Neuronal repair after spinal cord injury by in vivo astrocyte reprogramming mediated by the overexpression of NeuroD1 and Neurogenin-2.

BACKGROUND: As a common disabling disease, irreversible neuronal death due to spinal cord injury (SCI) is the root cause of functional impairment; however, the capacity for neuronal regeneration in the developing spinal cord tissue is limited. Therefore, there is an urgent need to investigate how defective neurons can be replenished and functionally integrated by neural regeneration; the reprogramming of intrinsic cells into functional neurons may represent an ideal solution. METHODS: A mouse model of transection SCI was prepared by forceps clamping, and an adeno-associated virus (AAV) carrying the transcription factors NeuroD1 and Neurogenin-2(Ngn2) was injected in situ into the spinal cord to specifically overexpress these transcription factors in astrocytes close to the injury site. 5-bromo-2´-deoxyuridine (BrdU) was subsequently injected intraperitoneally to continuously track cell regeneration, neuroblasts and immature neurons marker expression, neuronal regeneration, and glial scar regeneration. In addition, immunoprotein blotting was used to measure the levels of transforming growth factor-ß (TGF-ß) pathway-related protein expression. We also evaluated motor function, sensory function, and the integrity of the blood-spinal cord barrier(BSCB). RESULTS: The in situ overexpression of NeuroD1 and Ngn2 in the spinal cord was achieved by specific AAV vectors. This intervention led to a significant increase in cell regeneration and the proportion of cells with neuroblasts and immature neurons cell properties at the injury site(p < 0.0001). Immunofluorescence staining identified astrocytes with neuroblasts and immature neurons cell properties at the site of injury while neuronal marker-specific staining revealed an increased number of mature astrocytes at the injury site. Behavioral assessments showed that the intervention did not improve The BMS (Basso mouse scale) score (p = 0.0726) and gait (p > 0.05), although the treated mice had more sensory sensitivity and greater voluntary motor ability in open field than the non-intervention mice. We observed significant repair of the BSCB at the center of the injury site (p < 0.0001) and a significant improvement in glial scar proliferation. Electrophysiological assessments revealed a significant improvement in spinal nerve conduction (p < 0.0001) while immunostaining revealed that the levels of TGF-ß protein at the site of injury in the intervention group were lower than control group (p = 0.0034); in addition, P70 s6 and PP2A related to the TGF-ß pathway showed ascending trend (p = 0.0036, p = 0.0152 respectively). CONCLUSIONS: The in situ overexpression of NeuroD1 and Ngn2 in the spinal cord after spinal cord injury can reprogram astrocytes into neurons and significantly enhance cell regeneration at the injury site. The reprogramming of astrocytes can lead to tissue repair, thus improving the reduced threshold and increasing voluntary movements. This strategy can also improve the integrity of the blood-spinal cord barrier and enhance nerve conduction function. However, the simple reprogramming of astrocytes cannot lead to significant improvements in the striding function of the lower limbs.

Glycine and N-Acetylcysteine (GlyNAC) Combined with Body Weight Support Treadmill Training Improved Spinal Cord and Skeletal Muscle Structure and Function in Rats with Spinal Cord Injury.

Skeletal muscle atrophy is a frequent complication after spinal cord injury (SCI) and can influence the recovery of motor function and metabolism in affected patients. Delaying skeletal muscle atrophy can promote functional recovery in SCI rats. In the present study, we investigated whether a combination of body weight support treadmill training (BWSTT) and glycine and N-acetylcysteine (GlyNAC) could exert neuroprotective effects, promote motor function recovery, and delay skeletal muscle atrophy in rats with SCI, and we assessed the therapeutic effects of the double intervention from both a structural and functional viewpoint. We found that, after SCI, rats given GlyNAC alone showed an improvement in Basso-Beattie-Bresnahan (BBB) scores, gait symmetry, and results in the open field test, indicative of improved motor function, while GlyNAC combined with BWSTT was more effective than either treatment alone at ameliorating voluntary motor function in injured rats. Meanwhile, the results of the skeletal muscle myofiber cross-sectional area (CSA), hindlimb grip strength, and acetylcholinesterase (AChE) immunostaining analysis demonstrated that GlyNAC improved the structure and function of the skeletal muscle in rats with SCI and delayed the atrophication of skeletal muscle.

Mechanical stress regulates autophagic flux to affect apoptosis after spinal cord injury.

Increased mechanical stress after spinal cord injury (SCI) expands the scope of nerve tissue damage and exacerbates nerve function defects. Surgical decompression after SCI is a conventional therapeutic strategy and has been proven to have neuroprotective effects. However, the mechanisms of the interaction between mechanical stress and neurons are currently unknown. In this study, we monitored intramedullary pressure (IMP) and investigated the therapeutic benefit of decompression (including durotomy and piotomy) after injury and its underlying mechanisms in SCI. We found that decreased IMP promotes the generation and degradation of LC3 II, promotes the degradation of p62 and enhances autophagic flux to alleviate apoptosis. The lysosomal dysfunction was reduced after decompression. Piotomy was better than durotomy for the histological repair of spinal cord tissue after SCI. However, the autophagy-lysosomal pathway inhibitor chloroquine (CQ) partially reversed the apoptosis inhibition caused by piotomy after SCI, and the structural damage was also aggravated after CQ administration. An antibody microarray analysis showed that decompression may reverse the up-regulated abundance of p-PI3K, p-AKT and p-mTOR caused by SCI. Our findings may contribute to a better understanding of the mechanism of decompression and the effects of mechanical stress on autophagy after SCI.

Therapeutic effects of rapamycin and surgical decompression in a rabbit spinal cord injury model.

Surgical decompression after spinal cord injury (SCI) is a conventional treatment. Although it has been proven to have clinical effects, there are certain limitations, such as the surgical conditions that must be met and the invasive nature of the treatment. Therefore, there is an urgent need to develop a simple and maneuverable therapy for the emergency treatment of patients with SCI before surgery. Rapamycin (RAPA) has been reported to have potential as a therapeutic agent for SCI. In this study, we observed the therapeutic effects of rapamycin and surgical decompression, in combination or separately, on the histopathology in rabbits with SCI. After combination therapy, intramedullary pressure (IMP) decreased significantly, autophagic flux increased, and apoptosis and demyelination were significantly reduced. Compared with RAPA/surgical decompression alone, the combination therapy had a significantly better effect. In addition, we evaluated the effects of mechanical pressure on autophagy after SCI by assessing changes in autophagic initiation, degradation, and flux. Increased IMP after SCI inhibited autophagic degradation and impaired autophagic flux. Decompression improved autophagic flux after SCI. Our findings provide novel evidence of a promising strategy for the treatment of SCI in the future. The combination therapy may effectively improve emergency treatment after SCI and promote the therapeutic effect of decompression. This study also contributes to a better understanding of the effects of mechanical pressure on autophagy after neurotrauma.

Melatonin treatment protects against acute spinal cord injury-induced disruption of blood spinal cord barrier in mice.

The spinal cord microcirculation plays a critically important role in maintaining the normal function of spinal cord neurons, glial cells, and axons. Previous researches were largely focused on improved neurological manifestations of spinal cord injury (SCI) while ignoring to improve spinal cord microcirculation disorder after melatonin treatment. Therefore, the mechanism of melatonin that affects blood spinal cord barrier (BSCB) integrity and microcirculation in SCI remains unclear. The present study was performed to investigate the effect of melatonin on the BSCB in a SCI mice model. Melatonin (5, 10, 25, 50, 100 mg/kg i.p.) was administered to mice immediately following SCI. Compared to the 48 h post-SCI group, mice treated with melatonin (50 mg/kg) exhibited significantly reduced BSCB permeability. Additionally, melatonin treatment restrained microvessel loss; attenuated edema; protected the tight junction proteins, endothelial cells, and pericytes; decreased the number of cell apoptosis; and reduced MMP3/AQP4/HIF-1α/VEGF/VEGFR2 expression after SCI. Above all, our results clearly demonstrated that melatonin could stabilize microvascular barrier function and microcirculation of SCI, whose mechanism was to promote the repair of the damaged BSCB.