The Mechanism Research Of Calcitonin Gene-related Peptide In Extraocular Muscular Atrophy

Objective:Strabismus is a common ophthalmic disease,which is an abnormal movement of the extraocular muscles caused by the functional imbalance of the nerves related to the movement of the ocular muscles,the pathogenesis is still unclear now.Calcitonin gene-related peptide(CGRP)is a neuropeptide released by motor neurons.But,its mechanism in extraocular muscle is still unknown.To investigate the relationship between the extraocular muscle and CGRP and find the possible mechanism of action by researching the pathology and protein expression in patients with concomitant exotropia and rat model construction.Methods:The study included patients from June 2019 to June 2020 in the first affiliated Hospital of Fujian Medical University.According to age divided into infant concomitant exotropia group(3～6 years old,including3 and 6)(N=11),children concomitant exotropia group(7～18 years of age,including 7,excluding18)(N=17),adult concomitant exotropia group(≥18 years old)(N=11).The tissue sections of surgical operation were stained to detect the expression levels of extraocular muscle fibrosis through Hematoxylin-Eosin staining and Masson staining.The neurofibrin marker protein S-100β was detected byimmunohistochemistry and the expression level of CGRP was detected by Western blot.After CGRP-KO rat model construction,HE staining,Masson staining and Sirus-Red staining were performed on the extraocular muscles of rats to detect the extraocular muscle atrophy and expression of the muscle function related index such as collagen III.Tunel test and Western blot were used to detect apoptosis levels in the extraocular muscles of rats.The phosphorylation level of AKT/CREB in the extraocular muscles of patients with concomitant exotropia was verified at the same time.Results:We found that the muscle area of the extraocular muscles in concomitant exotropia at all age groups were decreased than that in the control group through the HE staining and Masson staining.The results of immunohistochemistry showed that S-100β positive cells in the extraocular muscles of the patients with concomitant exotropia and the control group were mainly distributed in the muscle space,and the expression level was relatively low.S-100β expression level in concomitant exotropia group at all age groups were lower than that in control group,while the expression of CGRP increased through the Western blot.In CGRP-KO rats,it was further found that the atrophy of muscle.The muscle cross-sectional area and density of CGRP-KO extraocular muscles were lower than that of the control group in HE staining.Masson staining showed that the red muscle fibers of the extraocular muscles of CGRP-KO rats at all ages were significantly lower than those of the control group.Under light microscope,the muscle fibers in the extraocular muscles of the rats at all ages were decreased,meanwhile in the polarized light,the expression level of collagen III in the extraocular muscles of the rats were lower than the control group.The level of apoptosis in the extraocular muscles of CGRP-KO rats and patients were significantly higher than that in the control group(p<0.001)through the Western blot,and the phosphorylation level of AKT/CREB in the extraocular muscles of patients with concomitant exotropia was higher than that in the control group.Conclusion:1.Concomitant exotropia patients have obvious extraocular muscle atrophy in weak lateral and increased CGRP expression.2.CGRP knockout may cause atrophy of extraocular muscle in rat,which may be associated with the excessive activation of the apoptosis pathway in extraocular muscle cells.3.The apoptosis in patients with concomitant exotropia may be mediated through AKT/CREB pathways.