A Study Of Drug-loaded Supramolecular Hydrogel In The Treatment Of Spinal Cord Injury

【Objective】 Spinal cord injury(SCI)often leads to the loss of sensory motor function of the limbs below the injury stage,which seriously affects the quality patients’ life.As the leading role of secondary injury,inflammation is the main cause of neurological deterioration after SCI.Therefore,regulating the local inflammation of the injury and reducing its harmful effects may be beneficial to the repairment of SCI.Non-steroidal anti-inflammatory drugs(NSAIDs)can reduce the production of inflammatory factors by suppressing cyclooxygenase,and are widely used in the treatment of various inflammation-related diseases.In the treatment of SCI,NSAIDs,through suppressing Rho A pathway,can reduce neuronal apoptosis,promote axon regeneration,and ultimately improve neural function after SCI.Naproxen(Npx),as a classic NSAIDs,can improve the motor function of SCI mice after oral administration,and can also reduce the neurodegeneration after SCI in rats by inhibiting Rho A pathway.Although Npx has a certain effect on SCI repair,the efficacy is weak,which may be related to its low selectivity for COX-2 and the low drug concentration in the injury site.By linking naproxen with D-configuration amino acids,it can self-assemble into a supramolecular hydrogel in water,which can increase the selectivity for COX-2 by 20 times,and can stably and durably release bioactive gel factors in local application.This study will explore the effects of local application of drug-loaded supramolecular hydrogel on the inflammatory response,macrophage polarization state at spinal cord epicenter,and neurological function improvement after SCI.【Methods】 1.Animals: 16-week-old Wister rats weighing about 220 grams were randomly divided into 4 groups: control(sham)group,injury(Injury)group,naproxen(Npx)group,Drug-loaded supramolecular hydrogel(Nano-Naproxen,Nano-Npx)group,8 rats in each group.2.Animal model: Sham rats only received T10 laminectomy.The other three groups of animals were established a spinal cord contusion model with IMPACTOR MODEL- impactor.The mass of the impact hammer is 10 g and the impact height is 25 mm.3.Intervention: After successful modeling,the same volume of drug-loaded supramolecular hydrogel,naproxen or saline was applied to the epidural of the surgical site of the rats of the Nano-Npx group,Npx group,Injury group and Sham group.4.Functional and histopathological evaluation: Rats were assigned BBB scores at 1,7,14,21,and 28 days after spinal cord contusion;MRI imaging of the spinal cord of rats was performed at 4 weeks after SCI to evaluate the area of lesion area.After completing the MRI scan,rats in each group were sacrificed,and spinal cord tissue was obtained.The obtained tissues were stained with HE,LFB,and Nissl to evaluate the area of spinal cavity,the number of effective nerve fibers,and neurons.5.Evaluation of inflammation: Glial fibrillary acidic protein(GFAP),Neuronal core antigen(Neu N),CD68(macrophage-specific antigen),Ionized calcium binding adapter molecule-1(IBA-1),inducible nitric oxide synthase(i NOS),arginase-1(Arg-1),COX-2 were subjected to immunofluorescence staining,to evaluate the local inflammation and macrophage activation status of epicenter.【Results】 1.Behavioral evaluation: From 1 week after injury,the BBB scores of rats in NanoNpx group and Npx group were significantly higher than those in Injury group(P <0.001,P <0.05),and the scores of Nano-Npx group rats was significantly higher than the Npx group(P <0.01),and showed the same trend in the following three weeks.2.MRI evaluation: the high signal area of T2 of spinal tissue in Nano-Npx group and Npx group at week 4 after injury was significantly smaller than Injury group(P <0.001,P <0.05).In addition,the high signal area of T2 in Nano-Npx group was significantly smaller than that of the Npx group(P <0.05).3.Histopathological evaluation: HE and GFAP immunofluorescence staining results showed that,compared with the Injury group,drug-loaded supramolecular hydrogel can significantly reduce the cavity area and the formation of colloidal scar after SCI(P <0.05).The results of LFB and Nissl staining showed that the number of effective nerve fibers and the number of surviving neurons in Nano-Npx group were significantly higher than those in Npx group and Injury group(P <0.01,P <0.001).4.Repair mechanism: Compared with Npx group and Injury group,Nano-Npx significantly reduced the number of macrophages,microglia and reactive astrocytes(P <0.05)of the lesion core,and down-regulated the expression of COX-2(P <0.05)of each cell.In addition,Nano-Npx down-regulated the number of M1 and upregulated the number of M2 in the epicenter.【Conclusion】 1.This study finds that drug-loaded supramolecular hydrogels can reduce neural degeneration and improve motor function of spinal cord injured rats.2.Drug-loaded supramolecular hydrogels can reduce the number of activated microglia,macrophages and reactive astrocytes,down-regulate the expression of COX-2 of the inflammatory cells,and promote macrophages to M2 activation,inhibit its activation to M1,thereby reducing the inflammatory response and promoting the repair of injured spinal cord.3.This study provides experimental evidence for drug-loaded supramolecular hydrogels in the repair of the central nervous system and a new strategy for the treatment of spinal cord injury.