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
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.360942
Source of Support: None, Conflict of Interest: None
Keywords: NEP1–40, NT-3, spinal cord injury, transplantation
Spinal cord injury (SCI) is a devastating clinical disorder and often results in permanent motor or sensory dysfunctions., 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.,, 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.,, 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.
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. Among the species, these proteins have an identical structure of 66 amino acid residues at their carboxyl terminus, called Nogo-66. Nogo-66 binds to its receptor (NgR), triggering the Rho signaling pathway and restricting axonal regeneration. 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. Wang et al.'s previous study has confirmed that NEP1–40-gene-transduced NSCs can promote axonal outgrowth and recovery from SCI,, but NEP1–40 gene transfection causes no induction of cell differentiation. 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., 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. 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.
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.
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. 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) 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.
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.
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.
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.
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]].
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].
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].
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 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.
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].
Because of the multidifferentiation potential of NSCs, their transplantation for the repair of SCI models has been a hotspot of medical research., Some investigators have confirmed that NSCs transplantation is effective for recovery from SCI in animals.,,,, However, because of a low survival rate, a lack of axonal outgrowth, and an uncertain differentiation tendency, 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., 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., However, they found that NEP1–40 cannot significantly induce directional differentiation after NSCs transplantation. 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., 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. 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. 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.
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., 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., 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., 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. 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. 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. 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. 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.
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.
We would like to express our thanks to Elsevier Language Editing for editing the language of our manuscript.
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Conflicts of interest
There are no conflicts of interest.
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