The Effects And Mechanisms Of CDNF-BMSCs Transplantation In The Treatment Of Spinal Cord Injury Of Rats

BackgroundSpinal cord injury (SCI) is a worldwide refractory disease resulting in paraplegia or quadriplegia, which has a significant impact on quality of life, life expectancy and economic burden. Traumatic injury of spinal cord causes neuron death and axonal damage, leading to the loss of motor and sensory function and showing limited regeneration because of an adverse environment such as inflammation, ischemia, lipid peroxidation, or glial scarring. To date, there is no safe and effective therapy to treat traumatic SCI. Bone marrow-derived mesenchymal stem cells (BMSCs) are candidates for cell therapies because of their relative accessibility and therapeutic benefit to injured spinal cord. However, these effects and axonal regeneration are limited due to inflammation and adverse microenvironment, which are initiated rapidly in the nervous tissue after injury. At present there is no effective treatment for spinal cord injury, however, cerebral dopamine neurotrophic factor (CDNF), may provide a potential therapy. CDNF was reported to protect midbrain dopaminergic systems induced by 6-OHDA and promote nerve regeneration in injured peripheral nerve system (PNS). Moreover, microglia are important resident immune cells in the central nervous system (CNS) and involved in the neuroinflammation caused by CNS disorders, our previous study showed that CDNF alleviated the inflammatory injury in microglia. Although the receptor, signaling pathways and exact mechanisms of CDNF are still unclear, it may provide a novel therapeutic method to promote nerve regeneration and functional recovery by its anti-inflammation potential and neuroprotective roles in the injured spinal cord.ObjectivesIn this study, we established transgenic BMSCs overexpressing CDNF by lentivirus (plenti-CDNF) and transplanted these cells into the lesion site of the spinal cord. Then we investigated the effect of this cell therapy on the neuroinflammation and morphological and functional recovery using an rat model of spinal cord contusion. The in vitro part of this study aims to investigate the protective role of CDNF in the lipopolysaccharide-induced inflammatory models of microglial cells and illustrate the mechanisms of anti-inflammatory property of CDNF.Materials and MethodsThis study includes two parts:PART Ⅰ. To determine the transduction efficiency of CDNF lentiviral vector into BMSCs in vitro, the intracellular immunostaining of CDNF in BMSCs was detected by flow cytometry and the expression of CDNF protein was detected by Western blot and ELISA. After the construction of transgenic CDNF-BMSCs, the cell transplantation was performed in the spinal cord injury (SCI) models of adult female Wistar rats. The rats were randomly assigned to four groups: plenti-CDNF-BMSCs transplantation (CDNF-BMSCs group), plenti-his-BMSCs transplantation (Vehicle-BMSCs group), SCI control group, Sham control group. The locomotor and sensorimotor function of the hindlimbs of the rats in each group was assessed before (baseline) and post injury by the Basso, Beattie and Bresnahan (BBB) locomotor rating scale, BBB subscore scale, and horizontal ladder walking test. In vivo BMSCs survival and CDNF secretion after transplantation were detected by immunohistochemistry and Elisa assays. We examined the histology and immunohistochemistry of the spinal cord to determine the anatomical basis of observed functional recovery. The immunohistochemistry of Neurofilaments (NF) was performed to detect the regenerated neural fibers in the spinal cord. Remyelination of the regenerating neural fibers were detected at 6 week by a transmission electron microscope. Thoracic cord neurons were labeled by retrograde tracer HRP, which was administrated in the sciatic nerve and transported through the site of injury, reflecting the nerve regeneration in the injured spinal cord. We also quantified the expression of PGE2, TNF-a, IL-1β in the injured tissues by RT-PCR to investigate the effect of CDNF-BMSCs transplantation on neuroinflammation in spinal cord induced by contused trauma.PART Ⅱ. Primary microglial cells were isolated from cerebral cortices of one-day neonatal rats and the inflammatory models of microglia were induced by LPS. To observe the levels of CDNF affected by various concentrations of LPS, microglia were exposed to LPS with different concentrations for 24 h. Then the expression of CDNF was detected by Western blot. To investigate the effect of CDNF on microglial inflammation, the cells were pre-treated with different dose of CDNF and then stimulated by LPS. Then the immunoreactivity of the pro-inflammatory cytokines (PGE2 and IL-1β) were determined and the levels of these secreted cytokines in microglia culture medium were detected by ELISA. To measure the extent of inflammatory damage to the cells, the stable cytosolic enzyme resulting from cell lysis, lactate dehydrogenase (LDH) was measured in the cell culture medium using a Cytotoxicity Detection Kit Plus. To explore the mechanisms of the anti-inflammatory property of CDNF in LPS induced microglia, the immunoreactivity of MAPKs signalings, inluding JNK, P38 and ERK1/2, were determined by Western blot.ResultsPART Ⅰ. In the present study, we have investigated and compare the restorative efficiency of transgenic cells transplants after spinal cord injury. Firstly the construction of transgenic CDNF-BMSCs in vitro was successful, which was confirmed by Flow cytometry, Western blot and ELISA assays. The locomotor and sensorimotor function improvement was assessed and CDNF-BMSCs group showed significantly better locomotor function recovery compared with the Vehicle-BMSCs treatment and SCI control groups after 3 weeks post-injury. To explore the anatomical basis underneath the functional recovery, the histology and immunohistochemistry were performed at 6 week after SCI. The results showed that the cavitary lesions after SCI were limited in a minor volume by CDNF-BMSCs transplantation compared to Vehicle-BMSCs and SCI control groups. And the regenerated neural fibers in injured spinal cord were promoted in CDNF-BMSCs group. Consistently, the nerve regeneration and remyelination at the injured site of spinal cord observed by a transmission electron microscope showed a notable improvement in CDNF-BMSCs transplanted group, comparing with SCI and Vehicle-BMSCs control. Moreover, we utilized the retrograde tracers HRP to detect the retrograde axoplasmic transport through the site of injury, which reflected the structural and functional repair of the injured spinal cord. There were significantly more HRP-positive neurons observed in CDNF-BMSCs group, indicating a better regeneration of neural fibers in the lesions. These results showed beneficial effects of CDNF-BMSCs transplantation on repair of the spinal cord trauma, which may be attributed to the limited inflammatory lesions and improved microenvironment. We also found that CDNF gene transfer promoted the surviving BMSCs in lesion sites, along with the secretion of CDNF significantly higher than other groups post-transplantation. And the subsequent data showed that CDNF-BMSCs inhibited local neuroinflammation and decreased the production of proinflammatory cytokines after SCI, suggesting a promising therapy to repair the injured spinal cord and promote the neural regeneration and functional restoration.PART II. Our study explores the hypothesis that conserved dopamine neurotrophic factor (CDNF), a secretory neurotrophic factor, may provide a novel therapy for associated with neuroinflammation related to the microglia. We observed that CDNF was upregulated in rat primary microglia treated with Lipopolysaccharide (LPS), an inflammatory inducer. Thus we hypothesize that CDNF may play a regulating role, in the inflammation in microglial cells induced by LPS. Finally, our data showed that CDNF significantly attenuated the production of proinflammatory cytokines (PGE2 and IL-1β) and remarkably alleviated the cytotoxicity (percentage of LDH released) in the LPS-induced microglia by suppressing the phosphorylation of JNK, but not the P38 or ERK pathways.ConclusionsPART Ⅰ.In summary, we have shown the neuroprotective effects of a cell therapy using CDNF-BMSCs against spinal cord injury. The locomotor function and nerve regeneration of the injured spinal cord were promoted by the transplantation of the transgenic CDNF-BMSCs constructed by lentiviral vectors. The durable and stable expression of CDNF plays a key role in suppressing neuroinflammation and decreasiing the production of proinflammatory cytokines after SCI, providing beneficial microenvironment for regeneration of neural fibers. Our results suggest a new therapeutic potential of the CDNF-BMSCs transplantation therapy to treat spinal cord injury.PART Ⅱ. Our data show that CDNF protects the microglia against inflammatory injuries and alleviates the productions of proinflammatory cytokines, suggesting that CDNF might be a potential novel agent for the treatment of neuroinflammation in the CNS disorders. We also demonstrated that CDNF might owe its anti-inflammatory activity to JNK signalling, which are not fully understood yet. However, it was clarified that CDNF significantly inhibits phosphorylation of JNK, which might be critical in further studies on the anti-neuroinflammatory property of CDNF.