Nerve regeneration in adult mammalian spinal cord is poor because of the lack of intrinsic regeneration of neurons and extrinsic factors - the glial scar is triggered by injury and inhibits or promotes regeneration. Recent technological advances in spatial transcriptomics (ST) provide a unique opportunity to decipher most genes systematically throughout scar formation, which remains poorly understood. Here, we first constructed the tissue-wide gene expression patterns of mouse spinal cords over the course of scar formation using ST after spinal cord injury from 32 samples. Locally, we profiled gene expression gradients from the leading edge to the core of the scar areas to further understand the scar microenvironment, such as neurotransmitter disorders, activation of the pro-inflammatory response, neurotoxic saturated lipids, angiogenesis, obstructed axon extension, and extracellular structure re-organization. In addition, we described 21 cell transcriptional states during scar formation and delineated the origins, functional diversity, and possible trajectories of subpopulations of fibroblasts, glia, and immune cells. Specifically, we found some regulators in special cell types, such as Thbs1 and Col1a2 in macrophages, CD36 and Postn in fibroblasts, Plxnb2 and Nxpe3 in microglia, Clu in astrocytes, and CD74 in oligodendrocytes. Furthermore, salvianolic acid B, a blood-brain barrier permeation and CD36 inhibitor, was administered after surgery and found to remedy fibrosis. Subsequently, we described the extent of the scar boundary and profiled the bidirectional ligand-receptor interactions at the neighboring cluster boundary, contributing to maintain scar architecture during gliosis and fibrosis, and found that GPR37L1_PSAP, and GPR37_PSAP were the most significant gene-pairs among microglia, fibroblasts, and astrocytes. Last, we quantified the fraction of scar-resident cells and proposed four possible phases of scar formation: macrophage infiltration, proliferation and differentiation of scar-resident cells, scar emergence, and scar stationary. Together, these profiles delineated the spatial heterogeneity of the scar, confirmed the previous concepts about scar architecture, provided some new clues for scar formation, and served as a valuable resource for the treatment of central nervous system injury.
Mice ; Animals ; Gliosis/pathology* ; Cicatrix/pathology* ; Spinal Cord Injuries ; Astrocytes/metabolism* ; Spinal Cord/pathology* ; Fibrosis ; Mammals ; Receptors, G-Protein-Coupled

Objective To explore the effect of shionone(SHI)on motor function in the mouse model of spinal cord injury(SCI)and probe into the underlying molecular mechanism.Methods C57BL/6 mice were treated to induce the SCI model and then assigned into a model group(SCI group),a SCI+SHI group,and a sham surgery(control)group.The Basso mouse scale(BMS)score was determined to evaluate the recovery of motor function in SCI mice.Hematoxylin-eosin(HE)staining,Nissl staining,and immunofluorescence staining were employed to examine the fibrosis,morphological changes of neurons,and neuron apoptosis in the spinal cord tissue of SCI mice,respectively.The mouse hippocampal neuronal cell line HT22 was cultured in vitro and then classified into tumor necrosis factor α(TNF-α)induction and SHI groups.Western blotting was employed to determine the expression of apoptosis-associated proteins.Network pharmacology,gene ontology annotation,and Kyoto Encyclopedia of Genes and Genomes pathway enrichment were employed to predict the possible molecular targets and signaling pathways of SHI in promoting functional recovery from SCI.Furthermore,the prediction results were verified by in vitro and in vivo experiments.Results Compared with the SCI group,the SCI+SHI group showed increased BMS score on days 21,28,35,and 42(P=0.003,P=0.004,P=0.023,and P=0.007,respectively),reduced area of spinal cord fibrosis(P=0.021),increased neurons survived(P=0.001),and down-regulated expression of cleaved cysteine aspastic acid-specific protease 3(cleaved Caspase-3)(P=0.017).Compared with the TNF-α group,the SHI group presented down-regulated expression levels of cleaved Caspase-3 and Bax(P=0.010,P=0.001)and up-regulated expression level of Bcl-2(P=0.001).The results of bioinformatics analysis showed that SHI might improve the motor function of SCI mice via the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway.The results of in vivo and in vitro experiments showed that SHI inhibited the phosphorylation of PI3K and Akt in SCI mice or HT22 cells exposed to TNF-α(all P<0.05).The number of apoptotic HT22 cells after treatment with insulin-like growth factor 1 was higher than that in the SHI group(P=0.003).Conclusion SHI may inhibit neuron apoptosis via the PI3K/Akt signaling pathway,thereby promoting the recovery of motor function in SCI mice.
Mice ; Animals ; Proto-Oncogene Proteins c-akt/metabolism* ; Caspase 3/metabolism* ; Phosphatidylinositol 3-Kinases ; Tumor Necrosis Factor-alpha/metabolism* ; Mice, Inbred C57BL ; Spinal Cord Injuries ; Apoptosis ; Neurons/pathology* ; Fibrosis

Evidence suggested that glycogen synthase kinase-3β (GSK-3β) is involved in Nogo-66 inhibiting axonal regeneration in vitro, but its effect in vivo was poorly understood. We showed that stereotactic injection of shRNA GSK-3β-adeno associated virus (GSK-3β-AAV) diminished syringomyelia and promoted axonal regeneration after spinal cord injury (SCI), using stereotactic injection of shRNA GSK-3β-AAV (tested with Western blotting and RT-PCR) into the sensorimotor cortex of rats with SCI and by the detection of biotin dextran amine (BDA)-labeled axonal regeneration. We also determined the right position to inject into the sensorimotor cortex. Our findings consolidate the hypothesis that downregulation of GSK-3β promotes axonal regeneration after SCI.
Animals ; Axons ; drug effects ; metabolism ; Dependovirus ; genetics ; Glycogen Synthase Kinase 3 beta ; genetics ; metabolism ; Humans ; Nerve Regeneration ; genetics ; RNA, Small Interfering ; administration & dosage ; genetics ; Rats ; Sensorimotor Cortex ; drug effects ; pathology ; Spinal Cord Injuries ; genetics ; pathology ; therapy ; Syringomyelia ; genetics ; pathology ; therapy

OBJECTIVETo observe the effects of mecobalamin on the expression of apoptosis-related proteins, Bax and Bcl-2, in neurons after peripheral nerve injury, and to explore the role of neuron apoptosis in peripheral nerve regeneration after injury.

METHODSThirty healthy adult male wistar rats were randomly divided into sham-operation group, model group, and mecobalamin group, with 10 rats in each group. A rat model of left sciatic nerve semi-injury was established using forceps. Rats in the mecobalamin group were fed mecobalamin, while rats in the sham-operation group and model group were given the same dose of normal saline. The protein expression of Bax and Bcl-2 in neurons was measured at 14 days after operation. A semi-quantitative analysis of Bax and Bcl-2 proteins was performed by image analysis technology.

RESULTSThe model group had significantly increased Bax protein expression and significantly reduced Bcl-2 protein expression in spinal cord anterior horn motor neurons and ganglion sensory neurons compared with the sham-operation group (P<0.05). Compared with the model group and sham-operation group, the mecobalamin group had significantly reduced Bax protein expression and significantly increased Bcl-2 protein expression in spinal cord anterior horn motor neurons and ganglion sensory neurons (P<0.05).

CONCLUSIONMecobalamin has anti-apoptotic effect, and it contributes to neurological function recovery possibly by inhibiting the death of injured neurons.

Animals ; Apoptosis ; Male ; Neurons ; cytology ; drug effects ; Peripheral Nerve Injuries ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; Rats ; Rats, Wistar ; Sciatic Nerve ; pathology ; Spinal Cord ; cytology ; Vitamin B 12 ; analogs & derivatives ; pharmacology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo observe the pathological changes in rabbits with spinal cord injury induced by decompression sickness (DCS), and to investigate the role of tumor necrosis factor-alpha (TNF-α) in spinal cord injury induced by DCS.

METHODSRabbits were randomly divided into normal control group, DCS group, and safe decompression group. The rabbit model of DCS was established. Light microscopy, real-time PCR, and immunohistochemical method were used to observe the pathomorphological changes in the thoracolumbar spinal cord and the mRNA and protein expression of TNF-α, respectively. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to observe the apoptosis in the spinal cord.

RESULTSIn the DCS group, cavities formed in the white matter of spinal cord and gliosis occurred around necrotic areas. Moreover, the mRNA and protein expression of TNF-α was significantly higher in the DCS group than in the normal control group and the safe decompression group (P<0.01). The results of TUNEL showed that the number of positive apoptotic cells was significantly larger in the DCS group than in the normal control group and the safe decompression group (P<0.05).

CONCLUSIONApoptosis plays an important role in spinal cord injury induced by DCS. In the early stage of DCS, the massive release of TNF-α initiates apoptosis and contributes to the pathological changes in spinal cord injury induced by DCS.

Animals ; Apoptosis ; Decompression Sickness ; metabolism ; pathology ; Disease Models, Animal ; In Situ Nick-End Labeling ; RNA, Messenger ; Rabbits ; Spinal Cord ; pathology ; Spinal Cord Injuries ; metabolism ; pathology ; Tumor Necrosis Factor-alpha ; metabolism

Spinal cord injury (SCI) causes not only loss of sensory and motor function below the level of injury but also chronic pain, which is difficult and challenging of the treatment. Repetitive transcranial magnetic stimulation (rTMS) to the motor cortex, of non-invasive therapeutic methods, has the motor and sensory consequences and modulates pain in SCI-patients. In the present study, we studied the effectiveness of rTMS and the relationship between the modulation of pain and the changes of neuroglial expression in the spinal cord using a rat SCI-induced pain model. Elevated expressions of Iba1 and GFAP, specific microglial and astrocyte markers, was respectively observed in dorsal and ventral horns at the L4 and L5 levels in SCI rats. But in SCI rats treated with 25 Hz rTMS for 8 weeks, these expressions were significantly reduced by about 30%. Our finding suggests that this attenuation of activation by rTMS is related to pain modulation after SCI. Therefore, rTMS might provide an alternative means of attenuating neuropathic pain below the level of SCI.
Animals ; Astrocytes/*cytology ; Calcium-Binding Proteins/metabolism ; Disease Models, Animal ; Immunohistochemistry ; Male ; Microfilament Proteins/metabolism ; Microglia/*cytology ; Nerve Tissue Proteins/metabolism ; Neuralgia/etiology ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries/complications/pathology/*therapy ; *Transcranial Magnetic Stimulation

OBJECTIVETo observe the change of behavior, pathological change of the spinal cord,and expression of brain-derived neurotrophic factor (BDNF) and brain-derived neurotrophic factor (NGF) on rats with spinal cord injury in order to explore the optimal time of BMSCs transplantation.

METHODSEighty health SD rats were randomly divided into 8 groups (group A,B,C,D,E,F,G,H), 10 rats in each group. According to the modified Allen method,the rat model of spinal cord injury was built. Group A as non-injured group only exposed the spinal cord but not result in blast injury. BMSCs of vitro culture were respectively infunded the region of spinal cord injury in group C, D, E, F, G, H (as transplantation groups) at the 0 h, 6 h, 24 h,3 d,5 d,7 d after model made. Group B as single model group was infunded the equal cell culture fluid. BBB score was used to evaluate the function of spinal cord at the 1st,2nd and 4th weeks after injury. The morphological changes of the tissue of spinal cord injury were observed by HE stain and the expression of BDNF and NGF were detected by Elisa method at the 4th weeks after BMSCs transplantation.

RESULTSIn non-injured group,BBB score was highest than that of other 7 groups at the 1st, 2nd and 4th weeks after injury (P<0.01). There was no significant difference in BBB score between single model group and transplantation groups at the 1st week after BMSCs transplantation (P>0.05). BBB score in transplantation groups were higher than that of single model group at the 2nd and 4th weeks after BMSCs transplantation (P<0.05). At the 2nd week after injury, BBB score from high to low was group F,E,G,D,H,C,but there was no significant difference among the groups (P>0.05). At the 4th week after injury,there was significant differences in BBB score between group F and other transplantation groups (group C,D,E,G,H)(P0.05). The levels of BDNF and NGF in group F was highest at the 4th week after BMSCs transplantation (P<0.05). In non-injured group,HE staining showed the struction of spinal cord was clear and complete,no neutrophil was found. But in other 7 groups,HE staining showed obviously oedema at local tissue,juncture of gray and white matter was undefined,and different gliocyte proliferation and inflammatory cell infiltrate cound be found.

CONCLUSIONAllogeneic BMSCs transplantation is effective to stimulate the recovery of spinal cord function in rats with spinal cord injury,and the optimal time of BMSCs transplantation maybe at the 3 d after injury.

Animals ; Behavior, Animal ; Brain-Derived Neurotrophic Factor ; genetics ; Female ; Male ; Mesenchymal Stem Cell Transplantation ; Nerve Growth Factor ; genetics ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; metabolism ; pathology ; therapy ; Transplantation, Homologous

OBJECTIVETo study the effect of adenovirus-mediated basic fibroblast growth factor(FGF-2) gene transfer in vivo on oligodendrocyte cell numbers throughout ventrolateral white matter following spinal cord injury in rats.

METHODSThirty-two adult female Sprague Dawley rats were injured with the Infinite Horizon Impactor, and then were randomly assigned to four groups: FGF-2-Adts high-titer group (1.27x10(7) pfu/rat), FGF-2-Adts intermediate-titre group (6.37x10(6) pfu/rat), FGF-2-Adts low-titer group (3.18 x 10(6) pfu/rat), and green fluorescent protein (GFP)-Adts group (5.9x10(7) pfu/rat). The transgenic expression in vivo was detected with fluorescence microscopy. The locomotor function of the hindlimbs of rats was evaluated using Rivlin plate. Slides mounted with tissue sections were processed for immunohistochemical detection and quantification of oligodendrocytes (CC1(+)) in the ventral lateral funiculi (VLF) of injured spinal cords.

RESULTSOne week after spinal cord injury, GFP showed that many cells had expressed objective gene in vivo and the angles of the occlusal plane of rats in FGF-2 groups were significantly higher than in GFP-Adts group. Also, there was a significant difference among the FGF-2-Adts treatment groups for the volume of gray matter sparing. However, there were no significant differences for total white matter sparing. Stereological quantification of total CC1(+) cell numbers in the spared VLF showed a significant reduction in numbers with GFP controls compared to all other groups 4 weeks after injury. In contrast, the FGF-2 Adts intermediate-titer group had significantly more CC1(+) cells when compared to both the FGF-2-Adts high- and low-titer groups.

CONCLUSIONAdenovirus-mediated FGF-2 gene transfer can promote the functional recovery of the injured spinal cord by enhancing the proliferation and/or differentiation of oligodendrocytes.

Adenoviridae ; genetics ; Animals ; Disease Models, Animal ; Female ; Fibroblast Growth Factor 2 ; genetics ; metabolism ; Genetic Therapy ; Oligodendroglia ; pathology ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; metabolism ; physiopathology ; therapy ; Transfection

OBJECTIVETo investigate the effects of complement inhibiting component of Ephedra sinica on immunological inflammation following acute spinal cord injury (SCI) in rats.

METHODSThe complement inhibiting component of Ephedra sinica was isolated by multiple precipitation steps and thin layer chromatography, and then the activity was analyzed. Fifty healthy SD rats were selected and randomly divided into the control group and the experimental group, 25 in each group. Induction of SCI was performed following a modified Allen's weight-drop method. The complement inhibiting component from Ephedra sinica (15 mg/kg) dissolving in 5 mL normal saline was immediately administered by gastrogavage after SCI, once daily. Equal volume of normal saline was administered to rats in the control group by gastrogavage. Hematoxylin and eosin (H&E) staining and C3 immunohistochemical staining were performed in SCI tissue at 12 h, day 1, 3, 7, and 14 after SCI. C3 positive expressions and myeloperoxidase (MPO) activity were assessed. Intercellular adhesion molecule-1 (ICAM-1) mRNA expression level was evaluated by Real-time PCR technique.

RESULTSC3 positive expression, MPO activity, and ICAM-1 mRNA level were significantly weaker in the Ephedra sinica group than in the control group at all time points (12 h, day 1, day 3, day 7, and day 14 after SCI) (P < 0.01, P < 0.05).

CONCLUSIONSThere existed complement system activation following acute SCI. The complement inhibiting component of Ephedra sinica significantly reduced immunological inflammation after SCI, and played an important role in secondary SCI.

Animals ; Complement Activation ; drug effects ; immunology ; Complement Inactivating Agents ; pharmacology ; Ephedra sinica ; chemistry ; Inflammation ; immunology ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; immunology ; metabolism ; pathology

OBJECTIVETo detect the expression of telomerase in glial scar and its correlation with glial scar.

METHODSThere were 120 Sprague Dawley rats were randomly divided into non-interference group of telomerase, interference group of telomerase and control group. Non-interference group and interference group were for spinal cord injury, which adopted Allen's Weight Dropping to make molding; control group was for sham operation to open the vertebral plate and expose spinal marrow, in which spinal cord injury would not be caused. The expression of telomerase and glial fibrillary acidic profein (GFAP) was detected by PCR-ELISA and Western blot, and the formation of glial scar was observed by immunofluorescence on the 1st, 3rd, 5th, 7th, 14th, 28th, 42 th and 56th day after the spinal injury, and analyzed its relativity.

RESULTSThe expression of telomerase in non-interference group was (0.180 ± 0.004 - 1.217 ± 0.072), which was significantly higher than those in interference group (0.028 ± 0.007 - 0.092 ± 0.004, χ(2) = 28.753 - 37.518, P < 0.05) and control group (0.072 ± 0.007 - 0.075 ± 0.004, χ(2) = 18.618 - 41.093, P < 0.05) at all the time, with statistical significance. The expression of GFAP in non-interference group was (1.98 ± 0.15 - 19.40 ± 0.55) which was significantly higher than those in interference group (1.10 ± 0.13 - 16.64 ± 1.02, χ(2) = 14.538 - 37.366, P < 0.05) and control group (0.44 ± 0.05 - 0.48 ± 0.04, χ(2) = 16.733 - 34.041, P < 0.05) at all the time, with statistical significance. The expression of GFAP showed a linear correlation with that of telomerase in non-interference group, and with statistical differences (r = 0.755, P < 0.01). The expression of telomerase in interference group and control group were always negative. Glial scar observed by immunofluorescence in non-interference group was heavier than that in interference group, and control group showed no formation of glial scar.

CONCLUSIONSTelomerase shows a dynamic expression in glial scar and has positive correlational linear relationship with GFAP which shows the formation of glial scar. And the telomerase may be an important factor in promoting the formation of glial scar.

Animals ; Cicatrix ; enzymology ; Glial Fibrillary Acidic Protein ; metabolism ; Neuroglia ; enzymology ; pathology ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; enzymology ; pathology ; Telomerase ; metabolism