| Objective:To establish a radiation-injured model of lacrimal gland in mice with various doses of X-ray radiation on orbital region and explore its mechanism of pathophysiology. Further explore whether Gadd45a play a role in the repairment of radiation-injured lacrimal gland and its associated signaling pathways.Method:The first part:Fifty healthy female C57BL/6 mice were randomly divided into five groups:one control group and four experimental groups. The irradiated mice (n= 40) were exposed to dosages of 12,15,18 and 21Gy, respectively; the sham-irradiated controls (n= 10) were anesthetized in parallel with the irradiated rats but not irradiated. The mice were placed laterally and covered with a bolus material, then the X-ray irradiation was performed under general anaesthesia from above towards the mice’ orbital region. The schirmer I/II tests, Single-photon emission computed tomography/Computed tomography (SPECT/CT), HE, immunohistological, and ultrastructural examinations were conducted prior and 3days,7days as well as 30days after irradiation with various dosages. The second part:ten healthy female C57BL/6 mice were randomly divided into two groups:the control group and experimental group. The control group was not irradiated. The experimental group were under general anesthesia with irradiation of 15Gy.3 days after irradiation,the left lacrimal gland were surgical removed and used for Quantitative PCR、 Immunohistochemistry、scratch-wound healing assay、Cell proliferation assay、 Western blot analysis to observe the expression level of Gadd45a in radiation-injured lacrimal gland tissue.7 days after irradiation,the right lacrimal gland were also removed and subjected to the same test procedure.Results:At a total dose of 15Gy, the lacrimal gland acinar cells decreased after 3days. Schirmer I test showed significantly reduced lachrymal secretion and the difference was statistically significant. Disturbance in the tracer uptake as well as reduction of the lacrimal ejection fraction was assessed under SPET/CT test. Immunohistochemical findings include a significant loss in the expression of α-SMA, aquaporin (AQP-5), and an excessive expression of Tenascin-C and CK8, which was statistically significant. Ultrastructural changes including a retention of secretory granules and an increase of apoptotic acinar nuclei as well as macrophage phagocytosis. This radiation dosage didn’t cause obvious eye complications (cataract, radiation retinopathy, etc.). At the dosages of 18 and 21Gy, the lacrimal gland inflammation and tissue apoptosis expand obviously. At a dose of 12Gy, there is no statistically significant decline in tear secretion. These changes were still evident 30days after irradiation but less intensive. Growth arrest and DNA damage-inducible 45 a (Gadd45a) was observed overexpressed in the irradiated lacrimal gland. Moreover, overexpressed Gadd45a may impair lacrimal gland repair by inhibiting lacrimal gland epithelial cell migration and proliferation. Further signalling pathway analyses indicated that Gadd45a overexpression suppresses Akt (protein kinase B, PKB), P38 and JNK phosphorylation.Conclusions:The model of radiation-injured lacrimal gland was successfully constructed. Pathophysiological manifest the impaired structures of lacrimal gland cells and infiltration of inflammatory cells, as well as damaged function of tear secretion. These changes may prerequisites for further study on radio protection of lacrimal glands during radiotherapy of the peri orbital region for orbital tumors. Gadd45a may be a therapeutic target in radiation-induced lacrimal gland injury. |
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