Induced ablation of scleral TGF-β signaling in mutant mice increases susceptibility to IOP-induced optic nerve damage

Abstract

Purpose : Axonal optic nerve (ON) damage in glaucoma is characteristically associated with increased amounts of active Transforming Growth Factor-β2 (TGF-β). Data from Quigley et al., (2015) indicate that TGF-β-signaling promotes IOP-induced ON damage, an effect that involves remodeling of the sclera. In the present study we investigated the functional role of scleral TGF-β-signaling in glaucoma. To this end we induced ocular hypertension (OHT) in mice with an inducible deficiency of TGF-β receptor type II (Tgfbr2) in scleral fibroblasts.

Methods : Tgfbr2^fl/fl (T2^fl/f) mice with a floxed allele of Tgfbr2 were crossed with Col1a2-CreER (Col1) mice. The mouse Col1a2 promoter is specifically active in fibroblasts, including those of the sclera. 14 days after Tamoxifen (TX) treatment OHT was induced in one eye, using the magnetic-microbead-model. IOP was measured by rebound tonometry. After six weeks of OHT, ON axons were PPD-stained and quantified. Somata of retinal ganglion cells (RGC) were quantified on whole mounts by RBPMS-staining. TGFBR2 and its mRNA were analyzed by western blotting and real time RT-PCR. IOP data was analyzed using one-way ANOVA, mRNA expression, protein synthesis, axon loss (Δaxons) and RGC loss (ΔRGC) were compared using two-tailed students t-tests.

Results : Following TX-treatment, scleral TGF-β RII mRNA and protein were significantly decreased (p ≤ 0.05). IOP was significantly elevated up to six weeks after injection. No difference in IOP of injected and un-injected eyes could be found between T2^fl/fl and Col1xT2^fl/fl animals at any timepoint. After six weeks of OHT, reduced numbers of ON axons (11±2%, n=17, p≤0.001) were seen in OHT eyes in comparison to contralateral eyes. Moreover, OHT also led to a decrease of retinal ganglion cell somata (22,87±9,87%; n = 10; p ≤ 0.001). Axon loss was significantly higher in mice with a fibroblast specific deficiency of TGF-β RII in comparison to control animals (Δaxons T2^fl/fl = 9.29±5.76%; n=9; Δaxons Col1xT2^fl/fl = 14.89±4.85%; n=8; p≤0.01). ΔRGC/mm² was not changed in Col1xT2^fl/fl mice (ΔRGC/mm² T2^fl/fl = 22.21±6.49%; n=5; ΔRGC/mm² Col1xT2^fl/fl = 22.37±15.09%; n=5; p=0.88).

Conclusions : We conclude that the ablation of scleral TGF-β signaling increases the susceptibility to IOP-induced optic nerve damage. Scleral TGF-β signaling in mutant mice appears to be beneficial for ON axon survival in experimentally induced OHT.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.