IOP-induced blood-retinal barrier compromise contributes to RGC death in glaucoma

The integrity of the blood-retinal barrier (BRB) has been largely unexplored in glaucoma. We reveal that elevated intraocular pressure (IOP) partially compromises the BRB in two human-relevant inherited mouse models of glaucoma (DBA/2J and Lmx1bV265D). Experimentally increasing IOP in mouse eyes further confirms this. Notably, the compromise induces subtle leakage, happening without bleeding or detected endothelial cell junction disruption, and it precedes neurodegeneration. Leakage occurs from peripheral veins in the retinal ganglion cell layer with a concomitant loss of the transcytosis inhibitor MFSD2A. Supporting this, bulk RNA sequencing of retinal veins revealed upregulated vesicular transport and downregulated Wnt signaling in hypertensive versus control veins. Transmission electron microscopy confirmed intact tight junctions in D2 glaucoma mice, while increased vesicles suggest enhanced transcytosis. In human glaucoma eyes, albumin immunostaining showed BRB leakage in retinal veins, mirroring our mouse findings. Importantly, stabilizing ß-catenin in retinal endothelial cells prevents both vascular leakage and neurodegeneration in the DBA/2J model. The occurrence of leakage in all 3 high IOP models and in human glaucoma indicates that BRB compromise may be a common, yet overlooked, mechanism in glaucoma. These findings suggest that IOP-induced BRB compromise plays a critical role in glaucoma, offering a new therapeutic target. Overall design: 9 months old DBA/2J and DBA/2J-Gpnmb+/SjJ were intraperitoneally injected with 0.33 mg/g body weight sodium fluorescein (AK-FLUOR, Akorn). 5 minutes after injection, mice were euthanized by cervical dislocation, eyes enucleated and retinal veins were dissected using super fine forceps (Dumont, NC9497215) under the fluorescent dissection scope. 4 major veins from the same mouse, including main branches were combined as one sample. Total RNA was extracted using RNeasy Micro kit (Qiagen, 74004). RNA concentration and quality (RIN) was measured by Bioanalyzer. About 100 pg total RNA (RIN>9) were reverse transcribed to cDNA using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara). The library was prepared using Nextera XT (Illumina) and sequenced by Aviti Element. Bulk RNA-seq data was aligned to GRCm38 and gencode M23 and quantified by Kallisto. Transcript quantifications were imported into R framework for downstream analysis.