Leveron&Nexovas
Neurology India
menu-bar5 Open access journal indexed with Index Medicus
  Users online: 12083  
 Home | Login 
About Editorial board Articlesmenu-bullet NSI Publicationsmenu-bullet Search Instructions Online Submission Subscribe Videos Etcetera Contact
  Navigate Here 
 Search
 
  
 Resource Links
  »  Similar in PUBMED
 »  Search Pubmed for
 »  Search in Google Scholar for
 »Related articles
  »  Article in PDF (3,148 KB)
  »  Citation Manager
  »  Access Statistics
  »  Reader Comments
  »  Email Alert *
  »  Add to My List *
* Registration required (free)  

 
  In this Article
 »  Abstract
 » Methods
 » Results
 » Discussion
 » Conclusions
 »  References
 »  Article Figures

 Article Access Statistics
    Viewed216    
    Printed18    
    Emailed0    
    PDF Downloaded7    
    Comments [Add]    

Recommend this journal

 


 
Table of Contents    
ORIGINAL ARTICLE
Year : 2022  |  Volume : 70  |  Issue : 8  |  Page : 251-258

Transplantation of NEP1-40 and NT-3 Gene-Co-Transduced Neural Stem Cells Improves Function and Neurogenesis after Spinal Cord Injury in a Rat Model


Department of Orthopedic Surgery and Orthopedic Research Institute, West China Hospital, Sichuan University, No. 37, Guoxue Road, Chengdu, Sichuan 610041, People's Republic of China

Date of Submission09-Sep-2021
Date of Decision27-Jun-2022
Date of Acceptance01-Aug-2022
Date of Web Publication11-Nov-2022

Correspondence Address:
Lei Wang
Department of Orthopedic Surgery and Orthopedic Research Institute, West China Hospital, Sichuan University, No. 37 Guoxue Road, Chengdu, Sichuan - 610041
People's Republic of China
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0028-3886.360942

Rights and Permissions

 » Abstract 


Background: Spinal cord injury (SCI) generally results in necrosis, scarring, cavitation, and a release of inhibitory molecules of the nervous system, which lead to disruption of neurotransmission and impede nerve fiber regeneration. This study was intended to evaluate the therapeutic efficacy rates of the transplantation of NEP1–40- and NT-3 gene-co-transduced neural stem cells (NSCs) in a rat model of SCI.
Methods: Ninety Sprague–Dawley rats were subdivided randomly into six groups: sham-operated, SCI model, SCI + NSCs-NC, SCI + NEP1-40-NSCs, SCI + NT-3-NSCs, and SCI + NEP1-40/NT-3-NSCs. Motor function at different time points was evaluated using the Basso, Beattie, and Bresnahan locomotor activity scoring system (BBB). At 8 weeks post-transplantation, histological analysis, a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, immunofluorescent assay, immunocytochemical staining, and cholera toxin subunit B (CTB) retrograde tracing were performed.
Results: BBB scores of the co-transduction group significantly surpassed those of other transplantation groups and of the SCI-model group after 2 weeks post-transplantation. The apoptotic rate of neurocytes was significantly lower in the co-transduction group than in other experimental groups. Expression of NF-200, MBP, and ChAT was significantly higher in the SCI + NEP1-40/NT-3-NSCs group than in other transplantation groups, whereas the expression of GFAP and GAD67 was the second lowest after the sham-operated group. CTB retrograde tracing showed that CTB-positive neural fibers on the caudal side of the hemisected site were more numerous in the SCI + NEP1-40/NT-3-NSCs group than in other experimental groups.
Conclusion: Transplantation of NEP1–40- and NT-3-gene-co-transduced NSCs can modify the protein expression following acute SCI and promote neuron formation and axonal regeneration, thus having a neuroprotective effect. Furthermore, this effect surpasses that of transplantation of single-gene-transduced NSCs. Transplantation of NEP1–40- and NT-3-gene-co-transduced NSCs is effective at the neural recovery of the rat model of SCI and may be a novel strategy for clinical treatment of SCI.


Keywords: NEP1–40, NT-3, spinal cord injury, transplantation
Key Message: Transplantation of NEP1–40- and NT-3-gene-co-transduced NSCs can modify the protein expression following acute SCI and promote neuron formation and axonal regeneration, thus having a neuroprotective effect.


How to cite this article:
Chen F, Zhang Z, Wang Ln, Yang X, Zhou CG, Zhu C, Wang L, Liu Lm, Song Ym. Transplantation of NEP1-40 and NT-3 Gene-Co-Transduced Neural Stem Cells Improves Function and Neurogenesis after Spinal Cord Injury in a Rat Model. Neurol India 2022;70, Suppl S2:251-8

How to cite this URL:
Chen F, Zhang Z, Wang Ln, Yang X, Zhou CG, Zhu C, Wang L, Liu Lm, Song Ym. Transplantation of NEP1-40 and NT-3 Gene-Co-Transduced Neural Stem Cells Improves Function and Neurogenesis after Spinal Cord Injury in a Rat Model. Neurol India [serial online] 2022 [cited 2022 Dec 3];70, Suppl S2:251-8. Available from: https://www.neurologyindia.com/text.asp?2022/70/8/251/360942




Spinal cord injury (SCI) is a devastating clinical disorder and often results in permanent motor or sensory dysfunctions.[1],[2] Efficacy of present clinical treatments is limited. Routine medications, surgical decompression, and other treatments can only prevent secondary injury of the spinal cord, and it is difficult to fundamentally restore the function of a damaged spinal cord.[3],[31],[32] Therefore, nerve fiber regeneration after SCI has become a major research topic worldwide.

Neural stem cells (NSCs) can differentiate into a variety of cells within the nervous system, indicating that NSCs transplantation may be an effective treatment of SCI.[4],[33],[34] However, NSCs transplantation alone is not sufficient for recovery from SCI, mainly because of the limited survival of transplanted NSCs, blockage of axonal outgrowth, and a lack of certain differentiation potentials.[5]

Nogo A, one type of protein product of the Nogo gene, expressed on the surface of oligodendrocytes, has been proved to be the key inhibitor of axonal outgrowth.[6] Among the species, these proteins have an identical structure of 66 amino acid residues at their carboxyl terminus, called Nogo-66.[7] Nogo-66 binds to its receptor (NgR), triggering the Rho signaling pathway and restricting axonal regeneration.[6] Nogo-66 extracellular peptide residues 1–40 (NEP1–40)—the region derived from the Nogo-66 sequence—can competitively bind to NgR and block the Rho signaling pathway.[6] Wang et al.'s previous study has confirmed that NEP1–40-gene-transduced NSCs can promote axonal outgrowth and recovery from SCI,[8],[9] but NEP1–40 gene transfection causes no induction of cell differentiation.[8] Neurotrophin-3 (NT-3), one of the most successfully used nerve growth factors, can enhance neural regeneration and survival, stimulate directional differentiation, and promote axonal growth.[10],[11] The aforementioned advantages of the NT-3 gene easily make up for a deficiency of the NEP1–40 protein.

To our knowledge, no study has evaluated the joint effect of these two proteins. We hypothesized that by means of genetic engineering, co-transduction of NT-3 and NEP1–40 genes into NSCs through a viral vector may result in simultaneous production of NT-3 and NEP1–40 and may promote neural recovery. In a previous study by Wang et al., NEP1–40- and NT-3-gene co-transduction into NSCs has been proven to be feasible, and the expression of the two target genes has been stable.[12] Therefore, this study was intended to evaluate the therapeutic efficacy of NEP1–40- and NT-3-gene-co-transduced NSCs in a rat model of SCI.


 » Methods Top


Subjects

The study protocol was approved by the Institutional Medical Experimental Animal Care Committee of the authors' affiliated institution. One Sprague–Dawley (SD) rat on gestational day 15 and 90 adult female SD rats (weighting 250 ± 20 g) were provided by the Center of Experimental Animals of our institution.

NSC isolation and culturing

All procedures performed on animals were approved by the Biomedical Research Ethics Committee of West China Hospital of Sichuan University and were conducted in accordance with the National Institutes of Health guidelines.

NSCs were derived from the fetal brains of embryonic day 15 rats, which were extracted from pregnant Sprague–Dawley rats. The uterus was removed and immersed in pre-cooled 0.1% phosphate buffer for 5 min. Fetal mice were extracted and both cerebral cortices were exposed under a microscope. The hippocampi were isolated by blunt dissection and digested with accutase (Sigma-Aldrich, USA), and then centrifugation was performed at 800 rpm/min for 5 min. Cells were resuspended with NSC complete culture medium (mixture ratio of NSC basal medium and NSC proliferation supplement was 9:1; STEMCELL, USA). These cells were then placed in a T75 culture flask and were cultured in an NSC complete medium containing 20 ng/mL epidermal growth factor, 20 ng/mL basic fibroblast growth factor, 2% B27, 1% N2 (all from Abcam, UK), 10,000 U/L streptomycin, and 100,000 U/L penicillin. These cells were cultured at 37°C and 5% CO2 and were passaged in the medium described above.

Mouse anti-nestin (1:200, Abcam, UK) was used to identify the purity of NSCs. Cells were digested into single cells by trypsin, and then cell smears were prepared. Immunofluorescent staining was performed after the smear was completely dried. The numbers of DAPI + and DAPI+/nestin + cells were counted under a microscope, and the ratio reveals the positive survival rate of NSCs. The results proved that more than 90% of cells were nestin-positive, demonstrating a fairly high purity of NSCs.

Transduction

NSCs were plated at a concentration of 1.5 × 105 cells/well in six-well plates and transduced with the control lentiviruses. Previously, Wang et al.[12] successfully transduced NEP1–40 and NT-3 genes into NSCs. By means of real-time fluorescence PCR and western blot assays, the co-transduced NSCs had been proven to be feasible and can yield stable expression. Cells were harvested for injection of the SCI model after transduction.

The animal model of SCI and cell transplantation

Ninety adult female SD rats were randomly distributed into six groups: sham-operated group, SCI-model group (underwent hemisection of the spinal cord and injected phosphate-buffered saline [PBS]), SCI + NSCs-NC group (injection of NSCs without gene transduction), SCI + NEP1-40-NSCs group (injection of NSCs with NEP1–40 gene transduction), SCI + NT-3-NSCs group (injection of NSCs with NT-3 gene transduction), and SCI + NEP1-40/NT-3-NSCs group (injection of NSCs subjected to NEP1–40- and-NT-3-gene-co-transduction). The SD rat model of SCI was established via the following steps:

The rats received intraperitoneal anesthesia with 30 mg/kg sodium pentobarbital and were fixed on an operating table in the prone position. The T8 spinal-process-centered incision was made to expose the T6–T10 segments. Laminectomy of T8–T10 was performed. In the sham-operated group, the wound was flushed and closed. In the experimental groups, the spinal cord was hemisected at the ninth thoracic level (right side) to establish the SCI model. After the surgical procedure, the rats were set on a constant-temperature electric blanket (40°C) until recovery from anesthesia and were well taken care of without limitations on food or water. Bladders were manually emptied until bladder function returned. Penicillin (5 × 104 IU/kg) was injected intramuscularly for 7 consecutive days to prevent infection.

After 7 days, the injured spinal cord segment was exposed again. Each group received a subdural injection of 5 μL cell suspension (~1 × 105 cells/μL) at a speed of 0.5 μL/min via a microsyringe at both 2 mm cephalad and caudad of the hemisected site. The needle was kept in place for another 10 min after the infusion to prevent cell leakage upon withdrawal. Finally, the incisions were seamed stepwise.

Identification of rat nerve functions

Rat behavioral assessment was performed according to the Basso, Beattie, and Bresnahan locomotor rating scale (BBB)[13] at the following time points: before transplantation, 3 days, and 1, 2, 4, 6, and 8 weeks post-transplantation. The range of BBB scores was between 0 (complete paralysis) and 21 (norm). The BBB scores were obtained by two individuals, and the averages were recorded.

Tissue harvesting and hematoxylin–eosin staining

After the behavioral assessment, 10 rats randomly selected from each group received intraperitoneal anesthesia with 30 mg/kg sodium pentobarbital. Spinal cord tissues of the injury region were taken out and preserved in a freezer at −70°C. The rats were then sacrificed by the decapitation method. Spinal cords for staining were post-fixed for 4 h at 4°C in paraformaldehyde (4%). The fixed tissues were placed in 2 mL cryogenic vials and stored in a refrigerator at 4°C. The rostral, scar, and caudal segments of the spinal cord were then sliced into 5-μm-thick sections for staining. After that, the sections of two rats from each group were subjected to hematoxylin–eosin staining for general histological examination.

Apoptosis in the rat spinal cord

The apoptotic rate of neurocytes was measured by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay. The cells were quantified by means of the TUNEL reaction mixture (45 μL of equilibration buffer +1 μL of biotin-11-dUTP + 4 μL of TdT; Nanjing Keygen Biotech Co., Nanjing, China). The frozen sections of four rats from each group were incubated in a wet box in the dark at 37°C for 60 min, washed three times in PBS, counterstained by Hoechst staining (Hoechst 33258, Beyotime Biotechnology Co.), and imaged using a fluorescence microscope.

Immunofluorescent staining

Frozen sections of four rats from each group were inactivated by 3% H2O2 in PBS and washed in PBS three times. An anti-glial fibrillary acidic protein (GFAP) antibody (1:250, Abcam), anti-myelin basic protein (MBP) antibody (1:250, Abcam), or anti-neurofilament protein 200 (NF-200) antibody (1:500, Abcam) served as primary antibodies and were incubated with tissue sections at 4°C overnight, and then the slides were washed three times with 2 mL of PBS. One drop of the corresponding secondary antibody (1:200) was added to sections and incubated at 37°C for 30 min. The sections were then counterstained by Hoechst staining and imaged using the fluorescence microscope.

Immunohistochemical analysis of spinal-cord tissues

The rest of the frozen sections were inactivated with 3% H2O2 and washed in PBS. An anti-glutamic acid decarboxylase 67 (GAD67) antibody (1:100, Affinity Biosciences, Cincinnati, OH, USA) or an anti-choline acetyltransferase (ChAT) antibody (1:100, Affinity Biosciences) served as a primary antibody and was incubated with the slides at 4°C overnight. The corresponding secondary antibody was then added to the tissue sections and incubated at 37°C for 2 h. Hematoxylin was added for staining. Frozen sections were imaged using a microscope.

Retrograde tracing

After transplantation for 8 weeks, five rats from each group received intraperitoneal anesthesia. An incision on the hip was made to expose the right-side sciatic nerve and was injected with cholera toxin subunit B (CTB, 1%, 5mL, Thermo Fisher Scientific) on the caudal side. The animals were then maintained for 7 days before sacrifice. The injured spinal cord was then fixed with formaldehyde and sliced into 5-μm-thick frozen sections. CTB was visualized by avidin-Cy3 (Thermo Fisher Scientific) staining. The rostral side of the injured spinal cord was visualized under a microscope to compare tracing conditions.

Statistical analyses

Results were analyzed statistically by Mann–Whitney U-test in SPSS 23.0 software (SPSS Inc, Chicago, IL, USA). A P value of less than 0.05 indicated a significant difference.


 » Results Top


BBB grading

The experimental groups received fairly low BBB scores (~0.5) after hemisection of the spinal cord. In the first week after transplantation, BBB scores in these groups barely changed. After 2 weeks, BBB scores in the SCI + NSCs-NC, SCI + NEP1-40-NSCs, SCI + NT-3-NSCs, and SCI + NEP1-40/NT-3-NSCs groups began to improve, and as time went on, the recovery accelerated. The SCI + NEP1-40/NT-3-NSCs group manifested the most significant increase in BBB scores, reaching 16.3 at 8 weeks post-transplantation, statistically significantly higher than the scores of other experimental groups [P < 0.05; [Figure 1]].
Figure 1: BBB scores in the six groups at different time points (sham-operated group, SCI-model group, SCI + NSCs-NC group, SCI + NEP1-40-NSCs group, SCI + NT-3-NSCs group, and SCI + NEP1-40/NT-3-NSCs group). *P < 0.05 as compared with the selected group, **P < 0.01 as compared with the selected group

Click here to view


Hematoxylin–eosin staining

Hematoxylin–eosin staining of the sham-operated group showed that the spinal cord structure was intact and nerve cells were normal and evenly distributed. SCI-model group revealed obvious multifocal cavities present in the injury zone, with spinal cord tissue damage, scar-free healing, and a structural disorder. Cavities were smaller in the SCI + NSCs-NC, SCI + NEP1-40-NSCs, and SCI + NT-3-NSCs groups. Typical morphological changes in nerve cells were also observed. In the SCI + NEP1-40/NT-3-NSCs group, the injured spinal cord tissue was similar to normal tissue, and typical nerve cell-like morphological changes were observed, with nearly no cavities appearing [Figure 2].
Figure 2: Hematoxylin-eosin (HE) staining results in the six groups (sham-operated group, SCI-model group, SCI + NSCs-NC group, SCI + NEP1-40-NSCs group, SCI + NT-3-NSCs group, and SCI + NEP1-40/NT-3-NSCs group) at 8 weeks post-transplantation (×200 original magnification)

Click here to view


The TUNEL assay

This assay was performed to measure the apoptosis in the SCI region. Compared with the sham-operated group, the number of apoptotic neurocytes in the SCI-model group increased significantly, with these cells scattered all around the injury region according to fluorescence microscopy. Significantly fewer apoptotic cells were found in the SCI + NSCs-NC, SCI + NEP1-40-NSCs, SCI + NT-3-NSCs, and SCI + NEP1-40/NT-3-NSCs groups [Figure 3]a. Compared with other experimental groups, the apoptotic rate was significantly lower in the co-transduction group [P < 0.05; [Figure 3]b].
Figure 3: (a) Apoptosis in the SCI region of different groups (sham-operated group, SCI-model group, SCI + NSCs-NC group, SCI + NEP1-40-NSCs group, SCI + NT-3-NSCs group, and SCI + NEP1-40/NT-3-NSCs group) as measured by the TUNEL assay. DAPI nuclear staining was conducted for nuclear localization (×200 original magnification); (b) Apoptotic rates of the six groups. (c) Expression of NF-200 in the six groups according to immunofluorescent staining; (d) OD intensity of NF-200 expression in all groups. (e) Expression of MBP in the six groups as evidenced by immunofluorescent staining; (f) OD intensity of MBP expression in all groups. (g) Expression of GFAP in the six groups according to immunofluorescent staining; (h) OD intensity of GFAP expression in all groups. *P < 0.05 as compared with the selected group, **P < 0.01 as compared with the selected group

Click here to view


Expression levels of NF-200, MBP, and GFAP

The expression of NF-200, MBP, and GFAP in all groups is shown in [Figure 3]c, [Figure 3]e, and [Figure 3]g, and calculated by optical density (OD) intensity.

The expression of the sham-operated group was the highest. By contrast, in the SCI-model group, the lowest NF-200 expression was found. Fluorescence signals were significantly stronger in the SCI + NSCs-NC, SCI + NEP1-40-NSCs, and SCI + NT-3-NSCs groups than in the SCI-model group to various degrees (P < 0.05). Expression was the highest in the SCI + NEP1-40/NT-3-NSCs group among the transplantation groups [P < 0.05; [Figure 3]d].

Similar to the expression of NF-200, the fluorescence signal of MBP was the lowest in the SCI-model group. Compared to the SCI + NSCs-NC, SCI + NEP1-40-NSCs, and SCI + NT-3-NSCs groups, the expression of MBP was significantly higher in the SCI + NEP1-40/NT-3-NSCs group [P < 0.05; [Figure 3]f].

Low expression of GFAP was seen in the sham-operated group. By contrast, the expression of GFAP was the highest in the SCI-model group and decreased to some extent in the transplantation groups (P < 0.05). GFAP expression in the SCI + NEP1-40/NT-3-NSCs group was close to the sham-operated group [Figure 3]h.

Immunohistochemical results

Immunohistochemical staining expression of ChAT and GAD67 is shown in [Figure 4]a and [Figure 4]c. OD intensity was calculated and compared among all groups.
Figure 4: (a) Expression of ChAT at the SCI lesion in the six groups as determined by immunohistochemical staining (×200 original magnification). (b) OD intensity of ChAT expression in all groups. (c) Expression of GAD67 at the SCI lesion in the six groups according to immunohistochemical staining (×200 original magnification). (d) OD intensity of GAD67 expression in all groups. *P < 0.05 as compared with the selected group, **P < 0.01 as compared with the selected group

Click here to view


The ChAT expression was significantly lower in the SCI-model group than in the sham-operated group (P < 0.05). The expression of ChAT in the SCI + NEP1-40/NT-3-NSCs group was found to be the highest among the experimental groups [P < 0.05; [Figure 4]b].

On the contrary, the expression of GAD67 was significantly the highest in the SCI-model group (P < 0.05). The SCI + NEP1-40/NT-3-NSCs group had a lower expression rate when compared with the other transplantation groups [P < 0.05; [Figure 4]d].

CTB retrograde tracing

This tracing was performed to test whether transplantation of NSCs with a target gene can rebuild the relevant signal transduction pathway. The expression of different groups is shown in [Figure 5]a. The results indicated that in the sham-operated group, the signal transduction pathway was intact, and a large amount of labeled neural fibers were seen on the rostral side of the injured spinal cord. In contrast, barely any CTB-positive neural fibers were found in the SCI-model group 9 weeks after SCI. In the SCI + NSCs-NC, SCI + NEP1-40-NSCs, and SCI + NT-3-NSCs groups, the number of CTB-positive neurons was to some extent higher than the SCI-model group (P < 0.05). The SCI + NEP1-40/NT-3-NSCs group had a large quantity of CTB-positive nerve fibers at the rostral side, significantly more than other transplantation groups [P < 0.05; [Figure 5]b].
Figure 5: (a) CTB-positive nerve fibers on the rostral side of the injured spinal cord in the six groups according to the examination of CTB retrograde tracing (×200 original magnification). (b) Percent of CTB-positive neural fibers in all six groups. *P < 0.05 as compared with the selected group, **P < 0.01 as compared with the selected group

Click here to view



 » Discussion Top


Because of the multidifferentiation potential of NSCs, their transplantation for the repair of SCI models has been a hotspot of medical research.[14],[15] Some investigators have confirmed that NSCs transplantation is effective for recovery from SCI in animals.[16],[17],[35],[36],[37] However, because of a low survival rate, a lack of axonal outgrowth, and an uncertain differentiation tendency,[5] transplantation of NSCs alone is not sufficient for SCI repair. Transplantation of genetically modified NSCs can improve the local microenvironment, thus promoting cell survival, proliferation, and directional differentiation.[18],[19] Previously, Wang et al. successfully transduced the NEP1–40 gene into NSCs and confirmed that NEP1–40-gene-transduced NSCs can promote recovery from SCI, better than transplantation of regular NSCs.[8],[9] However, they found that NEP1–40 cannot significantly induce directional differentiation after NSCs transplantation.[8] NT-3 gene had been transduced into NSCs in a previous study and proved to be effective in promoting NSCs survival, proliferation, and neuronal differentiation in vitro.[9],[20] Furthermore, NT-3 has been proven to enhance the survival and proliferation of oligodendrocyte precursor cells and can promote myelination of oligodendrocytes both in vitro and in an injured spinal cord.[21] The combination of NEP1–40 and NT-3 may have an additive effect on neural recovery. Thus, we built and tested whether NEP1–40- and-NT-3-gene-co-transduced NSCs transplantation has recovery effect in a SCI rat model.

It has been reported that cavity formation is a characteristic of progressive tissue necrosis, which follows the initial destruction after SCI.[22] Therefore, a reduction in the structural injury or cavity volume proves a therapeutic effect. In our study, histological results from different groups at 8 weeks post-transplantation showed that the SCI + NEP1-40/NT-3-NSCs group had hardly any cavities and manifested typical nerve-cell-like morphological changes, similar to the normal spinal cord tissue. This result indicated that transplantation of NEP1-40/NT-3-NSCs had a regenerative effect after SCI.[38]

Normally, traumatic SCI evolves in two phases: the primary insult (mechanical injury) and the secondary injury. The pathophysiology of the second phase includes necrotic and apoptotic cell death.[23],[39] Although cell death appears at the site of injury immediately after the insult, apoptotic death of oligodendroglia occurs days later. This apoptosis next contributes to sensorimotor deficits through demyelination of neural pathways.[24],[40] Thus, a reduction in the apoptotic rate may to some extent protect neural function. In this study, we conducted the TUNEL assay to measure the apoptotic rate of neurocytes. We observed that at 8 weeks post-transplantation, the apoptotic rate was significantly lower in the co-transduction group than in the SCI-model group and other transplantation groups. Transplantation of NEP1–40- and-NT-3-gene-co-transduced NSCs had an additive neuroprotective effect, better than non-transduction or single gene transduction of NSCs.

The neurofilament protein plays an important role in the maintenance of neuron function and axoplasmic transport and can be detected in normal neurons and axons.[25],[26] NF-200 can reflect the morphology and functional state of neurons. MBP is synthesized by oligodendrocytes and can promote the formation or regeneration of the myelin sheath and restore the ability of axons to transfer signals.[27] GFAP is mainly present in astrocytes of the central nervous system and is a specific marker of astrocytes. The number of astrocytes increases after SCI, and glial scars form; this process hinders the growth and regeneration of axons.[28] In our study, the expression of NF-200 and MBP was significantly higher in the SCI + NEP1-40/NT-3-NSCs group than in other transplantation groups, whereas GFAP expression was the lowest. This finding means that co-transduction of the two target genes may modify the protein expression after acute SCI and promote neuron formation and axonal myelination and then reduce glial-cell hyperplasia and glial scar formation in the spinal cord at the later stage of SCI. In addition, the effect of co-transduction was significantly better than that of non-transduction and single-gene transduction.

ChAT is a synthetase of acetylcholine, which is an important neurotransmitter in the nervous system. ChAT distribution is similar to that of acetylcholine and has served as an indirect indicator to estimate acetylcholine content.[29] GAD67 is an important rate-limiting enzyme in the synthesis of γ-aminobutyric acid (GABA), which is an inhibitory neurotransmitter in the central nervous system. The expression level of GAD67 can directly reflect the amount of GABA in the spinal cord.[30] The co-transduced group had a much higher expression of ChAT and lower expression of GABA than did the other experimental groups; this finding proved the possibility of neurotransmission recovery after SCI. Through CTB retrograde tracing, we observed CTB-positive neural fibers on the caudal side of the hemisected site of the spinal cord in the transplantation groups. The signal was significantly stronger in the SCI + NEP1-40/NT-3-NSCs group than in other experimental groups; this result confirmed the feasibility of promotion of axonal regeneration.

The BBB grading revealed that transplantation of either NT-3-gene-transduced or NEP1–40-gene-transduced NSCs facilitated behavioral recovery from injuries, whereas the combination of these two genes had a significantly better effect than that of each gene alone. We inferred that the recovery was achieved mostly via neuron formation and differentiation and axonal regeneration.

To date, there are concerns about the unpredictable adverse effects in the host after stem cell transplantation, such as tumor formation, infection, immune reaction, or even death. We reckon that with further improvement in biosafety of transgenic-NSCs transplantation, transplantation of NEP1–40- and-NT-3-gene- co-transduced NSCs could be a new strategy for treating patients with SCI.


 » Conclusions Top


In this study, we found that transplantation of NEP1–40- and-NT-3-gene-co-transduced NSCs may promote neuronal survival, proliferation, and differentiation and axonal regeneration at an injury site, thus restoring neural function in the SD rats with SCI. The effect of neural recovery was better than that of transplantation of single-gene-transduced NSCs. This therapy may provide a novel strategy for the clinical treatment of SCI.

Acknowledgements

We would like to express our thanks to Elsevier Language Editing for editing the language of our manuscript.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 » References Top

1.
Furlan JC, Sakakibara BM, Miller WC, Krassioukov AV. Global incidence and prevalence of traumatic spinal cord injury. Can J Neurol Sci 2013;40:456-64.  Back to cited text no. 1
    
2.
van den Berg ME, Castellote JM, de Pedro-Cuesta J, Mahillo-Fernandez I. Survival after spinal cord injury: A systematic review. J Neurotrauma 2010;27:1517-28.  Back to cited text no. 2
    
3.
Tederko P, Krasuski M, Kiwerski J, Nyka I, Białoszewski D. Strategies for neuroprotection following spinal cord injury. Ortop Traumatol Rehabil 2009;11:103-10.  Back to cited text no. 3
    
4.
Furuya T, Hashimoto M, Koda M, Okawa A, Murata A, Takahashi K, et al. Treatment of rat spinal cord injury with a Rho-kinase inhibitor and bone marrow stromal cell transplantation. Brain Res 2009, 1295:192-202.  Back to cited text no. 4
    
5.
Kabatas S, Teng YD. Potential roles of the neural stem cell in the restoration of the injured spinal cord: review of the literature. Turk Neurosurg 2010;20:103-10.  Back to cited text no. 5
    
6.
Dupuis L, Pehar M, Cassina P, Rene F, Castellanos R, Rouaux C, et al. Nogo receptor antagonizes p75NTR-dependent motor neuron death. Proc Natl Acad Sci 2008;105:740-5.  Back to cited text no. 6
    
7.
Steward O, Sharp K, Yee KM, Hofstadter M. A re-assessment of the effects of a Nogo-66 receptor antagonist on regenerative growth of axons and locomotor recovery after spinal cord injury in mice. Exp Neurol 2008;209:446-68.  Back to cited text no. 7
    
8.
Wang L, Song Y, Yuan H, Liu L, Gong Q, Kong Q, et al. Influence of Nogo extracellular peptide residues 1-40 gene modification on survival and differentiation of neural stem cells after transplantation. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 2013;27:1368-74.  Back to cited text no. 8
    
9.
Wang L, Song YM, Liu LM, Tao LI, Gong Q, Yang X, et al. Effect of NEP1-40 gene modified neural stem cell transplantation on the behavior recovery of rats after spinal cord injury. West China Med J 2014;29:2006-11.  Back to cited text no. 9
    
10.
Hagg T, Baker KA, Emsley JG, Tetzlaff W. Prolonged local neurotrophin-3 infusion reduces ipsilateral collateral sprouting of spared corticospinal axons in adult rats. Neuroscience 2005;130:875-87.  Back to cited text no. 10
    
11.
Lu HX, Hao ZM, Jiao Q, Xie WL, Zhang JF, Lu YF, et al. Neurotrophin-3 gene transduction of mouse neural stem cells promotes proliferation and neuronal differentiation in organotypic hippocampal slice cultures. Med Sci Monit 2011;17:Br305-311.  Back to cited text no. 11
    
12.
Wang L, Song Y, Liu L, Yang X, Feng G, Zhou C, et al. Experimental study of lentivirus-mediated Nogo extracellular peptide residues 1-40 gene and neurotrophin 3 gene co-transduction in neural stem cells. Chinese journal of reparative and reconstructive surgery 2018;32:420-7.  Back to cited text no. 12
    
13.
Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 1995;12:1-21.  Back to cited text no. 13
    
14.
Hwang DH, Shin HY, Kwon MJ, Choi JY, Ryu BY, Kim BG, et al. Survival of neural stem cell grafts in the lesioned spinal cord is enhanced by a combination of treadmill locomotor training via insulin-like growth factor-1 signaling. J Neurosci 2014;34:12788-800.  Back to cited text no. 14
    
15.
Wu MF, Zhang SQ, Gu R, Liu JB, Li Y, Zhu QS, et al. Transplantation of erythropoietin gene-modified neural stem cells improves the repair of injured spinal cord. Neural Regen Res 2015;10:1483-90.  Back to cited text no. 15
[PUBMED]  [Full text]  
16.
Fan WL, Liu P, Wang G, Pu JG, Xue X, Zhao JH, et al. Transplantation of hypoxic preconditioned neural stem cells benefits functional recovery via enhancing neurotrophic secretion after spinal cord injury in rats. J Cell Biochem 2018;119:4339-51.  Back to cited text no. 16
    
17.
Ruff CA, Wilcox JT, Fehlings MG. Cell-based transplantation strategies to promote plasticity following spinal cord injury. Exp Neurol 2012;235:78-90.  Back to cited text no. 17
    
18.
He Z, Koprivica V. The Nogo signaling pathway for regeneration block. Annu Rev Neurosci 2004;27:341-68.  Back to cited text no. 18
    
19.
Xu J, He J, He H, Peng R, Xi J. Comparison of RNAi NgR and NEP1-40 in acting on axonal regeneration after spinal cord injury in rat models. Mol Neurobiol 2017;54:8321-31.  Back to cited text no. 19
    
20.
Zhang YQ, He LM, Xing B, Zeng X, Zeng CG, Zhang W, et al. Neurotrophin-3 gene-modified Schwann cells promote TrkC gene-modified mesenchymal stem cells to differentiate into neuron-like cells in poly (lactic-acid-co-glycolic acid) multiple-channel conduit. Cells Tissues Organs 2012;195:313-22.  Back to cited text no. 20
    
21.
Bambakidis NC, Miller RH. Transplantation of oligodendrocyte precursors and sonic hedgehog results in improved function and white matter sparing in the spinal cords of adult rats after contusion. Spine J 2004;4:16-26.  Back to cited text no. 21
    
22.
Park SI, Lim JY, Jeong CH, Kim SM, Jun JA, Jeun SS, et al. Human umbilical cord blood-derived mesenchymal stem cell therapy promotes functional recovery of contused rat spinal cord through enhancement of endogenous cell proliferation and oligogenesis. J Biomed Biotechnol 2012;2012:362473.  Back to cited text no. 22
    
23.
Li L, Guo JD, Wang HD, Shi YM, Yuan YL, Hou SX, et al. Prohibitin 1 gene delivery promotes functional recovery in rats with spinal cord injury. Neuroscience 2015;286:27-36.  Back to cited text no. 23
    
24.
Crowe MJ, Bresnahan JC, Shuman SL, Masters JN, Beattie MS. Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys. Nat Med 1997;3:73-6.  Back to cited text no. 24
    
25.
Perrot R, Berges R, Bocquet A, Eyer J. Review of the multiple aspects of neurofilament functions, and their possible contribution to neurodegeneration. Mol Neurobiol 2008;38:27-65.  Back to cited text no. 25
    
26.
Uchida K, Baba H, Maezawa Y, Kubota C. Progressive changes in neurofilament proteins and growth-associated protein-43 immunoreactivities at the site of cervical spinal cord compression in spinal hyperostotic mice. Spine 2002;27:480-6.  Back to cited text no. 26
    
27.
Cayre M, Bancila M, Virard I, Borges A, Durbec P. Migrating and myelinating potential of subventricular zone neural progenitor cells in white matter tracts of the adult rodent brain. Mol Cell Neurosci 2006;31:748-58.  Back to cited text no. 27
    
28.
Doetsch F, Caillé I, Lim DA, García-Verdugo JM, Alvarez-Buylla A. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 1999;97:703-16.  Back to cited text no. 28
    
29.
Satomoto M, Satoh Y, Terui K, Miyao H, Takishima K, Ito M, et al. Neonatal exposure to sevoflurane induces abnormal social behaviors and deficits in fear conditioning in mice. Anesthesiology 2009;110:628-37.  Back to cited text no. 29
    
30.
Ying Z, Roy RR, Edgerton VR, Gómez-Pinilla F, et al. Exercise restores levels of neurotrophins and synaptic plasticity following spinal cord injury. Exp Neurol 2005;193:411-9.  Back to cited text no. 30
    
31.
S Divyalasya TV, Kumar AK, Sahana Bhat NR, Lakhan R, Agrawal A. Quality of Life after Surviving a Spinal Cord Injury: An Observational Study in South India. Neurol India 2021;69:861-866.  Back to cited text no. 31
    
32.
Tariq MB, Wu OC, Agulnick MA, Kasliwal MK. The 100 Most-Cited Papers in Traumatic Injury of the Spine. Neurol India 2020;68:741-759.  Back to cited text no. 32
[PUBMED]  [Full text]  
33.
Amitkumar M, Singh PK, Singh KJ, Khumukcham T, Sawarkar DP, Chandra SP, Kale SS. Surgical Outcome in Spinal Operation in Patients Aged 70 Years and Above. Neurol India 2020;68:45-51.  Back to cited text no. 33
[PUBMED]  [Full text]  
34.
R Soliman MA, Alkhamees AF, Khan A, Shamisa A. Instrumented Four-Level Anterior Cervical Discectomy and Fusion: Long-Term Clinical and Radiographic Outcomes. Neurol India 2021;69:937-943.  Back to cited text no. 34
    
35.
Singh S, Srivastava AK, Baranwal AK, Bhatnagar A, Das KK, Jaiswal S, Behari S. Efficacy of Silicone Conduit in the Rat Sciatic Nerve Repair Model: Journey of a Thousand Miles. Neurol India 2021;69:318-325.  Back to cited text no. 35
[PUBMED]  [Full text]  
36.
Sharma M. Nerve Guidance Conduits: Journey of a Thousand Miles in Search of a Destination. Neurol India 2021;69:326-327.  Back to cited text no. 36
[PUBMED]  [Full text]  
37.
Martinez-Perez R, Ganau M, Rayo N, Alemany VS, Boese CK, Moscote-Salazar LR. Prognostic Value of Age and Early Magnetic Resonance Imaging in Patients with Cervical Subaxial Spinal Cord Injuries. Neurol India 2020;68:1345-1350.  Back to cited text no. 37
[PUBMED]  [Full text]  
38.
Meena R, Doddamani RS, Agrawal D, Chandra PS. Dorsal Root Entry Zone (DREZ) Lesioning for Brachial Neuralgia. Neurol India 2020;68:1012-1015.  Back to cited text no. 38
[PUBMED]  [Full text]  
39.
Martinez-Perez R, Joswig H, Rayo N, Moscote-Salazar LR, Gomez PA. Subacute Management of a Dislocated Hangman Fracture, What Happens Afterwards? A Long-Term Follow Up. Neurol India 2020;68:959-960.  Back to cited text no. 39
[PUBMED]  [Full text]  
40.
Zhao D, He X, Liu L, Liu Q, Xu H, Ji Y, Zhu L, Wang G, Xu J, Wang Y. Correlation between Arteriole Membrane Potential and Cerebral Vasospasm after Subarachnoid Hemorrhage in Rats. Neurol India 2020;68:327-332.  Back to cited text no. 40
[PUBMED]  [Full text]  


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]



 

Top
Print this article  Email this article
   
Online since 20th March '04
Published by Wolters Kluwer - Medknow