differential genes were mainly concentrated in the signaling pathways related to nerve regeneration and tissue damage, and that these signaling pathways could promote the high expression of βⅢ-tubulin, which was the result of a dynamic and complex combination of effects.

After the scar tissue was resected, the hind limb motor function of the rats recovered to varying degrees. We found the expression of βⅢ-tubulin in the reformed scar tissue, and the expression level was related to the degree of hind limb motor function recovery. Immunofluorescence results showed the formation of scar tissue from beginning to stable maturation is achieved within 2 weeks. High-throughput sequencing analysis showed the number and significance of differential genes were the most obvious at 7 days before and after scar tissue resection. Extracellular matrix molecules in the injured area will be regulated after SCI, resulting in the gradual accumulation of some key inhibitory molecules, such as chondroitin sulfate proteoglycan, within a week, and it won’t stop until the chronic phase [28]. However, in the early stage of injury, inhibiting the formation of scar tissue is not conducive to the repair of SCI, but exacerbates expansion of the lesion and tissue denaturation [23]. Therefore, in scar tissue intervention, the integrity of scar tissue should be maintained within one week to ensure that it can give full play to its role in protecting peripheral nerve tissue and regulating inflammatory response. It can also be judged from the comparison before and after the scar resection on the 10th and 15th days that one week after the SCI, the factors affecting neuronal and tissue repair almost disappear, and the growth process of scar tissue has gradually entered a stable phase from an active stage. Compared with previous studies, the results of our study suggests that scar tissue resection in the 2nd week after injury is the most effective intervention period to remove the inhibitory effect of scar tissue.

Transparent scar formation was seen on the 5th day after SCI of rats, and the scar was stable on the 15th day. After SCI of rats, cell apoptosis occurred immediately in the tissue of the injured area, and within 24 hours, neurons rapidly atrophied and died. The motor function of the rats with SCI after scar resection was improved to varying degrees, and it could be seen that the expression of βⅢ-tubulin increased in the re-formed scars. The degree of recovery of motor function of rats was positively correlated with the expression of βⅢ-tubulin, and correlated with the time point of scar resection. The recovery of scar resection was the most significant in the 2nd week after SCI. High-throughput sequencing analysis suggested that the recovery mechanism might be an integration of these functions such as promoting axons regeneration, promoting nerve signal transmission, forming functional blood vessels, protecting neuronal survival, and reducing scarring. The Tubb3 and Tubb6 genes expression is significantly different in the tissue before and after early scar resection, which are the targets of axons regeneration intervention in SCI.

Experimental animals and ethics

Female SD rats (n = 150) weighing 200–220 g were used as the experimental animals for this study. The experimental animals were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. All experimental animals are raised in Experimental Animal Barrier Environment, temperature 26℃, humidity 50%. The experimental protocol was approved by the Ethics Committee of Xuanwu Hospital Capital Medical University.

Complete transection injury of the T8-T9 spinal cord

Female SD rats were successful anesthesia by intraperitoneal injection of 1% pentobarbital (50 mg/Kg). A 2 mm-long gap was made between T8-T9 and suture the lesion. After operation, intraperitoneal injection of penicillin (80,000 units/Kg) was given for 7 days. After the operation, the bladder was manually massaged to assist urination twice a day until the rats recovered their autonomous urination function.

Scar resection of the rats after SCI

After successful anesthesia, micro scissors were used to sever the connection between the scar and normal spinal tissue respectively at both ends of the scar in the injured area. After operation, intraperitoneal injection of penicillin (80,000 units/Kg) was given for 7 days. After the operation, bladder-assisted urination was performed twice daily manual massage until the rats recovered their autonomous urination function.

BBB scale

The BBB (Basso Beattie Bresnahan) rating was performed in each group every 7 days in an open field with a diameter of 2 m after surgery. Each experimental animal received a continuous 5-minute double-blind assessment of motion characteristics [13–16].

Electrophysiological examination

On the 120th modeling-postoperative day, three experimental animals in each group were randomly selected for electrophysiological examination under anesthesia. Cortical motor evoked potential (CMEP) and cortical somatosensory evoked potential (CSEP) of each rat were tested. The latency and amplitude of each group of rats were collected by the Keypoint EMG/evoked potential instruments and used Keypoint EMG/EP system for data processing (Dandy Company, Denmark). The electrodes were placed with reference to the placement of the International 10–20 system electrodes. The stimulation intensity was 25 mV and 3.5 mA.

Histological and immunofluorescent staining

The spinal cord tissues were taken out and fixed in 4% paraformaldehyde for 72 hours, and then immersed in 20% and 30% sucrose respectively for 24 hours. The frozen tissue sections were cut at 20 µm (Leica CM1950).

HE (Beyotime C0105M), Nissl (Beyotime C0117) and Tunel (Beyotime C1088) staining tissue sections were stained according to the kit [17–22]. The images were captured with a digital slide scanner (3DHISTECH PANNORAMIC).

For immunofluorescence, the tissue sections were treated by 0.3% PBST and blocked for 1 hour with 10% BSA. The primary antibodies were incubated overnight at 4°C in GFAP (1:800, Sigma), Tuj1 (β-Ⅲ tubulin, 1:500, Sigma) and the secondary antibodies incubated at RT (room temperature) for 2 hours. The tissue sections were scanned with a confocal microscope (Leica TCS SP5 CARS).

RNA-seq analysis

We divided into 9 groups according to the resection time point and the sampling time point, with 3 experimental animals in each group: S27-Day 7 group, S210-Day 10 group, S215-Day 15 group, S230-Day 7 group, S220-Day 10 group, S210-Day 15 group, S17-Day 7 group, S110-Day 10 group, S115-Day 15 group. Spinal cord tissues of 3 normal female rats were used as blank control. Samples from 10 groups of 30 animals were collected. Total RNA from samples were extracted following the standard protocol of TRIZOL Reagent (Invitrogen). The samples was using a customer sequencing service (OE Biotech Co., Ltd, Shanghai, China).

Statistical analysis

GraphPrism 7.0 software was used for quantitative data mapping and primary statistical analysis. SPSS 27.0 statistical analysis software was used for statistical analysis. The difference was statistically significant at p < 0.05.

Acknowledgements

Thanks to the guidance of rat electrophysiology in Functional Laboratory of the China Academy of Chinese Medical Sciences.

Authors’ contributions

Conception and design of the experiments: DS, SL and BL. Data collection: BL. Analysis and interpretation of data: BL, SL. Drafting the article and revising it critically for intellectual content: BL, SL and DS. All authors read and approved the final manuscript.

Funding

This work was supported by the National Science Foundation for Young Scientists of China (31900740).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

The experimental protocol was approved by the Ethics Committee of Xuanwu Hospital Capital Medical University. Our animal experiments complied with the Basel Declaration fundamental principles and the International Council for Laboratory Animal Science (ICLAS) ethical guidelines. All methods were reported in accordance with ARRIVE guidelines.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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No competing interests reported.

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Resection of scar tissue in rats with spinal cord injury can promote the expression of βⅢ-tubulin in the injured area

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Abstract

Figures

Background

Results

Ethological and histopathological Characteristics of SCI model rats

Resected the scar tissue of the SCI model rats can promotes functional recovery

Neuronal and axonal regeneration in the re-formed scar

High-throughput sequencing analysis suggest that the recovery mechanism is related to changes in the scar tissue environment

Discussion

Conclusion

Materials And Methods

Experimental animals and ethics

Complete transection injury of the T8-T9 spinal cord

Scar resection of the rats after SCI

BBB scale

Electrophysiological examination

Histological and immunofluorescent staining

RNA-seq analysis

Statistical analysis

Declarations

References

Additional Declarations

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