Influences of Glabridin Treatment on Motor Function Recovery after Spinal Cord Injury / Influencia del Tratamiento con Glabridina en la Recuperación de la Función Motora después de una Lesión de la Médula Espinal

SUMMARY: Spinal cord injury (SCI) usually arises from compression due to traffic accidents and falls, resulting in varying degrees of movement, sensory loss, and possible paralysis. Glabridin (Gla) is a natural compound derived from licorice. It significantly affects drug development and medicine because of its anti-inflammatory, anti-oxidative, anti-tumoral, antibacterial, bone protective, cardiovascular protective, neuroprotective, liver protective, anti-obesity, and anti-diabetic properties. Various methods were employed to administer Gla to SCI mice in order to investigate its impact on the recovery of motor function. The mice were allocated into four cohorts using a randomization procedure. In the sham cohort, solely the lamina of vertebral arch was surgically exposed without causing any harm to the spinal cord tissue. Conversely, the injury cohort was subjected to spinal cord tissue damage and received no treatment thereafter. The mice in the remaining two cohorts received a dosage of 40 mg/kg Gla every two days via either intraperitoneal or intrathecal injection for a duration of 42 d following spinal cord injury. We conducted behavioral tests utilizing the Basso Mouse Scale score and gait analysis techniques. Magnetic resonance imaging and hematoxylin and eosin were employed to evaluate scar tissue formation. Systemic inflammation in mice was evaluated by employing an enzyme-linked immunosorbent assay. Gla promoted motor function recovery in mice following SCI and improved the pathological environment in the damaged area. These alterations were more evident in mice subjected to the intrathecal injection method. Intraperitoneal injections appear to be more beneficial for controlling systemic inflammatory responses. Although more intensive studies are required, Gla exhibits promising clinical potential as a cost-effective dietary phytochemical.

La lesión de la médula espinal (LME) generalmente surge de la compresión producto de caídas y accidentes de tránsito, lo que resulta en alteraciones del movimiento, pérdida sensorial y posible parálisis. La Glabridina (Gla) es un compuesto natural derivado del regaliz, constituyéndose en un aporte significativo para el desarrollo de fármacos y la medicina debido a sus propiedades antiinflamatorias, antioxidantes, antitumorales, antibacterianas, osteoprotectoras, cardioprotectoras, neuroprotectoras, hepatoprotectoras, antidiabéticas y contra la obesidad. En el presente trabajo se emplearon varios métodos para administrar Gla a ratones con lesión medular con el fin de investigar su impacto en la recuperación de la función motora. Los ratones fueron distribuidos en cuatro grupos mediante un procedimiento de aleatorización. En el grupo simulado, únicamente se expuso quirúrgicamente la lámina del arco vertebral sin causar ningún daño al tejido de la médula espinal. Por el contrario, el grupo lesionado fue sometido a daño del tejido de la médula espinal, sin recibir tratamiento posterior. Los ratones de los dos grupos restantes recibieron una dosis de 40 mg/kg de Gla cada dos días mediante inyección intraperitoneal o intratecal durante 42 días después de la lesión de la médula espinal. Fueron realizadas pruebas de comportamiento utilizando la puntuación de la escala Basso Mouse y técnicas de análisis de la marcha. Se emplearon imágenes por resonancia magnética y se aplicaron tinciones histológicas (Hematoxilina & Eosina) en muestras para evaluar la formación de tejido cicatricial. La inflamación sistémica en ratones se evaluó mediante el empleo de un ensayo inmunoabsorbente ligado a enzimas. Gla promovió la recuperación de la función motora en ratones después de una lesión medular y mejoró el entorno patológico en el área dañada. Estas alteraciones fueron más evidentes en ratones sometidos al método de inyección intratecal. Las inyecciones intraperitoneales parecen ser más beneficiosas para controlar las respuestas inflamatorias sistémicas. Aunque se requieren estudios más intensivos, Gla exhibe un potencial clínico prometedor como fitoquímico dietético rentable.

Functional effects of microRNA-211-5p-targeted inhibition of erythropoietin hepatocyte kinase receptor and ligand B2 signaling pathway on spinal cord nerve injury / 中国医师进修杂志

Objective:To detect the expression of microRNA (miR)-211-5p, erythropoietin hepatocyte kinase receptor B2 (EphB2) and erythropoietin hepatocyte kinase ligand B2 (ephrin B2) in spinal cord tissues as well as nerve cells after spinal cord injury (SCI), and to explore their mechanisms and effects on neurological recovery in SCI rats.Methods:The study was conducted from May 2020 to June 2021 using Sprague Dawley (SD) rats and PC12 cells. SD rats were divided into sham-operated group and SCI group of 30 rats each, and Basso-Beattie-Bresnahan (BBB) score were performed at different postoperative time points (1, 3, 7, 14, 21 and 28 d), and the relative expression of miR-211-5p and Eph/ephrin B2 mRNA was measured by quantitative real-time polymerase chain reaction (qPCR); the SCI rats were divided into recombinant lentiviral vector LV-miR-211-5p group (group A), empty lentiviral vector LV-eGFP (group B) and saline group (group C), with 15 rats in each group, respectively. The recombinant lentiviral vector, empty lentiviral vector and saline were injected on the cephalic and caudal sides of the spinal cord injury, and the relative expression of miR-211-5p and Eph/ephrin B2 mRNA in the spinal cord tissue was measured at 1, 7 and 14 d after surgery. In addition, a PC12 injury cell line model was established with 150 μmol/L hydrogen peroxide (H 2O 2), and the apoptosis rate and apoptosis-related proteins and contents of different cell lines were detected by flow cytometry and Western blot, respectively. MiR-211-5p was verified to target EphB2 by dual luciferase reporter gene. Results:The results of the animal experiments showed that at different postoperative time points, the miR-211-5p levels in the SCI group were lower than those in the SHAM group: 0.70 ± 0.03 vs. 1.00 ± 0.10, 0.60 ± 0.04 vs. 1.00 ± 0.05, 0.45 ± 0.10 vs. 1.00 ± 0.12, 0.30 ± 0.06 vs. 1.00 ± 0.15, 0.20 ± 0.05 vs. 1.00 ± 0.13, 0.10 ± 0.02 vs. 1.00 ± 0.07. In contrast, levels of Eph/ephrin B2 were higher in the SCI group compared to the SHAM group: 1.10 ± 0.05 vs. 1.00 ± 0.01, 1.80 ± 0.01 vs. 1.00 ± 0.08, 2.30 ± 0.01 vs. 1.00 ± 0.10, 2.60 ± 0.01 vs. 1.00 ± 0.05, 2.80 ± 0.01 vs. 1.00 ± 0.06, 3.00 ± 0.01 vs. 1.00 ± 0.07 and 1.20 ± 0.05 vs. 1.00 ± 0.02, 1.60 ± 0.01 vs. 1.00 ± 0.03, 2.10 ± 0.10 vs. 1.00 ± 0.01, 2.40 ± 0.11 vs. 1.00 ± 0.09, 2.70 ± 0.13 vs. 1.00 ± 0.05, 2.90 ± 0.12 vs. 1.00 ± 0.03 ( P<0.05). At 14 d after surgery, Group A exhibited higher BBB scores than Groups B and C: (14.0 ± 1.1) points vs. (8.0 ± 1.1) and (8.2 ± 1.2) points, while miR-211-5p levels were higher than those in Groups B and C: 1.90 ± 0.10 vs. 0.40 ± 0.01 and 0.50 ± 0.02, and Eph/ephrin B2 levels were lower than those in Groups B and C: 0.70 ± 0.10 vs. 1.80 ± 0.04 and 1.90 ± 0.06, 0.60 ± 0.03 vs. 2.00 ± 0.04 and 2.10 ± 0.05 ( P<0.05). Immunofluorescence staining showed that the levels of GAP-43 and synaptophysin in group A were higher than those in groups B and C at 14 d after surgery ( P<0.05). Cellular assays showed that overexpression of miR-211-5p inhibited the apoptosis rate of H 2O 2-induced PC12 cells and the expression of the apoptosis-related gene Cleaved-caspase3 ( P<0.05). Knockdown of miR-211-5p increased the apoptosis rate of H 2O 2-induced PC12 cells and the expression of the apoptosis-related gene Cleaved-caspase3 ( P<0.05). Dual luciferase reporter gene assay confirmed that EphB2 was a target gene of miR-211-5p and overexpression of EphB2 antagonized the inhibitory apoptosis effect of miR-211-5p on H 2O 2-induced PC12 cells. Conclusions:This study showed that miR-211-5p could promote neurological repair in SCI by inhibiting the expression of Eph/ephrin B2 signaling pathway, suggesting that using miR-211-5p as a target to inhibit Eph/ephrin B2 signaling pathway may have a protective effect on SCI.

The effect of magnetic stimulation of the sacral nerve on detrusor weakness after spinal cord injury/ / 中国康复医学杂志

Objective:To investigate the effect of the magnetic stimulation of the sacral nerve on detrusor weakness after spinal cord injury. Method:36 individuals with detrusor weakness after spinal cord injury were divided into experimental group and control group after randomization.Both groups received conventional rehabilitation treatment,besides,the experimental group received repetitive functional magnetic stimulation(rFMS)on S3 nerve roots.Before treat-ment and after 4-weeks treatment,the simple bladder capacity,bladder ultrasound,self-rating anxiety scale were observed. Result:The maximum automatic micturition volume,average automatic micturition volume,residual urine vol-ume,initial uriesthesis,and anxiety self-evaluation score of the two groups were improved after intervention.And the improvements are more in experimental group(P＜0.05). Conclusion:The effect of the rFMS on individuals with detrusor weakness after spinal cord injury is signifi-cantly better than conventional intervention,which provides a new idea for the clinical treatment of neurogenic bladder with detrusor weakness.

Protective effect of Periplaneta americana powder on rats with spinal cord injury via the TLR4/MyD88/NF-κB pathway / 中国实验动物学报

Objective To explore the protective effect and possible mechanism of Periplaneta americana powder on rats with spinal cord injury.Methods Forty-eight male SD rats were randomly divided into sham operation,saline,Periplaneta americana powder,and Toll-like receptor 4(TLR4)inhibitor groups.Except for the sham operation group,a rat spinal cord hemi-transection injury model was established in the other three groups.The sham operation group received no treatment after the operation,saline and drug groups were subjected to intragastric administration of equal volumes of normal saline and Periplaneta americana powder(630 mg/kg),respectively,and the TLR4 inhibitor group was administered an intraperitoneal injection of TLR4 inhibitor(3 mg/kg).On days 1,3,7,and 14 after the operation,the motor function of rat hind limbs was evaluated by the Basso,Beattie,and Bresnahan(BBB)score.Histopathological changes of the spinal cord were observed by hematoxylin-eosin staining.Changes in the number of neurons were observed by immunohistochemistry.The levels of inflammatory factors IL-1,IL-6,IL-10,and TNF-α were measured by ELISA,and expression of TLR4,myeloid differentiation factor 88(MyD88),and NF-κB p65 was detected by Western Blot.Results Compared with the sham operation group,the BBB score and number of neurons in the saline group were significantly decreased,while the degree of pathological damage,and IL-1,IL-6,TNF-α,TLR4,MyD88,and NF-κB p65 levels were significantly increased(P＜0.05).Compared with the saline group,periplaneta americana powder and TLR4 inhibitor groups showed an increase in BBB scores and the number of neurons,and decreases in the degree of pathological damage and IL-1,IL-6,TNF-α,TLR4,MyD88,and NF-κB p65 levels(P＜0.05).Compared with the TLR4 inhibitor group,the periplaneta americana powder group had better increases in the BBB score,number of neurons and decreases in the degree of pathological damage and expression of IL-1 and TNF-α.Conclusions Periplaneta americana powder reduces the production of inflammatory factors after spinal cord injury by inhibiting the TLR4/MyD88/NF-κB pathway,protects nerves,and promotes motor recovery.

Effects of Electroacupuncture on Urodynamics and Expression of ERK/CREB/Bcl-2 Pathway in Spinal Cord of Rats with Neurogenic Bladder after Suprasacral Spinal Cord Injury / 中国中医药信息杂志

Objective To observe the effects of electroacupuncture at"Ciliao","Zhongji","Sanyinjiao"and"Dazhui"on urodynamics and expression of ERK/CREB/Bcl-2 pathway in spinal cord tissue of neurogenic bladder rats after suprasacral spinal cord injury.Methods Sixty female SD rats randomly selected 24 and divided into blank group and sham-operation group(12 rats in each group),the remaining 36 rats were subjected to surgical modeling.After modeling,rats were randomly divided into the model group and the electroacupuncture group,with 12 rats in each group.The electroacupuncture group received unilateral electroacupuncture stimulation at acupoints"Ciliao","Zhongji","Sanyinjiao",and"Dazhui"for 30 minutes each time,once a day,for 7 consecutive days.After administration,urodynamic testing was performed,HE staining was used to observe the morphology of bladder detrusor tissue,TUNEL method was used to detected apoptosis in spinal cord tissue,Western blot was used to detected expressions of p-ERK1/2,p-CREB,p-p90Rsk,CRE,Bcl-2,and Bax proteins in spinal cord tissue.Results Compared with the sham-operation group,the basal pressure,maximum pressure,and leakage point pressure of the bladder in the model group increased significantly(P<0.01),while the maximum capacity and compliance of the bladder decreased significantly(P<0.01);the structure of bladder smooth muscle cells was severely damaged and disorderly arranged,accompanied by a large amount of inflammatory cell infiltration;the apoptosis rate of spinal cord tissue cells significantly increased(P<0.01),and the expressions of p-ERK1/2,p-p90Rsk,p-CREB,CRE,and Bcl-2 proteins in spinal cord tissue were significantly decreased,while the expression of Bax protein significantly increased(P<0.01).Compared with the model group,the basal pressure,maximum pressure,and leakage point pressure of the bladder in the electroacupuncture group decreased significantly(P<0.05),while the maximum capacity and compliance of the bladder increased significantly(P<0.05,P<0.01);the integrity of bladder smooth muscle cells was enhanced,the degree of cell edema was reduced,and inflammatory cell infiltration was reduced;the apoptosis rate of spinal cord tissue cells was significantly reduced(P<0.05),and the expressions of p-ERK1/2,p-p90Rsk,p-CREB,CRE,and Bcl-2 proteins in spinal cord tissue significantly increased,while the expression of Bax protein was significantly decreased(P<0.05,P<0.01).Conclusion Electroacupuncture can promote the repair of bladder detrusor tissue in rats with neurogenic bladder model after suprasacral spinal cord injury,increase the maximum capacity and compliance of the bladder,alleviate the high pressure state in the bladder,and its mechanism is related to activating the ERK/CREB/Bcl-2 pathway,reducing secondary apoptosis of damaged neurons,effectively improving bladder innervation,and protecting bladder function.

A robotic brace for dynamic trunk support when evaluating seated trunk control after a spinal cord injury / 中华物理医学与康复杂志

Objective:To explore the utility of a robotic trunk support brace (a RoboBDsys) in evaluating seated trunk control after a spinal cord injury (SCI).Methods:Twenty wheelchair-dependent SCI patients were tested for trunk resistance while seated and wearing the RoboBDsys. In the test they were required to maintain an upright seated position for 1 minute while external forces were applied from the left, right, anterior and posterior directions. The system generated eight observations of swings of the center of pressure (COP), their mean velocity and their standard deviations. Sway area, sway path (SP), mean sway frequency (MF) and mean sway amplitude (MA) were also recorded along with the maximum voluntary contraction torque (MVC). The trunk control test (TCT), the Tinetti Balance Scale (Tinetti), the modified Functional Reach Test (mFRT) and Spinal Cord Independence Measure III (SCIM-III) were also administerd and their results were correlated with the RoboBDsys resistance test results.Results:All twenty of the patients completed the resistance test in the left and right directions, but only 11 did it in the anterior and posterior directions. In the left and right-direction resistance tests the average center of pressure deviation, sway area and MVC showed some significant correlation with the TCT, Tinetti, mFRT and SCIM-III results (| r| ranging from 0.46 to 0.74). The average MVC in the anterior-direction correlated moderately with the TCT, mFRT and SCIM-III results (| r|=0.63 to 0.67), while that in the posterior-direction had moderate correlation with the TCT and mFRT results only (| r|=0.63 to 0.67). Conclusion:The Robotic Brace for Dynamic Trunk Support system can be effective in assessing reactive postural control and functional independence after a spinal cord injury.

Study on the neuroprotective effect of curculigoside on rats with spinal cord injury / 中国药房

OBJECTIVE To investigate the neuroprotective effect of curculigoside （CUR） on rats with spinal cord injury （SCI） based on phosphatase and tensin homologue deleted on chromosome ten gene-induced putative kinase 1 （PINK1）/E3 ubiquitin ligase Parkin signaling pathway. METHODS Taking male SD rats as subjects， 15 rats were randomly selected as sham operation group； the rest rats were chosen to establish SCI model by spinal cord impact method， and then were divided into model group， CUR low-dose group （36 mg/kg CUR， gavage）， CUR high-dose group （72 mg/kg CUR， gavage） and CUR high-dose+3- methyladenine （3-MA） group （72 mg/kg CUR， gavage+20 mg/kg autophagy inhibitor 3-MA， intraperitoneal injection）， with 15 rats in each group. Rats in each group were given corresponding liquid/normal saline， once a day， for 28 consecutive days. Basso- Beattie-Bresnahan （BBB） score and Rivlin inclined plate experiment were performed on the 14th and 28th day after administration； the pathological changes of spinal cord tissue in rats were observed in each group； the apoptosis of spinal cord tissue， the levels of oxidative stress factors [malondialdehyde （MDA）， superoxide dismutase （SOD）， glutathione （GSH）]， and the protein expressions of brain-derived neurotrophic factor （BDNF）， glial fibrillary acidic protein （GFAP）， PINK1， Parkin， p62 and microtubule- associated protein light chain 3 （LC3） were all determined. RESULTS Compared with the sham operation group， obvious edema and bleeding in the spinal cord tissue of rats were observed in the model group， accompanied by a large number of inflammatory cell infiltration； BBB score and inclined plate angle， SOD and GSH levels， the protein expressions of BDNF， PINK1 and Parkin， and LC3Ⅱ/Ⅰ ratio were significantly reduced； the apoptosis rate， MDA level， the protein expressions of GFAP and p62 in spinal cord tissue were significantly increased （P＜0.05）. Compared with the model group， the edema， bleeding and infiltration of inflammatory cells in the spinal cord tissue of rats were reduced in the administration groups， and the above quantitative indicators had been significantly improved （P＜0.05）； 3-MA could significantly reverse the improvement effects of the above indexes by CUR （P＜0.05）. CONCLUSIONS CUR can promote the recovery of neurological and motor functions in SCI rats， improve the pathological injury of the spinal cord and inhibit apoptosis， which may be related to mitochondrial autophagy mediated by activating the PINK1/Parkin signaling pathway.

Long-term efficacy and complications of implantable diaphragm pacer in children with high cervical spinal cord injury: case report and literature review / 中华实用儿科临床杂志

The long-term efficacy and complications of implantable diaphragm pacer (IDP) in a child with cervical spinal cord injury (CSCI) in the Department of Spinal and Neural Functional Reconstruction, China Rehabilitation Research Center in September 2022 were retrospective analyzed.A male child had quadriplegia without an obvious cause at the age of 12 years, and he was then lived completely with the assistance of mechanical ventilation.At the age of 14 years, he could wean off the ventilator in unilateral diaphragmatic pacing mode.However, mechanical ventilation was re-given for months after 5 years due to pneumonia, and then the IDP was re-given with the self-felt decreased pacing effect.After hospitalization, the patient was examined with mild diaphragmatic atrophy, secondary flat chest, and mild scoliosis.After optimization of the transdiaphragmatic pacing threshold and rehabilitation, his respiratory function improved.IDP can be used in CSCI for long time, while flat chest and scoliosis that limited the expansion of the lungs should be considered.At the meantime, the increased abdominal spasm affected the abdominal compliance, leading to the decrease in the efficiency of the diaphragm.

Mechanism of Buyang Huanwu decoction in the treatment of spinal cord injury based on network pharmacology / 中国现代医生

Objective To verify the mechanism of Buyang Huanwu decoction in the treatment of spinal cord injury based on network pharmacology and molecular docking.Methods The active ingredients and targets of Buyang Huanwu decoction were screened out by TCMSP,SymMap,PubChem and Swiss Target Prediction databases.Spinal cord injury targets were retrieved from OMIM,GeneCards,TTD,and DrugBank databases.Through venny software,the intersection target of Buyang Huanwu decoction and spinal cord injury was obtained.The active ingredient-target network for the treatment of spinal cord injury was constructed with Cytoscape software.Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of common targets were carried out by DAVID,and the binding ability of drugs and targets was analyzed by molecular docking technology.Results A total of 106 active ingredients and 225 targets of Buyang Huanwu decoction,1315 targets of spinal cord injury and 112 targets of drug-disease intersection were obtained.The active ingredients of Buyang Huanwu decoction were quercetin,kaempferol,ellagic acid,luteolin and hederagenin in the treatment of spinal cord injury.Conclusion Buyang Huanwu decoction can achieve the purpose of treating spinal cord injury through various signal pathways.

Research Progress on Epidural Electrical Stimulation for Motor Disorders after Spinal Cord Injury / 中山大学学报(医学科学版)

In recent years， the incidence of spinal cord injury （SCI） has shown an increasing trend， and the resulting motor impairment cause significant harm to patients themselves and public health. Although the current clinical treatments for motor disorders after SCI are diverse， mainly focusing on improving dysfunction and improving patients' daily living ability through external stimulation， there is still a lack of radical solutions. Epidural electrical stimulation （EES） as an emerging treatment technology， has been shown in a number of international clinical studies on the SCI significant potential of motor function recovery， has significant curative effect， convenient， widely applicable， adjustable advantages， the rehabilitation of movement disorders after SCI has a lot of big benefits， in the spinal cord injury rehabilitation has great prospect. However， most of the domestic research are limited to the neurogenic bladder caused by abnormal autonomic nerve function after SCI， and mostly combine traditional Chinese and western medicine rehabilitation treatment， the emerging therapy of EES， this paper aims to review the basic principle of EES， potential mechanism of action and its c linical application progress in the treatment of movement disorders after SCI， in order to provide reference for the clinical application and scientific research of EES treatment of motor disorders after SCI.

Research Progress of Acupuncture in Improving Central Nervous System Diseases by Regulating BDNF/TrkB Signaling Pathway / 中山大学学报(医学科学版)

As a key regulator of neuronal growth， development and synaptic plasticity， BDNF/ TrkB signaling pathway is widely involved in the occurrence and development of central nervous system diseases， such as ischemic stroke， Alzheimer's disease， Parkinson's disease and spinal cord injury. Studies have shown that acupuncture can regulate the activity of BDNF/TrkB signaling pathway， and has significant therapeutic potential for these diseases. Its mechanism of action is related to participating in synaptic remodeling， inhibiting neuronal apoptosis， and promoting neurogenesis or synaptic regeneration. This article reviews the role of BDNF/TrkB signaling pathway in central nervous system diseases and the regulation mechanism of acupuncture on this pathway， in order to provide new ideas for clinical treatment. Future studies should further explore the precise regulatory mechanism of acupuncture on BDNF/TrkB signaling pathway in order to develop more efficient and safe treatment strategies.

Human umbilical cord mesenchymal stem cell-derived exosomes reduce the permeability of blood-spinal cord barrier after spinal cord injury / 中国组织工程研究

BACKGROUND:Endothelin has been found to be involved in the breakdown of the blood-spinal cord barrier after spinal cord injury,and stem cell-derived exosomes can reduce the permeability of the blood-spinal cord barrier and repair spinal cord injury. OBJECTIVE:To investigate whether exosomes produced by human umbilical cord mesenchymal stem cells can reduce the permeability of the blood-spinal cord barrier by inhibiting endothelin-1 expression,thus repairing spinal cord injury. METHODS:Exosomes were extracted from the cultured supernatant by the hyperspeed centrifugation method.The morphology of exosomes was observed by transmission electron microscope.The expression levels of tsg101 and CD63 were detected by western blot assay.Eighty SD rats were randomly divided into sham operation group,model group,exosome group,and endothelin-1 group(n=20).The modified Allen's method was used to create the rat model of spinal cord injury.In the endothelin-1 group,10 μL(1 μg/mL)endothelin-1 was injected directly into the injured area with a microsyringe.Immediately,1 day,2 days after operation,sham operation group and model group were injected with 200 μL PBS solution through the tail vein;the exosome group and endothelin-1 group were injected with 200 μL exosome(200 μg/mL)solution through the tail vein,respectively.Hind limb motor function scores were performed on days 1,3,7,14 and 21 after spinal cord injury.The blood-spinal cord barrier permeability was observed by Evans blue staining on day 7 after injury.The expression levels of tight junction proteins β-Catenin,ZO-1,Occludin and endothelin-1 in the spinal cord were detected by western blot assay. RESULTS AND CONCLUSION:(1)Basso-Beattie-Bresnahan score in the exosome group was significantly higher than that in the model group at 3-21 days after injury(P<0.05).Hematoxylin-eosin staining showed that spinal cord injury was greatly reduced in the exosome group compared with the model group.Basso-Beattie-Bresnahan score in the endothelin-1 group was significantly decreased compared with the exosome group(P<0.05).Spinal cord injury was more severe in the endothelin-1 group than that in the exosome group.(2)The expression of endothelin-1 in the model group was significantly increased compared with the sham operation group(P<0.05),and the expression of endothelin-1 in the exosome group was significantly decreased compared with the model group(P<0.05).(3)The blood-spinal cord barrier Evans blue exudate in the exosome group was significantly decreased compared with the model group(P<0.05).The expression levels of the tight junction proteins β-Catenin,Occludin and ZO-1 in the exosome group were increased(P<0.05);the Evans blue exudate in the endothelin-1 group was significantly increased compared with the exosome group(P<0.05).The expression level of tight junction protein was significantly decreased compared with the exosome group(P<0.05).(4)The results show that human umbilical cord mesenchymal cell-derived exosomes protect the permeability of the blood-spinal cord barrier by down-regulating the expression of endothelin-1 and play a role in the repair of spinal cord injury.

Single-cell RNA sequencing reveals the heterogeneity of astrocytes / 中国组织工程研究

BACKGROUND:Astrocytes are the most abundant cells in the central nervous system,and various subsets of astrocytes are heterogeneous,performing a variety of special functions.Single-cell RNA sequencing(scRNA-seq)technology developed in recent years has extended our understanding of astrocyte heterogeneity from the perspective of transcriptome profiling. OBJECTIVE:To summarize the heterogeneity of scRNA-seq technology in different time and space,and pathological states and expand our knowledge of astrocyte heterogeneity on both molecular and functional levels. METHODS:The relevant articles on astrocyte heterogeneity and scRNA-seq were searched on PubMed,Elsevier,and CNKI databases.The search terms were"astrocytes,scRNA-seq,heterogeneity,Alzheimer disease,spinal cord injury,multiple sclerosis"in Chinese and English.Finally,74 articles were selected for viewing after screening according to inclusion criteria. RESULTS AND CONCLUSION:scRNA-seq studies related to the heterogeneity of astrocytes have shown that astrocyte is significantly heterogeneous across four aspects:species,developmental stage,central nervous system region,and pathological state.(1)Unique expression of certain genes occurs in astrocytes of different species,and the discovery of species-specific genes is beneficial for the translation of clinical studies.(2)During astrocyte development,differential gene expression emerged in the cellular subtypes identified at each stage,which further refined the cellular lineage of astrocytes and laid the foundation for the study of astrocyte developmental trajectories and mechanisms.(3)The discovery of differential gene expression allows regional localization of different astrocyte subpopulations and assists in the diagnosis and treatment of neurological diseases.(4)Astrocyte heterogeneity revealed by scRNA-seq can provide specific markers at the time of disease diagnosis and identify potential therapeutic targets.(5)The heterogeneity of astrocytes exists in many aspects,interacts with each other and is complex.The mechanisms of its generation,maintenance and transformation remain unclear.At present,molecular research on the single-cell level is still lacking.Linking transcriptionally defined astrocyte subpopulations to cellular activity,behavior and disease markers in real time remains one of the great challenges in the field.

Tauroursodeoxycholic acid treats spinal cord injury by reducing apoptosis of spinal cord neurons under glucose and oxygen deprivation / 中国组织工程研究

BACKGROUND:Tauroursodeoxycholic acid is a hydrophilic bile acid derivative that has neuroprotective effects in a variety of neurological disease models.However,there are few reports on the effects of tauroursodeoxycholic acid on spinal cord injury. OBJECTIVE:To investigate the effect of tauroursodeoxycholic acid on apoptosis of spinal cord neurons under hypoglycemic and hypoxic conditions,as well as the effect on recovery of motor function in mice after spinal cord injury. METHODS:(1)In vitro experiment:Primary spinal cord neurons were isolated from C57 BL/6 mouse embryos at 13.5 days of gestation.After 72 hours of culture,the cells were divided into three groups.In the normal group,cells were cultured in Neurobasal complete medium that was incubated in a CO2 incubator(5%CO2 + 95%air)for 24 hours.In the oxyglucose-deprived group,sugar-free Neurobasal medium was added and incubated in a triple-gas incubator(94%N2+5%CO2+1%O2)for 12 hours,and then the medium was replaced with Neurobasal complete medium and incubated in a CO2 incubator for 12 hours.In the experimental group,the treatment procedure was approximately the same as that in the oxyglucose-deprived group,except that taurodeoxycholic acid was added along with the sugar-free Neurobasal medium.TUNEL staining was used to detect apoptosis,cell counting kit-8 assay was applied to detect cell activity,and immunofluorescence staining was performed to detect cellular β-microtubule protein expression.(2)Animal experiment:Sixty C57 BL/6 mice were randomly divided into sham-operated group,spinal cord injury group and experimental group,with 20 mice in each group.Animal models of T9-T10 spinal cord injury were established using Allen's percussion method in the spinal cord injury group and the experimental group.Starting from the 1st day after modeling,taurodeoxycholic acid solution was given by gavage in the experimental group and normal saline was given by gavage in the sham-operated and spinal cord injury groups once a day for 14 consecutive days.Spinal cord tissue repair was assessed using behavioral and histological methods. RESULTS AND CONCLUSION:In vitro experiment:TUNEL staining,cell counting kit-8 and immunofluorescence staining showed that compared with the normal group,the number of apoptotic cells was higher(P＜0.01),while cell activity and β-microtubule protein expression were lower in the oxyglucose-deprived group(P＜0.01);compared with the oxyglucose-deprived group,the number of apoptotic cells was lower(P＜0.01),while cell activity and β-microtubule protein expression were higher in the experimental group(P＜0.01).Animal experiment:The Basso-Beattie-Bresnahan scores in the open field test and hind limb footprint experiments showed that the mice in the experimental group had better recovery of walking and motor functions than those in the spinal cord injury group.Hematoxylin-eosin staining showed that significant deformities and cavities were observed at the site of spinal cord injury and the number of nerve cells was significantly reduced in the spinal cord injury group.Compared with the spinal cord injury group,the experimental group showed a significant reduction in the area of spinal cord injury,less spinal cord deformity,fewer cavities,and an increase in the number of nerve cells.Immunofluorescence staining showed that the number of neuronal nucleus-labeled neuronal cells in the spinal cord injury group was less than that in the sham-operated group(P＜0.01),and the number of neuronal nucleus-labeled neuronal cells in the experimental group was higher than that in the spinal cord injury group(P＜0.01).To conclude,tauroursodeoxycholic acid could effectively reduce glucose/oxygen deprivation-induced apoptosis of spinal cord neurons and axonal loss,and promote the recovery of motor function in mice with spinal cord injury.

Efficient strategies for microglia replacement in spinal cord injury models / 中国组织工程研究

BACKGROUND:As the incidence of spinal cord injury increases with the years and axon regeneration after spinal cord injury was very difficult.How to promote the recovery from spinal cord injury and improve the transplantation efficiency of stem cells and other therapeutic cells after spinal cord injury has been the focus of clinical and scientific research. OBJECTIVE:To establish the efficient transplantation and replacement of mouse spinal cord microglia in the spinal cord injury model. METHODS:CX3CR1 creER-/+::LSL-BDNF-/+-tdTomato mice,CX3CR1+/GFP mice,β-actin GFP mice and C57 BL/6J wild-type mice at 8-10 weeks of age were selected.According to the requirements of the experiment,they were randomly divided into six groups.(1)Sham operation group:eight C57 BL/6J wild-type mice were used when only the lamina was removed without injury.(2)Spinal cord contusion injury group:eight C57 BL/6J wild-type mice were used.(3)Spinal cord crush injury group:eight C57 BL/6J wild-type mice were used.(4)Conjoined symbiotic spinal cord strike injury group:β-actin GFP mice with green fluorescent blood were surgically stitched together with C57 BL/6J wild-type mice,using eight β-actin GFP mice and eight C57 BL/6J wild-type mice.(5)Mr BMT-X Ray group(using PLX5622 to eliminate the spinal microglia and bone marrow transplantation with X-ray radiation):Bone marrow cells from four CX3CR1 creER-/+::LSL-BDNF-/+-tdTomato mice were extracted and transplanted into eight C57 BL/6J wild-type mice for spinal cord injury modeling.(6)Mr BMT-Busulfan group(using PLX5622 to eliminate the spinal microglia and bone marrow transplantation with Busulfan):Bone marrow cells from four CX3CR1+/GFP mice were transplanted into eight C57 BL/6J wild-type mice.The percentage of cell transplantation replacement in this group was observed,and the spinal cord injury model was not established in this group.The sham operation group,spinal cord contusion injury group and spinal cord crush injury group were sampled by perfusion on day 14 after spinal cord injury.The conjoined symbiotic spinal cord strike injury group was sampled by perfusion on day 7 after spinal cord injury.Mr BMT-X Ray group was sampled by perfusion on day 28 after spinal cord injury.Mr BMT-Busulfan group was sampled by perfusion on day 28 after transplantation.The sampling site was a 1.2 cm long spinal cord with the T10 segment as the center.In the Mr BMT-X Ray group and Mr BMT-Busulfan group,additional mouse brain tissue was retained to see if it would lead to brain transplantation and replacement.The number and proportion of transplanted and replaced cells in the damaged area were measured using transgenic mice,symbiosis and immunofluorescence. RESULTS AND CONCLUSION:Compared with the traditional peripheral blood transplantation(9.8%)of mice in the conjoined symbiotic spinal cord strike injury group,the new transplantation methods,Mr BMT-X Ray and Mr BMT-Busulfan,could greatly improve the proportion of spinal microglia transplantation and replacement,which could reach 84.8%and 95.6%,respectively.The difference was significant(P<0.05).The results showed that Mr BMT-X Ray and Mr BMT-Busulfan could achieve efficient replacement of spinal microglia cells,and could improve the problems of low cell transplantation efficiency,few survival numbers and unclear differentiation of the traditional cell transplantation methods.In addition,Mr BMT-X Ray can only replace the microglia in the spinal cord,while Mr BMT-Busulfan could avoid brain inflammation and injury caused by X-ray radiation transplantation.

Riluzole interferes with the activation of NLRP3 inflammasome in microglia of rats with spinal cord injury / 中国组织工程研究

BACKGROUND:Previous animal studies have shown that riluzole can inhibit neuroinflammatory response after spinal cord injury and promote functional recovery in injured rats,but the study on whether it can regulate the expression of nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome in the acute stage is lacking. OBJECTIVE:To observe whether riluzole can reduce microglial pyroptosis and promote functional recovery after spinal cord injury by modulating NLRP3 inflammasome through animal experiments,histological experiments and molecular biology experiments. METHODS:Female SD rats were divided into sham operation,model and riluzole groups,with 12 rats in each group.In addition to the sham operation group,T10 spinal cord injury was conducted in rats.The model group was treated with intraperitoneal administration of riluzole with solvent cyclodextrin.The riluzole group was treated with a 4 mg/kg dose of riluzole injection.The effect of riluzole on motor function recovery was assessed using the BBB score and inclined plane test.The recovery of sensory-evoked potential and motor-evoked potential was measured by electrophysiology.Hematoxylin-eosin staining was used to evaluate spinal cord tissue repair.The regulatory effects of riluzole on NLRP3,Caspase-1 and gasdermin D protein expression in spinal cord tissues were detected by western blot assay.ELISA was utilized to detect the expression levels of inflammatory factors interleukin-1β and interleukin-18.The effects of riluzole on the expression of NLRP3,Caspase-1,gasdermin D and interleukin-1β in microglial cells of the injured spinal cord were determined by immunofluorescence staining. RESULTS AND CONCLUSION:(1)At 35 days after spinal cord injury,BBB score and inclined plane test score in the riluzole group were higher than those in the model group(P<0.05).(2)At 3 days after spinal cord injury,the protein expressions of NLRP3,cleaved Caspase-1,gasdermin D-N(N-terminal domain),interleukin-1β,and interleukin-18 in the spinal cord homogenate of the riluzole group were significantly lower than those of the model group(P<0.05).(3)At 3 days after spinal cord injury,the fluorescence intensity of NLRP3,Caspase-1,gasdermin D and interleukin-1β in the riluzole group was significantly lower than that in the model group(P<0.05).(4)At day 35 after spinal cord injury,hematoxylin-eosin staining showed that the area of spinal cord injury in the riluzole group was smaller than that in the model group.Electrophysiological tests showed that the latency periods of sensory-evoked potential and motor-evoked potential in the riluzole group were shorter than those in the model group,and the latency period of wave amplitude in the riluzole group was higher than that in the model group.(5)These results suggest that riluzole can promote the repair of injured spinal cord tissue,promote the repair of nerve conduction function,and further promote the recovery of motor function in rats with spinal cord injury,which may be achieved through the regulation of NLRP3 inflammasome and the reduction of microglial pyroptosis.

Effects of Periplaneta americana powder on motor function and autophagic protein Beclin-1 in rats undergoing spinal cord hemisection / 中国组织工程研究

BACKGROUND:Spinal cord injury involves mechanisms such as oxidative stress,inflammation,apoptosis and autophagy.Activation of autophagy can improve neuromotor function after spinal cord injury and play a protective role in the spinal cord. OBJECTIVE:To investigate the effects of Periplaneta americana powder on hindlimb motor function and the autophagy protein Beclin-1 in the injured site of rats after spinal cord hemisection. METHODS:Thirty Sprague-Dawley rats,6-8 weeks of age,were randomly divided into three groups(n=10 per group).In the sham-operated group,the lamina was just opened to exposure the spinal cord followed by suturing.Normal saline group and Periplaneta americana powder group both underwent left hemisection of the spinal cord to prepare animal models of spinal cord hemisection.The normal saline group was continuously gavaged with normal saline for 14 days,and the Periplaneta americana powder group was continuously gavaged with Periplaneta americana powder for 14 days.The Basso Beattie Bresnahan scale score was performed at the 6th hour,1st day,3rd day,7th day and 14th day after operation to observe the hindlimb motor function.After 14 days of administration,the rats were sacrificed and sampled.Immunohistochemistry,western blot and immunofluorescence were used to detect the expression of Beclin-1 in the injured site of the spinal cord after hemisection. RESULTS AND CONCLUSION:After operation,the Basso Beattie Bresnahan scale scores were gradually increased in the normal saline group and Periplaneta americana powder group.Compared with the sham-operated group,the Basso Beattie Bresnahan scale scores were significantly reduced in the normal saline group and Periplaneta americana powder group at the 6th hour,1st day,3rd day,7th day and 14th day after operation(P<0.05).The Basso Beattie Bresnahan scale scores in the Periplaneta americana powder group were significantly higher than those in the normal saline group at the 7th and 14th days after operation(P<0.05).Immunohistochemical staining showed that Beclin-1 was weakly positive in the sham-operated group,mainly expressed in the cytoplasm;in the normal saline group,Beclin-1 was mainly expressed in the cytoplasm and partially expressed in the nuclear membrane;in the Periplaneta americana powder group,Beclin-1 was mainly expressed in the cytoplasm and partially expressed in the nuclear membrane.The proportion of Beclin-1 positive cells was higher in the normal saline and Periplaneta americana powder groups than in the sham-operated group(P<0.05),while the proportion of Beclin-1 positive cells was higher in the Periplaneta americana powder group than in the normal saline group(P<0.05).Western blot assay and immunofluorescence staining showed that the Beclin-1 protein expression was higher in the normal saline and Periplaneta americana powder groups than in the sham-operated group(P<0.05),and moreover,the Beclin-1 protein expression was higher in the Periplaneta americana powder group than in the normal saline group(P<0.05).To conclude,Periplaneta americana powder could improve the hindlimb motor function of rats with spinal cord hemisection injury,and the mechanism may be that polysaccharides in the Periplaneta americana powder increase the expression of Beclin-1.

Characterization and repair effect of supramolecular conducting hydrogel carrying ligustrazine on spinal cord injury in rats / 中国组织工程研究

BACKGROUND:Based on the concept of the combination of medicine and industry and the advantages of traditional Chinese medicine treatment,the construction of a new composite material loaded with the effective active ingredient of traditional Chinese medicine is a hot research spot in the repair of spinal cord injury,and is expected to become an effective means to solve this problem. OBJECTIVE:To observe the effect of supramolecular conducting hydrogel carrying ligustrazine in repairing spinal cord injury in rats. METHODS:The supramolecular conducting hydrogel carrying ligustrazine was prepared and its microstructure,conductivity,rheology,swelling rate and in vitro release performance were characterized.45 SD rats were divided into 3 groups by random number table method,with 15 rats in each group:no spinal cord injury in the sham operation group;spinal cord injury model was established in the model group;and supramolecular conducting hydrogel carrying ligustrazine was injected into the spinal cord injury area after model establishment in hydrogel group.BBB score was used to evaluate the recovery of hind limb motor function of each group before and 1,7,14,21 and 28 days after modeling,respectively.28 days after the model establishment,the spinal cord tissues were collected and analyzed by hematoxylin-eosin staining,immunohistochemical staining and western blot assay. RESULTS AND CONCLUSION:(1)Under scanning electron microscopy,the supramolecular conducting hydrogel with ligustrazine displayed a three-dimensional micrometer-scale porous network structure with high porosity and a pore size of approximately 100 μm.The conductivity of the hydrogel was 7.66 S/m;the swelling rate was 3 764.42%,and it had certain mechanical stability and injection property.In vitro sustained release experiments demonstrated that the supramolecular conducting hydrogel with ligustrazine sustainably released ligustrazine for more than 800 hours.With the extension of time,the cumulative release of ligustrazine exhibited an increasing trend.(2)With the extension of modeling time,the hind limb motor function gradually recovered in the model group and the hydrogel group,and the hind limb motor function of the hydrogel group was better than that of the model group on 14,21,and 28 days after modeling(P<0.05).Hematoxylin-eosin staining demonstrated that the spinal cord tissue of the model group had cavities and a large number of inflammatory cells could be seen at the stump.In the hydrogel group,some inflammatory cells were infiltrated in the injured area of the spinal cord;the void area of the injured area was reduced;neuron cells appeared in the junction area,and the tissue arrangement was relatively neat.Immunohistochemical staining and western blot assay exhibited that the expression of tumor necrosis factor α and interleukin-6 protein in the rat spinal cord of the hydrogel group was lower than that in the model group(P<0.05),and the expression of neuronal nuclear antigen protein was higher than that in the model group(P<0.05).(3)These findings confirm that the supramolecular conducting hydrogel carrying ligustrazine can promote the repair of spinal cord injury.

Repetitive magnetic stimulation of S3 nerve root and M1 area for treating urinary retention after spinal cord injury / 中国组织工程研究

BACKGROUND:Repetitive magnetic stimulation of either S3 nerve root or M1 area can improve the urination function of patients with urinary retention after spinal cord injury,but there are few reports on the repetitive magnetic stimulation of both sites in patients with urinary retention after spinal cord injury. OBJECTIVE:To observe the effect of repetitive magnetic stimulation of both S3 nerve root and M1 area on urinary retention after spinal cord injury. METHODS:Forty patients with urinary retention after spinal cord injury were enrolled and were randomly divided into two groups(n=20 per group):group A(repetitive magnetic stimulation in both S3 nerve root and M1 area)and group B(repetitive magnetic stimulation in the S3 nerve root and sham stimulation in the M1 area).Patients in both groups were given 4-week repetitive magnetic stimulation based on conventional bladder function intervention.The stimulation time and duration of treatment were same in both groups,with a treatment time of 21 minutes daily,5 days per week,for 4 weeks in total.The urination diary and urodynamics were compared between two groups. RESULTS AND CONCLUSION:Before treatment,there were no statistically significant differences in the average daily catheterization times,average daily catheterization volume,average single urinary volume,urinary storage period(maximum bladder volume,bladder pressure),and urinary voiding period(detrusor pressure,residual urine volume)between the two groups(P>0.05).After 4 weeks of treatment,the average daily catheterization times in group A were lower than before treatment(P<0.05),while the average single urination volume in group A was higher than that before treatment(P<0.05);and the average daily catheterization times in group B were lower than before treatment(P<0.05).After 4 weeks of treatment,the average daily catheterization times in group A were lower than those in group B,and the average single urination volume was higher than that in group B(P<0.05).After 4 weeks of treatment,the maximum bladder volume and detrusor pressure during urination were increased in both groups compared with before treatment(P<0.05),while the bladder pressure and residual urine volume at the maximum volume of the two groups were decreased compared with those before treatment(P<0.05).Compared with group B,the maximum bladder volume and detrusor pressure during urination were higher in group A,while the bladder pressure and residual urine volume at maximum volume were lower in group A(P<0.05).To conclude,two treatments can both improve the urination function of patients with urinary retention after spinal cord injury,and repetitive magnetic stimulation of both S3 nerve root and M1 area is superior to repetitive magnetic stimulation of S3 nerve root alone.Repetitive magnetic stimulation of both S3 nerve root and M1 area can effectively improve the urination function of patients with urinary retention after spinal cord injury.

Regulatory mechanisms and therapeutic strategies for pyroptosis after spinal cord injury / 中国组织工程研究

BACKGROUND:Cell death and neuroinflammation are two important targets in the treatment of spinal cord injury.Pyroptosis is a programmed cell death closely related to neuroinflammation and targeted inhibition of pyroptosis after spinal cord injury is a promising therapeutic strategy. OBJECTIVE:To summarize the molecular mechanism,positive and negative regulatory factors and therapeutic strategies of pyroptosis in spinal cord injury. METHODS:The search terms were"spinal cord injury,pyroptosis,nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),Caspase,Gasdermin D(GSDMD),IL-1β,IL-18"and 93 English literatures included in PubMed and Web of Science were finally selected for review. RESULTS AND CONCLUSION:As a newly discovered programmed cell death,pyroptosis has been shown to play an important role in the secondary injury stage after spinal cord injury.Among the regulatory factors of pyroptosis after spinal cord injury,CD73,NRF2,GDF-11,dopamine,FANCC and miR-423-5P could inhibit pyroptosis,while TLR4 and Aopps could promote pyroptosis.In terms of treatment,the active ingredients of traditional Chinese medicine(paeonol,tripterine,betulinic acid,piperine,kaempferol,and camptothecin),exosomes of various cell origins,and some drugs(metformin,topotecan,lithium,zinc,and carbon monoxide-releasing molecule 3)can effectively inhibit pyroptosis and reduce secondary spinal cord injury,but the toxicity and specific dose of these drugs need to be further studied.The specific molecular mechanism by which pyroptosis aggravates spinal cord injury is still poorly understood.The role of non-classical pathways and other inflammasomes is worth further exploration.At present,the research on pyroptosis after spinal cord injury only stays at the animal experiment stage.There are no related clinical studies and no approved targeted therapeutic drugs.(6)The application of pyroptosis after spinal cord injury has great potential,and its specific regulatory mechanism should be further studied in the future to provide a new target for the treatment of spinal cord injury.