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To investigate the effect of Semaphorin3A(Sema3A) on axon growth of primary retinal ganglion cells (RGCs) in mice.
C57/BL6 mice within 24 hours after birth were sacrificed and the eyeballs were removed, RGCs was isolated from retina and cultured in vitro.The primary cultured RGCs was identified by Brn3a immunofluorescence staining.Seven days after culture, the RGCs was divided into control group, 0.05 μg/ml Sema3A group, 0.10 μg/ml Sema3A group and 0.50 μg/ml Sema3A group, and the processing time was 2 hours.Immunofluorescence staining was used to detect the expression of neuron-specific marker β3-tubulin and dendritic marker MAP2, β3-tubulin+/MAP2– was identified as axons.The length of axons was measured by Image J software.The axon lengths of 0.10 μg/ml Sema3A group and control group at 0.5, 1 and 2 hours after treatment were calculated.The primary cultured cells were divided into control group, Sema3A treatment group, Y27632 treatment group and combined treatment group according to different drugs.The average axon lengths were compared among the groups.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology (ARVO).
Brn3a was positively expressed in primary cultured RGCs as a specific cell marker.Seven days after culture, RGCs tended to mature, with complete elongated neuronal processes and branches.The axon lengths of 0.05, 0.10 and 0.50 μg/ml Sema3A groups were (69.35±1.49), (60.45±1.42) and (93.65±1.86)μm, which were significantly shorter than (109.80±2.29)μm of the control group (all at P<0.01). The axon length of 0.10 μg/ml Sema3A group was (165.00±4.39)μm and (97.63±2.79)μm at 1 hour and 2 hours after treatment, respectively, which was significantly lower than (210.40±4.44)μm and (199.00±4.36)μm of control group at corresponding time points (both at P<0.01). There was a significant difference in the axon length of control group, Sema3A treatment group, mixed treatment group and Y27632 treatment group (F=142.50, P<0.01). The RGC axon length of Sema3A treatment group was significantly lower than that of control group and combined treatment group (both at P<0.01).
Sema3A can inhibit axon growth of primary retinal RGCs in mice, and ROCK signaling pathway inhibitors can alleviate such restrain effect.