Molecular Pathological Mechanism Of Spinal Cord Injury Based On Differentiation Subtypes Of Fibroblasts

Spinal cord injury(SCI)is considered life-changing and catastrophic,often resulting in permanent impairment of motor and sensory functions.At present,conventional therapeutic strategies have been used in clinical practice for many years,most of which are focused on basic life support,and most of these therapeutic strategies aim to remove the factors that may cause further deterioration of SCI.Most treatments do not intervene in the pathophysiology of SCI,or do intervene in some pathophysiology,but fail to identify the optimal time window for treating,resulting in unstable efficacy(e.g.,Methylprednisolone(MP)).To date,there has been no effective treatment for SCI-induced neurological deficits in clinical practice,largely due to the incomplete understanding of the molecular pathogenesis of SCI.This is directly related to the unclear regulation mechanism of the local and systemic molecular pathological changes after SCI.In order to answer the above questions,we performed spatial transcriptome sequencing and single cell RNA sequencing(sc RNA-seq)containing time-series information on the spinal cord tissue of the SCI mouse model(Contusion model),drawing the first single-cell altas of SCI at the molecular pathology resolution and spatio-temporal level.In SCI samples collected at 7 dpi,fibroblasts accumulated in the center of the lesion site and formed fibrotic scar core(FSC),surrounded by astrocytes and macrophages,which are the major components of glial scars.In addition,a large number of microglia,oligodendrocytes,neural precursor cells(NPCs)and oligodendrocyte progenitor cells(OPCs)were recruited to the outside of the FSC.In 14 dpi SCI samples,fibroblasts still occupied the center position of the FSC,and there appeared to be more microglia and macrophage infiltration into the interior of the FSC than was observed in the 7 dpi SCI samples.Therefore,fibroblasts may be the hub of cellular communication in SCI,sending continuous communication signals to other neurocytes and immune cells,and inducing other cells to express high levels of fibroblast marker genes(e.g.Col1a2),which further facilitate the molecular pathological process of SCI.In order to further explore the potential regulatory mechanism of fibroblasts and their related molecular markers,evaluating their value of translational medicine,we have used the sc RNA-seq data of the spinal cord tissue of the SCI animal model and RNA-seq data of the peripheral blood mononuclear cells(PBMCs)from SCI patients with clinical prognosis information and conducted multi-omics analysis.The analysis results show that the expression of fibroblasts and their related molecular markers are significantly associated with the prognosis of SCI animal models and the clinical grade SCI patients.More importantly,based on genes related to different differentiation subtypes of fibroblasts,we firstly proposed Fibroblast Differentiation Based SCI Classification(FDBSC)based on the differentiation status of fibroblasts.FDBSC has three subtypes including MFSC(Median Fibroblast Score Classification),LFSC(Low Fibroblast Score Classification)and HFSC(High Fibroblast Score Classification).Among them,MFSC has the best prognosis.The marker genes of MFSC are mainly involved in angiogenesis,matrix remodeling,and inflammation response.The fibroblast infiltration MFSC is moderate,between LFSC and HFSC.We speculate that moderate inhibition of fibroblast infiltration and infiltration may promote the prognostic recovery of SCI while extremely high or low levels of fibroblast infiltration are not conducive to the prognostic recovery of SCI.In addition,we also predicted targeted drugs for the key regulatory molecules of MFSC,LFSC and HFSC.FDBSC is the first molecular classification proposed in the field of clinical treatment of SCI,which is significantly related to the prognosis of SCI patients,providing a theoretical basis for the precise treatment of SCI patients.Finally,in order to explore the translational medicine value of fibroblasts in the treatment of SCI,we also treat SCI mice with MP,which has been widely reported to inhibit fibroblast growth and commonly used in the clinical treatment of SCI but has unstable efficacy.The results show that MP treatment can not only effectively inhibit the cellular communication ability of fibroblasts,but also attenuat the reprogram ability of fibroblasts.At the same time,MP can also enhance biological processes of angiogenesis and extracellular matrix remodeling.It was preliminarily confirmed that MP intervention in mice at 6dpi can obtain obvious effects.These results provide a theoretical supplement for MP treatment of SCI at the molecular pathological mechanism level(that is,one of the molecular biological mechanisms of MP for treatment of SCI may include the transdifferentiation of fibroblasts from HFSC subtype to MFSC subtype.And the effects of MP intervention at 6dpi was more obvious.).Based on the multi-omics data integration technology,this study carried out a detailed exploration of the molecular pathology mechanism of SCI,drawing the first single-cell altas of SCI at the molecular pathology resolution and spatio-temporal level.Our analysis indicated that fibroblasts served as the hub of intracellular communication in the SCI site,sent continuous immune signals to neurocytes,and induced the other cell types to express high levels of the fibroblast marker gene.Furthermore,when the proportion of fibroblasts and their cellular communications were attenuated by MP treatment,the BMS and MEP performance of SCI mice increased significantly;moreover,the ratio of Col1a2+ cells decreased markedly,and a new subtype of fibroblasts expressing Arg1 appeared.Our results highlight the vital pathological position of fibroblasts and indicate it as a significant therapeutic target for SCI.Additionally,based on genes related to differentiation subtypes of fibroblasts,we propose the first molecular classification of SCI named FDBSC,which has good clinical risk identification capability and prognostic prediction value and provide a reference for clinicians to make decisions.However,translational medicine value of FDBSC still needs further clinical trials to evaluate and determine.