Hydralazine Promotes Central Nervous System Recovery after Spinal Cord Injury by Suppressing Oxidative Stress and Inflammation through Macrophage Regulation.

OBJECTIVE: This study aims to investigate the effects of hydralazine on inflammation induced by spinal cord injury (SCI) in the central nervous system (CNS) and its mechanism in promoting the structural and functional recovery of the injured CNS. METHODS: A compressive SCI mouse model was utilized for this investigation. Immunofluorescence and quantitative real-time polymerase chain reaction were employed to examine the levels of acrolein, acrolein-induced inflammation-related factors, and macrophages at the injury site and within the CNS. Western blotting was used to evaluate the activity of the phosphoinositide 3-kinase (PI3K)/AKT pathway to study macrophage regulation. The neuropathic pain and motor function recovery were evaluated by glutamic acid decarboxylase 65/67 (GAD65/67), vesicular glutamate transporter 1 (VGLUT1), paw withdrawal response, and Basso Mouse Scale score. Nissl staining and Luxol Fast Blue (LFB) staining were performed to investigate the structural recovery of the injured CNS. RESULTS: Hydralazine downregulated the levels of acrolein, IL-1ß, and TNF-α in the spinal cord. The downregulation of acrolein induced by hydralazine promoted the activation of the PI3K/AKT pathway, leading to M2 macrophage polarization, which protected neurons against SCI-induced inflammation. Additionally, hydralazine promoted the structural recovery of the injured spinal cord area. Mitigating inflammation and oxidative stress by hydralazine in the animal model alleviated neuropathic pain and altered neurotransmitter expression. Furthermore, hydralazine facilitated motor function recovery following SCI. Nissl staining and LFB staining indicated that hydralazine promoted the structural recovery of the injured CNS. CONCLUSION: Hydralazine, an acrolein scavenger, significantly mitigated SCI-induced inflammation and oxidative stress in vivo, modulated macrophage activation, and consequently promoted the structural and functional recovery of the injured CNS.

Loss of microRNA-124 expression in neurons in the peri-lesion area in mice with spinal cord injury.

MicroRNA-124 (miR-124) is abundantly expressed in neurons in the mammalian central nervous system, and plays critical roles in the regulation of gene expression during embryonic neurogenesis and postnatal neural differentiation. However, the expression profile of miR-124 after spinal cord injury and the underlying regulatory mechanisms are not well understood. In the present study, we examined the expression of miR-124 in mouse brain and spinal cord after spinal cord injury using in situ hybridization. Furthermore, the expression of miR-124 was examined with quantitative RT-PCR at 1, 3 and 7 days after spinal cord injury. The miR-124 expression in neurons at the site of injury was evaluated by in situ hybridization combined with NeuN immunohistochemical staining. The miR-124 was mainly expressed in neurons throughout the brain and spinal cord. The expression of miR-124 in neurons significantly decreased within 7 days after spinal cord injury. Some of the neurons in the peri-lesion area were NeuN(+)/miR-124(-). Moreover, the neurons distal to the peri-lesion site were NeuN(+)/miR-124(+). These findings indicate that miR-124 expression in neurons is reduced after spinal cord injury, and may reflect the severity of spinal cord injury.

Two monoclonal antibodies recognising aa 634-668 and aa 1026-1055 of NogoA enhance axon extension and branching in cultured neurons.

In a previous study, we generated two monoclonal antibodies (mAbs) in mice, aNogoA-N and aNogo-66 mAb, which were raised against recombinant N-terminal fragments of rat NogoA and Nogo-66, respectively. When compared with the commercial rabbit anti-rat NogoA polyclonal antibody (pAb), which can specifically recognise NogoA, the two mAbs were also specific for the NogoA antigen in immunofluorescence histochemical (IHC) staining and Western blot (WB) analysis. Serial truncations of NogoA covering the N-terminal region of NogoA (aa 570-691) and Nogo-66 (aa 1026-1091) were expressed in E. coli. The epitopes recognised by aNogoA-N and aNogo-66 are located in the aa 634-668 and aa 1026-1055 regions of NogoA, respectively. Both mAbs remarkably enhanced the axon growth and branching of cultured hippocampal neurons in vitro. These results suggest that the antibodies that bind to aa 634-668 and aa 1026-1055 of NogoA may have stimulatory effects on axon growth and branching. Additionally, the two mAbs that we generated are specific for NogoA and significantly block NogoA function. In conclusion, two sites in NogoA located within aa 634-668 and aa 1026-1055 are recognised by our two antibodies and are novel and potentially promising targets for repair after central nervous system (CNS) injury.

[Establishment of PD-L1 transgenic mouse model and recovery of the motion after spinal cord injury].

AIM: To establish a ubiquitously expressed PD-L1 transgenic mouse model and evaluate its recovery of motor function after spinal cord injury. METHODS: Clone and sequence the complete mouse PD-L1 cDNA and construct the pCAG-PD-L1 transgenic vector by inserting the PD-L1 cDNA into the pCAGGS vector. The PD-L1 transgenic (TgPD-L1) mice were established by pronuclear micro-injection with fertilized eggs from C57BL/6 mice and the genotypes were confirmed by PCR with tail genomic DNA. The expression of PD-L1 on T and B lymphocytes from mouse spleen were detected by flow cytometry. The expression of PD-L1 in peripheral tissues was displayed by immunohistochemistry. The expression level of PD-L1 in spinal tissue was evaluated by RT-PCR. The recovery of motor function was analyzed by Basso-Beattie-Bresnahan(BBB) locomotion testing system at 3, 7, 14, 21, 28 and 35 day after spinal severe crush with forceps in mice. RESULTS: Three lines of TgPD-L1 mice in C57BL/6 background were generated and the exogenous PD-L1 gene can be heritable steadily to offsprings. PD-L1 was highly exppressed in spinal tissue, peripheral tissues, T and B lymphocytes using RT-PCR, immunohistochemistry and flow cytometry respectively in TgPD-L1 mice. The BBB scores were obviously higher at 21 day post-injury in TgPD-L1 than those of in WT mice (P<0.05). CONCLUSION: The TgPD-L1 mice whose background are C57BL/6 were established successfully and high expression level of PD-L1 in tissues promotes locomotion recovery after spinal cord injury in TgPD-L1 mice.

[Identification of six kinds of monoclonal antibodies against rat Nogo-A molecule in the immunofluorescent histochemical staining].

AIM: To detect and characterize of 6 monoclonal antibodies (mAbs) against different epitopes of rat Nogo-A molecule in immunohistochemistry to decide their applications in futrue. Four mAbs against Nogo66 fragment are named Nogo66-1, Nogo66-2, Nogo66-3 and Nogo66-4. The rest of 2 mAbs against N-termial 570-691aa fragment are named NogoN-1 and NogoN-2. METHODS: The immunofluorescence staining was used to detect the reactivity and specificity of those 6 mAbs in spinal tissue sections of rat. RESULTS: All 6 mAbs were double-labelled with commercial rabbit anti-Rat Nogo-A polyclonal antibody (PcAb) in spinal cord sections respecitvely. All 6 mAbs were colocalization with MBP respectively. However Nogo66-3 and NogoN-1 could also be double-staining with GFAP respectively. CONCLUSION: Nogo66-1, Nogo66-2, Nogo66-4 and NogoN-2 could recognize specifically in Nogo-A protein of tissues in immunohistochemical methods.

[Recovery of movement after spinal cord injury in DO11.10 transgenic mouse].

AIM: To analysis the role of T lymphocytes in spinal cord regeneration by comparing the recovery of movement and the morphological changes of injury area between BALB/c and DO11.10 transgenic mice. METHODS: Producing a crush injury model of spinal cord with special forceps. Analyze the changes of spinal cord injury area with H&E and GFAP, CD11b and lymphocytic immunohistochemical staining. Evaluate the recovery of movement function with Basso-Beattie-Bresnahan (BBB) locomotion testing system at 0, 7, 14 and 21 day post-injury (dpi). RESULTS: There were thicker and fastened glial scar at 21 dpi in the BALB/c mice but not in DO11.10 mice. The number of macrophages/microglia infiltrated in spinal cord injury area were more in DO11.10 than that in BALB/c mice at 14 dpi. The numbers of T lymphocytes infiltrated in spinal cord injury area were less in DO11.10 than that in BALB/c mice at 21 dpi. In addition, compare to BALB/c mice, the locomotion movement recovery of DO11.10 mice were much more significant within 3 weeks after spinal cord injury by BBB scoring system. CONCLUSION: The infiltrated autoimmune activation T lymphocytes which specifically react to neural antigens are not beneficial to recovery of movement after spinal cord injury in mice.