Construction and evaluation of a modified stretch injury model of primary cultured neurons

LEI Jin; ZHANG Fu-chi; LI Yu; WAN Xue-yan; ZHAO Kai; NIU Hong-quan; SHU Kai; LEI Ting

doi:10.13798/j.issn.1009-153X.2023.09.010

Abstract：Objective To explore an improved method for establishing a primary neuronal stretch injury model and its feasibility.Methods Cortical tissues from SD rats within 24 hours of birth were extracted for primary neuronal culture.An improved method for the classic neuronal stretch injury model(CICⅡmodel)was developed using an intelligent vacuum negative pressure machine and elastic membrane culture plate.Cells were divided into control group(no stretch injury),mild injury group(deformation distance 7 mm),moderate injury group(deformation distance 10 mm),and severe injury group(deformation distance 13 mm)based on the elastic membrane deformation distance.Cell morphology changes were observed under a microscope,and NeuN immunofluorescent staining was used to identify cell purity.Apoptosis-related proteins(cleaved Caspase-3,Bcl-2)expression was detected by immunoblotting,and lactate dehydrogenase(LDH)activity in the culture medium was measured using a colorimetric assay.Results On the 5th day of cell culture,cells showed good growth under the microscope,with NeuN staining indicating neuronal purity above 95%.Immediately after moderate injury,axonal rupture and vacuolated cell morphology were observed under the microscope.Compared to the control group,the expression levels of cleaved Caspase-3 and LDH in the culture medium were significantly increased(P<0.05),while Bcl-2 expression was significantly decreased(P<0.05)in the injury group.The expression levels of cleaved Caspase-3 and LDH in the culture medium were positively correlated with the degree of injury(P<0.05),while Bcl-2 expression was negatively correlated with the degree of injury(P<0.05).Conclusions High purity of primary neurons was obtained from cultured cortical tissues of newborn rats.The improved model in this study can simulate neuronal injury,is convenient to use,and has good reproducibility,making it suitable for research on cranial injuries and cell mechanics.