The hypoxia marker CA-IX correlates with segmental outflow in the trabecular meshwork of mice

Abstract

Purpose : The anterior segment of the human eye is hypoxic, with an oxygen tension of 12 mmHg (Siegfried CJ, et al. IOVS, 2010). Hypoxia induces the expression of several genes, one of which is carbonic anhydrase 9 (CA-IX) that is commonly used as hypoxia marker. Previously, we have shown that segmental outflow patterns in the trabecular meshwork (TM) redistribute over time in mice (Reina-Torres E, et al. EER, 2022). We hypothesize that this redistribution is related to local hypoxia in the TM. To test this hypothesis, we examined the spatial co-localisation between CA-IX expression and segmental outflow in mouse eyes.

Methods : Segmental outflow patterns were labelled in the TM of live C57BL/6J mice (N=2 eyes from 2 mice; 9wks) by intracameral injection of fluorescent tracer microparticles (0.2µm; 10¹¹/ml) under anaesthesia. After 6 hours, eyes were enucleated and fixed overnight in 4% PFA. Anterior segments were dissected and immunolabelled for CA-IX. Sagittal 100µm cryosections through the iridocorneal angle were imaged by confocal microscopy (N=40 & 47 sections/eye). From each section, 15 consecutive confocal slices (0.5µm; 40x) were selected for analysis. Based on tracer fluorescence, we defined a 2D polygon in the TM and projected this polygon through the z-slices to create a 3D mask. We calculated the mean intensity values of tracer and CA-IX immunolabelling within the mask and performed two analyses. First, we examined the linear correlation between tracer and CA-IX intensity using ANCOVA. Second, we compared the CA-IX labelling intensity between sections corresponding to the upper, middle and lower thirds of tracer intensity using one-way ANOVA.

Results : CA-IX and tracer intensities were positively correlated for both eyes (r=0.66 & 0.70, p<0.001). The intensity of CA-IX immunolabelling was significantly different between high, medium and low-flow regions of the TM, with regions of higher tracer exhibiting greater CA-IX labelling (p<0.005, both eyes).

Conclusions : CA-IX expression co-localises with regions of active segmental outflow in the TM. This suggests that hypoxia-inducible genes may be involved in segmental outflow regulation and that oxygen tension within aqueous humour may be important for normal outflow function.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Correlations between tracer and CA-IX intensities. Shading represents 95% CI on linear fits.