| Spinal cord injury(SCI),the most severe complication of a spinal injury,often results in serious neurological dysfunction in the body parts below the level of injury where the paralysis below the injury site is the most typical presentation.Apart from causing physical disability and psychological impact to the patient,SCI is also an economic burden to a family and society.Along with the rapid modern socioeconomic development,the incidence of spinal cord injury especially trauma in traffic accidents,engineering construction and natural disasters,has risen.Despite various basic and clinical researches on SCI worldwide,the effective way on prevention and treatment in spinal cord injury is still a major problem in today's medical field.Because of the low regenerative capacity of central nervous system,SCI poses a high risk of permanent paralysis.The pathophysiology of SCI is very complex.Axonal regeneration and functional recovery after spinal cord injury are closely related to neurotropic factors,myelin-related inhibitors and scar formation in post injury microenvironment.It mainly involves the primary injury and secondary injury mechanism of tissue damage.The secondary injury mechanisms mainly include inflammation,scar formation and axonal degeneration.However,scar formation is the main reason that hinders the axonal regeneration and hence affect the functional recovery.Two types of scar tissue are formed at the lesion site according to formation mechanism and time:glial scar and fibrotic scar.Glial scar consists of reactive astrocytes,reactive microglia and glial precursor cells.Fibrotic scar is formed by endothelial cells and fibroblasts of the meninges and capillaries in the lesion.After SCI,the local environment undergoes profound biochemical and cellular changes that affect neurons,oligodendrocytes and astrocytes.Early reactive astrogliosis involves the proliferation and hypertrophy of astrocytes,and eventually leads to glial scar formation,which is beneficial for axonal regeneration.Our previous experiments have confirmed that:in vitro experimental stimulation of astrocytes with TGF-?1 can transform them into reactive astrocytes,further hypertrophy and secretion of nerve growth factor which limits the inflammation and promotes nerve regeneration.About 7 days after injury,fibroblasts invade the lesion site from adjacent meninges and vasculature,forming fibrotic scar.The fibrotic scar acts as a mechanical and biological barrier to synaptic regeneration and functional recovery:active hypertrophy and secretion of extracellular matrix,including type IV collagen,fibronectin and laminin,forming a mechanical barrier that hinders synaptic growth;secretion of a variety of synaptic growth inhibitory molecules like NG2 proteoglycan,tenosin C,semaphorin 3A and EphB2,etc.forming a biological barrier that hinders functional recovery.14 days after injury,a glial limiting membrane which is often formed between the glial scar and the fibrotic scar,further prevent axonal regeneration and functional recovery.There are studies showed that suppressing the formation of fibrotic scar may allow better axonal regeneration following injury,however,none have good effect in functional recovery.Therefore,under the premise of not hindering the beneficial effects of early glial scars,we find a way to inhibit the fibroblasts activation and fibrosis development,subsequently inhibit the formation of fibrous scars after spinal cord injury.It reduces the adverse effects of fibrous scar formation,and this can be a potential method to promote the axonal regeneration.This project aims to inhibit fibrotic scar formation and provide the best environment for axonal regeneration on the basis of fully utilize the beneficial effects of early glial scar.By selecting different treatment times,we intend to validate the optimal treatment time window to provide an effective theoretical basis for the precise treatment of functional recovery after spinal cord injury.MicroRNAs(miRs)are endogenous single-stranded noncoding RNA molecules of 21 to 25 nt in length,that negatively regulate the function of the target protein by degrading the target gene mRNA or inhibiting its translation process.miRs bind to targets in different organisms in different ways and often interact with a variety of proteins.This complex regulatory network allow regulation of the expression of multiple genes through a single miR,or combination of several miRs can be used to fine regulate the expression of a gene.This property determines that miRs can simultaneously regulate a variety of target proteins,thereby regulating the functional changes of cells.Recent studies have revealed a role of microRNA-21(miR-21)in fibrotic scar formation in the heart,lungs,kidneys and liver.Our previous studies showed that miR-21 was highly expressed in the injury site in upward trend.Therefore,we speculate that miR-21 may be a new potential molecular target in fibrosis.We believe that miR-21 is an important regulator of fibrosis.By interfering the expression of miR-21 at different times,adverse effects of fibrous scar can be inhibited while the beneficial effects of glial scar are fully exerted,and this will be beneficial for nerve regeneration and functional recovery.However,further research is still required for its regulation mechanism and time node.In this regard,we propose the following problems to be solved:a)possibility to regulate the activation,differentiation,proliferation,secretion and other processes of fibroblasts by regulating the expression level of miR-21;b)achieve the optimal therapeutic effect and treatment node time by regulating the expression of miR-21 at different times after establishing a spinal cord injury model.We intend to establish a model of spinal cord injury and research on the miR-21 pathway regulation on fibroblast activation mechanisms,mechanism of fibrotic scar formation and the downstream functional targets by study the animal,tissue,cellular and molecular level.The successful construction of an animal model of spinal cord injury has important clinical significance for the treatment of spinal cord injury and is conducive to the in-depth exploration of the mechanism of spinal cord injury.The commonly used spinal cord injury models made in the laboratory are divided into three categories,which are crush injury or contusion models,complete transection and partial dissection models,ischemic injury models and chemical injury models.Currently,the modeling methods of spinal cord injury mice models mainly include the heavy weight falling method,the spinal cord compression method and the clamping method,among which the heavy weight falling method mainly includes the standard Allen's impact molding device and the precision impact molding device.Since Allen first used the principle of weight free fall to implement the SCl model by vertical hitting spinal cord in 1911,the resulting weight hitting spinal cord instrument has been updated gradually.Based on the above principles,this research group mainly adopts the animal model made by advanced modified standard Allen's impact molding device,which is simple in operation,less traumatic,easy to control,cheap and easy available experimental consumables,good consistency and compatibility,convenient in observation and evaluation,and is an ideal mouse model of spinal cord injury.In this laboratory,the animal model was made by using standard Allen's impact molding device,and the recovery of postoperative motor function of mice was evaluated by using BMS standard score.By using H&E staining and other methods,the effect of establishing the animal model of spinal cord injury in mice was analyzed.Results show that the damage of mice movement function is impaired,postoperative biopsy neuromorphic see controls are normal,and compared with the control group,SCI group mice the structural integrity of the spinal cord were seriously damaged,and there is an obvious organization oppression,in contusion and suggest we successfully build the C57/the BL6 mice animal model of spinal cord injury.This study mainly used mice models to evaluate the efficacy of microRNA-21(miR-21 KD)knockout in the treatment of acute thoracic spinal contusion.40 C57/BL6 mice were randomly divided into 4 groups:Sham operation group(Sham)mice without spinal contusion received surgical treatment;after spinal cord contusion,the spinal cord injury(SCI)group mice received no surgery.After the mice in the miR-21KD group experienced spinal cord injury,a single dose of miR-21KD virus vector(1× 107TU)was injected under the dura.Mice in the negative control group received subdural injection of the same amount of NC virus vector(1×107TU)after experienced spinal cord injury.The Basso Mouse Scale(BMS)was used to evaluate the hind limb motor function of the mice.The pathological changes of spinal cord tissues were observed by using rapid blue staining with hematoxylin-eosin and Luxol.Enzyme linked immunosorbent assay(ELISA)was used to determine the serum tumor necrosis factor(TNF-?),transformed growth factor(TGF-?)and IL-1? in peripheral blood.mRNA levels of BDNF were detected by RT-PCR.Compared to the SCI group,the knockout miR-21(miR-21 KD)group effectively improved the BMS score on the 14th day after injury treatment(p<0.01).The axonal regeneration and the neuronal morphology of spinal cord tissue in the miR-21 KD group displayed the most overt histologic signs of recovery at day 14 post-surgery.Compared to the others control group,the expression levels of TGF-?1?TNF-? and IL-1? in serum of the miR-21 inhibition group were significantly reduced,while the gene expression of BDNF was significantly upregulated after the knockout of miR-21(p<0.01).Recent studies have shown that miR-21 seems to play a certain regulatory role in the PTEN/PI3K/AKT signaling pathway during the process of fibrosis scar formation.Based on the above results,it is speculated that miR-21 may be an effective target for regulating scar formation and promoting neurological recovery after spinal cord injury.In order to explore whether the AKT pathway is involved in the process of fibrosis scar formation after spinal cord injury,we conducted a semi-quantitative detection of phosphorylation AKT activity and p-akt/total AKT ratio in different treated samples.The results showed that the phosphorylation level of AKT in spinal cord tissues was increased after injury,and the phosphorylation level of AKT was significantly decreased after miR-21 inhibition.This suggests that the AKT pathway may be at least involved in the post-traumatic response associated with miR-21 in SCI.The above data suggest that inhibition of miR-21 level after spinal cord injury can reduce inflammatory response and enhance motor function recovery.Further studies revealed the relationship between miR-21 and the AKT signaling pathway,the role of miR-21 in inflammatory cytokine changes and scar formation after spinal cord injury,and the feasibility of using miR-21 in related clinical treatment,thus providing a new therapeutic target for the treatment of spinal cord injury. |
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