Abstract
Purpose :
Previously, we identified increased levels of NOD-like receptor protein 3 (Nlrp3) inflammasome-associated genes in the retina of R345W Efemp1 mice (a model of Doyne Honeycomb Retinal Dystrophy). We hypothesized that genetically eliminating this pathway could protect against the main ocular phenotype of this AMD-like model, basal laminal deposits (BLamDs).
Methods :
We crossed Nlrp3-/- and caspase 1 (Casp1)-/- mice with R345W+/+ Efemp1 mice (all C57BL/6 background), followed by aging cohorts up to 12 mo. At select ages, mice were primarily evaluated using transmission electron microscopy (TEM) to access BLamD accumulation (6 per group, 3M/3F). Mice were anesthetized (ketamine/xylazine) and transcardial perfused (4% PFA) prior to enucleation and TEM processing. Eyes were regionally marked (to distinguish sample orientation), fixed in Karnovsky’s buffer, and washed in 0.1M sodium cacodylate. Eye cups were incubated in 1% OsO4 buffer, en bloc-stained with uranyl acetate followed by ethanol washes, resin infiltration, and Epon embedding. Samples were then post-stained with uranyl acetate/lead citrate. TEM samples were imaged (JEOL 1400+, 2,500x mag) along the Bruch’s membrane every 6 fields of view (20 fields of view/eye). ImageJ was then used to manually outline the deposit area per field of view, summed, and subjected to pairwise treatment comparisons by t-test.
Results :
Nlrp3-/- and Nlrp3-/-R345W+/+ mice formed significantly fewer BLamDs than R345W+/+ mice (p = 0.0001, p = 0.0147). Nlrp3-/- mice also demonstrated lower average BLamD thickness than wild-type mice, albeit not to a significant extent (1.951µm2 v. 2.057 µm2/FOV). Surprisingly, mice deficient in Casp1 (downstream of Nlrp3) showed significantly increased BLamD formation (both Casp1-/- and Casp1-/-R345W+/+ compared to their respective controls, p = 0.0006, p = 0.0482). Large BLamDs were mainly observed in female mice. Yet, ERG, OCT, and histology revealed that genetic removal of Nlrp3 and Casp1 were generally well-tolerated in the mouse eye.
Conclusions :
Our data reinforce the idea that genetically or pharmacologically targeting Nlrp3 may protect against AMD-related pathophenomena, including BLamDs. However, caution must be exercised in such approaches since broadly inhibiting Casp1 activity may exacerbate these same harbingers of disease.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.