[Protective effects of BIM knockdown on RGCs and its association with inflammation-related gene expression changes in...

Objective: To investigate the role of BCL-2-interacting mediator of cell death (BIM), a pro-apoptotic molecule, in retinal ganglion cell (RGC) injury after optic nerve crush (ONC), and to analyze its association with changes in the expression of inflammation-related genes. Methods: This was an experimental study. The study was conducted from January 2025 to December 2025. Healthy male mice aged 6-8 weeks were randomly divided into four groups: control, ONC, AAV2-scramble, and AAV2-shBim, with 6 mice in each group. The control group received no intervention, the ONC group underwent ONC only, the AAV2-scramble group received intravitreal injection of AAV2-mediated scrambled negative control sequence after ONC, and the AAV2-shBim group received intravitreal injection of AAV2-mediated short hairpin RNA targeting the BIM gene after ONC. Immunohistochemical staining was used to detect the protein expression of Bim, complement component 3 (C3), and lipocalin 2 (Lcn2). Hematoxylin-eosin (HE) staining was used to observe retinal structural changes. Retinal flat-mount immunofluorescence staining was used to assess RGC survival. Optical coherence tomography (OCT) was used to measure ganglion cell complex (GCC) thickness. Flash visual evoked potential (F-VEP) and flash electroretinography (F-ERG) were used to evaluate visual electrophysiological function. RNA sequencing was performed to analyze retinal transcriptomic changes after BIM knockdown. Quantitative real-time PCR (qPCR) was used to validate inflammation-related differentially expressed genes. Independent-sample t-test and one-way analysis of variance were used for statistical analysis. Results: The proportions of Bim-positive RGCs in the peripheral and central retina were 0.56±0.06 and 0.63±0.06 in the ONC group, respectively, both of which were higher than those in the control group (0.00±0.00) (t=21.60, 24.61; both P<0.001). The numbers of RNA-binding protein with multiple splicing(RBPMS)-positive RGCs in the peripheral and central retina were 74.2±4.4 and 118.5±8.0 in the ONC group, respectively, both of which were lower than those in the control group (222.7±6.0 and 325.0±6.5, respectively) (t=48.94, 48.88; both P<0.001). Significant differences were observed among the four groups in ganglion cell complex thickness, the number of TUJ1-positive RGCs, F-VEP N2-P2 amplitude, and F-ERG b-wave amplitude (F=57.42, 1 216.78, 467.88, 423.76; all P<0.001). In the AAV2-shBim group, ganglion cell complex thickness, the number of TUJ1-positive RGCs, F-VEP N2-P2 amplitude, and F-ERG b-wave amplitude were 54.64±2.61 μm, 242.8±13.1, 11.13±0.80 μV, and 318.00±25.14 μV, respectively, all of which were higher than those in the ONC group [(44.29±1.95) μm, 140.0±5.3, (3.43±0.48) μV, and (190.68±25.50) μV, respectively] (all P<0.001). RNA sequencing showed that the expression levels of the inflammation-related genes C3, CCL12, LCN2, S100A9, CCL6, and ANGPTL4 were lower in the AAV2-shBim group than in the ONC group (t=10.21, 12.02, 8.98, 12.19, 7.33, 9.41; all P<0.001), and the quantitative polymerase chain reaction results were consistent with the RNA sequencing results. The C3-positive cell rates in the central and peripheral retina were 0.11±0.04 and 0.08±0.02 in the AAV2-shBim group, respectively, both of which were lower than those in the ONC group (0.64±0.06 and 0.57±0.05, respectively) (t=18.63, 21.04; both P<0.001). The Lcn2-positive cell rates in the central and peripheral retina were 0.08±0.03 and 0.09±0.02 in the AAV2-shBim group, respectively, both of which were lower than those in the ONC group (0.55±0.06 and 0.48±0.07, respectively) (t=17.19, 12.38; both P<0.001). Conclusions: BIM expression is upregulated after ONC. AAV2-mediated BIM knockdown alleviates RGC loss, retinal structural damage, and visual electrophysiological dysfunction, accompanied by downregulation of inflammation-related gene expression.