| BackgroundAs an important neurological disorder characterized by acute onset and high rate of disability,cauda equina syndrome(CES)occur more frequently in patients with lumbar spine disease with serious damage.Because fo its special anatomical structure,cauda euqina is vulnerable to mechanical compression injury.However,once the CES patients had saddle region sensory disturbance,intestinal and sexual dysfunction,their prognosis of surgical treatment is very poor.The majority of them had residual bladder or anal sphincter dysfunction.Considering the surgical treatment and drug treatment can only relieve part of the injury with poor prognosis,it is urgent to be an effective treatment to deal with CES and improve the prognosis.Histone deacetylases(HDAC)and histone deacetylases(HAT)regulate histone acetylation levels in nucleosomes.HDAC has been widely used in clinic because of its close relationship with gene transcription,expression,inhibition and cell proliferation,differentiation.At the same time,with the deep research of HDAC and HDAC i,it is shown that HDAC i has therapeutic effect on infectious diseases and central nervous system diseases.Previous studies have demonstrated the neuroprotective effects of histone deacetylase inhibitors(HDAC i),which affect synaptic activity and neuronal function.However,there is no report about the application of HDAC i in the cauda equina injury.We constructed and improved cauda equina injury model in rats in this research on the basic of past study.Then HDAC i was injected into acute cauda equina injury model in rats by subcutaneous,in order to restore the balance between histone acetylation and deacetylation and regulate gene transcriptional regulation disorder,so as to achieve the purpose of protecting the function of cauda equine.Finally,RN A-seq was applicated to research the changes of transcription and expression of genes after the treatment of VPA in cauda euqina injury.Through the analysis of the data,we wonder explore its deeper molecular mechanism,hope to find targets for HDAC i protective effect on cauda equina injury.ObjectiveThe purpose of this research was to establish and improve the cauda equina injury model in rats.In addition,it is confirmed that the protective effect of HDAC i on acute cauda equina injury in rats.RNA-seq method to detect the changes of transcription and expression of genes after the treatment of HDAC i on acute cauda equine injury.Through the analysis of the data,we explore its deeper molecular mechanism,provide experimental basis for further study of the mechanism of cauda equina injury and find the new research directions on the treatment of cauda equina syndrome in future.Methods1.Select about 20,age: six weeks,weighing 250 grams experimental SD rats.The rats were randomly divided into three groups: operation group,n = 8;sham group,n = 6;control group,n = 6.Each rat bite at 4 lumbar spine vertebrae,in addition to the bite site with an electric drill drilling a 1.2mm hole,drill through the lamina.A piece of silicone block(length:12mm,width:1-1.5mm)was implanted into the distal end o f the spinal canal through the lamina,and the L5 and L6 segments of cauda equina were depressed.In sham operation group,we drilled the L4 lamina without implanting a piece of silicone block.After 3 days from the establishing of CES model,BBB score and oblique board test were used to test the neurobehavioral performances.After setting the time point,HE staining,LFB staining,TUN EL staining and immunofluorescence staining were used to determine the histopathological changes of each experimental group,determined to build a successful model.2.Select about 30,age: six weeks,weighing 250 grams experimental SD rats.The rats were randomly divided into three groups: VPA group,n = 10;control group,n =10;sham group,n = 10.The rats of VPA group and control group were drilled a hole with an electric drill and implanted a piece of silicone block.While the rats of sham group were drilled a hole without implanted.Injection of VPA was given 2 h before surgery.The dosage of VPA was 300 mg/kg(dissolved in saline).After surgery,the VPA group received the injection of VPA(300mg/kg)twice a day for a week.For the control group,same volume of saline was injected.Neurobehavioral tests were measured for a week from 2 h before surgery to 7t h d after surgery.At 7t h d after surgery,the tissue from cauda equina underwent the HE staining,LFB staining,TUN EL staining and immunofluorescence staining,the tissue from DRG underwent the TUN EL staining and the tissue from conus medullaris underwent the TUN EL staining.3.With the help of high-throughput sequencing technology,RNA-Seq was used to detect gene expression differences of the corresponding segments of cauda equina tissue from VPA group and control group.Through the analysis of the data,we found preliminarily the key molecules and signaling pathway which was highly correlated with the mechanism of HDAC i protection on cauda equina injury.Results1.Establish and improve the cauda equina injury model in rats successfully.2.In this study,the results of neurological test about BBB score and oblique board test suggest that lower limb motor function in the VPA group were significantly improved compared with the control after VPA implantation.Futhermore,the histopathological results showed that nerve fibers of Wallerian degeneration and demyelinating lesions reduced significantly in VPA group,while the VPA group own increased myelination compared with the control group by immunofluorescence staining.TUNEL staining showed that the number of apoptotic cells in the spinal cord anterior horn neurons and DRG neurons was significantly decreased in the VPA group.3.Through the analysis and preliminary validation on differential gene of rat cauda equina injury after application of HDAC i,we screened the differently expressed genes such as HDAC11,IL18,FGF1,FGF5,which the expression of HDAC11 in VPA was high,the expression of IL18 was decreased after VPA,the expression of FGF1 was high in the presence of VPA,and the expression of FGF5 decreased in the presence of VPA.Through the KEGG Pathway enrichment analysis,we found the following highly relevant signaling pathways: MAPK-JNK,p38 MAPK,calcium channel,GRB2-Ras-MAPK-Erk,Neuroactive Ligand-Receptor Interaction.ConclusionIn this experiment,the rat model of cauda equina injury is reasonable,easy to operate and reproducible.It can be used to simulate the pathogenesis of cauda equina injury and to study the pathogenic process of lumbar disc herniation.We confirmed the protective effect of HDAC i on the treatment of cauda equina injury model in rats.It can improve nerve motor dysfunction,Masson reduce neuronal apoptosis and promote nerve regenerat ion after injury.Therefore,HDAC i is a kind of drug therapy with great potential to improve the prognosis of patients.Through bioinformat ics analys is by RN A-seq and preliminary validation,combined with K EGG Pathway enrichment ana lys is,we screened the key molecules and signaling pathway which was highly correlated with the mechanism of HDAC i protection on cauda equina injury: HDAC11,IL18,FGF1,FGF5,MAPK-JNK,p38 MAPK,calcium C hanel,GRB2-Ras-MAPK-Erk,Neuroactive Ligand-Receptor Interaction.These findings may help us to understand the mechanism of the cauda equina injury and the protective mechanism of HDAC i in the treatment of cauda equina injury.At the same time,these will be our next step in the direction of indepth research,we hope to find the effective treatment target on cauda equina injury,develop the precise molecular therapy to benefit the majority of patients with cauda equina syndrome. |
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