Endothelin receptor-A antagonist attenuates retinal vascular and neuroretinal pathology in diabetic mice.

PURPOSE: We sought to determine the effects of atrasentan, a selective endothelin-A receptor antagonist, on the retinal vascular and structural integrity in a db/db mouse, an animal model of type 2 diabetes and diabetic retinopathy. METHODS: Diabetic mice, 23 weeks old, were given either atrasentan or vehicle treatment in drinking water for 8 weeks. At the end of the treatment period, eyes underwent trypsin digest to assess the retinal vascular pathology focusing on capillary degeneration, endothelial cell, and pericyte loss. Paraffin-embedded retinal cross sections were used to evaluate retinal sublayer thickness both near the optic nerve and in the retinal periphery. Immunohistochemistry and TUNEL assay were done to evaluate retinal cellular and vascular apoptosis. RESULTS: Compared with untreated db/db mice, atrasentan treatment was able to ameliorate the retinal vascular pathology by reducing pericyte loss (29.2% ± 0.4% vs. 44.4% ± 2.0%, respectively, P < 0.05) and capillary degeneration as determined by the percentage of acellular capillaries (8.6% ± 0.3% vs. 3.3% ± 0.41%, respectively, P < 0.05). A reduction in inner retinal thinning both at the optic nerve and at the periphery in treated diabetic mice was also observed in db/db mice treated with atrasentan as compared with untreated db/db mice (P < 0.05). TUNEL assay suggested that atrasentan may decrease enhanced apoptosis in neuroretinal layers and vascular pericytes in the db/db mice. CONCLUSIONS: Endothelin-A receptor blockade using atrasentan significantly reduces the vascular and neuroretinal complications in diabetic mice. Endothelin-A receptor blockade is a promising therapeutic target in diabetic retinopathy.

A pilot quantitative study of topographic correlation between reticular pseudodrusen and the choroidal vasculature using en face optical coherence tomography.

PURPOSE: To analyze the topographic correlation between reticular pseudodrusen (RPD) visualized on infrared reflectance (IR) and choroidal vasculature using en-face volumetric spectral-domain optical coherence tomography (SD-OCT). METHODS: A masked observer marked individual RPD on IR images using ImageJ (NIH, Bethesda, MD). Using the macular volume scan (Cirrus, Carl Zeiss Meditec Inc, Dublin, CA), the RPE slab function was used to generate a C-scan of the most superficial choroidal vasculature. An independent masked grader created a topographic binary map of the choroidal vasculature by thresholding the en-face image, which was overlaid onto the IR map of RPD. For each IR image, ImageJ was used to generate a random set of dots as "control lesions". RESULTS: 17 eyes of 11 patients (78±13.7 years) with RPD were analyzed. The average number of RPD lesions identified on IR images was 414±71.5, of which 49.6±4.3% were located overlying the choroidal vasculature, compared to 45.4±4.0% in controls (p = 0.014). 50.4±4.3% of lesions overlay the choroidal stroma, of which 76.5±3.1% were ≤3 pixels from the choroidal vessels. The percentage of RPD lesions located within ≤3 pixels from the choroidal vasculature was significantly greater than the percentage located ≥7 pixels away. (p<0.0001). Compared to controls (71.6±3.8%), RPD were more likely to be located ≤3 pixels away from choroidal vessels (p = 0.014). In contrast, control lesions were more likely to be ≥7 pixels away from choroidal vessels than RPD (9.1±1.9% vs. 4.8±1.2%, respectively, p = 0.002). CONCLUSIONS: Our analysis shows that RPD lesions follow the underlying choroidal vasculature. Approximately half the RPD directly overlay the choroidal vessels and the majority of the remaining lesions were ≤3 pixels (≤30 microns) from the vessel edge, supporting the hypothesis that RPD maybe related to pathologic changes at the choroidal level.

Sildenafil attenuates vaso-obliteration and neovascularization in a mouse model of retinopathy of prematurity.

PURPOSE: We sought to determine the effect of sildenafil on retinal vascular changes in a mouse model of oxygen-induced retinopathy (OIR). METHODS: Vascular defects in OIR mice were quantified by measuring vaso-obliteration at postnatal days 12 and 17 (P12 and P17) and neovascularization at P17 to compare sildenafil-treated to dextrose-treated OIR mice. Retinal HIF1α protein expression was quantified by Western blotting and normalized to that of ß-actin. Right ventricular hypertrophy was measured by Fulton's index as a surrogate for hyperoxia-induced pulmonary hypertension. RESULTS: At P12, OIR mice treated with sildenafil demonstrated a 24% reduction in vaso-obliteration (P < 0.05), whereas at P17, treated animals showed a 50% reduction in neovascularization (P < 0.05) compared to dextrose-treated controls. Sildenafil-treated OIR mice had stabilization of retinal HIF1α at P12, immediately after hyperoxia. At P17, sildenafil-treated OIR mice had decreased HIF1α relative to untreated mice. OIR mice developed right ventricle hypertrophy that was significant compared to that in room air controls, which was abrogated by sildenafil. CONCLUSIONS: Sildenafil treatment significantly decreased retinal vaso-obliteration and neovascularization in a mouse OIR model. These effects are likely due to sildenafil-induced HIF1α stabilization during hyperoxia exposure. Furthermore, we confirm disease overlap by showing that OIR mice also develop hyperoxia-induced right ventricular hypertrophy, which is prevented by sildenafil. This study is a first step toward delineating a potential therapeutic role for sildenafil in OIR and further suggests that there may be common pathophysiologic mechanisms underlying hyperoxia-induced retinal and pulmonary vascular disease.

Trypsin digest protocol to analyze the retinal vasculature of a mouse model.

Trypsin digest is the gold standard method to analyze the retinal vasculature (1-5). It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations (6,7). In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.

Hyperinduction of host beta interferon by a Listeria monocytogenes strain naturally overexpressing the multidrug efflux pump MdrT.

Many pathogens regulate or modify their immune-stimulating ligands to avoid detection by their infected hosts. Listeria monocytogenes, a facultative intracellular bacterial pathogen, interacts with multiple components of mammalian innate immunity during its infection cycle. During replication within the cytosol of infected cells, L. monocytogenes utilizes two multidrug efflux pumps, MdrM and MdrT, to secrete the small nucleic acid second messenger cyclic-di-AMP (c-di-AMP). Host recognition of c-di-AMP triggers the production of type I interferons, including beta interferon (IFN-ß), which, surprisingly, promote L. monocytogenes virulence. In this study, we have examined the capacity of multiple laboratory and clinical isolates of L. monocytogenes to stimulate host production of IFN-ß. We have identified the L. monocytogenes strain LO28 as able to hyperinduce IFN-ß production in infected cells ∼30-fold more than the common laboratory clone L. monocytogenes strain 10403S. Genomic analyses determined that LO28 contains a naturally occurring loss-of-function allele of the transcriptional regulator BrtA and correspondingly derepresses expression of MdrT. Surprisingly, while derepression of MdrT resulted in hyperstimulation of IFN-ß, it results in significant attenuation in multiple mouse models of infection. While type I interferons may promote L. monocytogenes virulence, this study demonstrates that unregulated expression of the c-di-AMP-secreting efflux pump MdrT significantly restricts virulence in vivo by an unknown mechanism.