The Effects of Ad5.CMV.hTGFβ2C226/228S on AHD in Mice
Stevenson, Cooper H.
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Elevated intraocular pressure (IOP) is a key risk factor for the development of primary open-angle glaucoma (POAG), a leading cause of blindness in people over the age of 40 years. Transforming growth factor beta-2 is a cytokine known to contribute to the pathogenesis of POAG due to its deleterious effects on aqueous humor outflow via the conventional, or trabecular, outflow pathway in the eye. However, its effects on the rate of aqueous outflow (Fu) via the unconventional or uveoscleral outflow pathway, rate of aqueous humor production (Fin), and episcleral venous pressure (Pe) are unknown. Further, effects of euthanasia and enucleation in our hands on TGFβ2-mediated effects on Fu are also unknown. The goal of the present study was to quantify the impact of over-expression of TGFβ2 on aqueous humor dynamics (AHD) in the mouse eye, with special emphasis on Fu, Fin, and Pe in the mouse eye. To simulate TGFβ2 over-expression, left (OS) eyes were injected intravitreally (IVT) with a mutant form of TGFβ2 (Ad5.CMV.hTGFβ2C226/228S, 2×10⁷pfu in 2μL), while right (OD) eyes were injected IVT with a null virus (Ad5.CMV.null, same titer and volume). Following 14 days, after which time mean IOP (determined tonometrically in conscious mice) had become elevated in TGFβ2-injected eyes (84.29% increase in IOP, P < 0.001), Fu was determined directly by cannulating the anterior aqueous chamber (AC) and perfusing it with fluorescein isothiocyanate-dextran (1×10⁻⁹ M), followed by dissection of the retina/choroid/iris-ciliary body/scleral shell, homogenization, and measurement of each sample's fluorescence, and then inference of flow rate using a standard curve. Those perfusion were performed in living eyes, also in eyes in situ in the animal immediately following euthanasia, and enucleated eyes perfused in vivo either (i) exposed to air, or (ii) submerged in PBS. In a further group of experiments in living animals aqueous humor outflow conductance (C) (also known as aqueous humor outflow facility), and Pe were measured, and then Fin and Fu were calculated using a constant flow infusion method. Further, we sought to determine whether IOP elevation would lead to a reduction in RGC numbers in the retina, so retinal flat mounts from both treated and untreated eyes from 5 of our animals were prepared and RGC counts were made. For eyes perfused in-vivo, Fu was reduced in OS (0.0048 ± 0.0017 μL/min) compared to OD (0.0987 ± 0.0126 μL/min, P = 0.025). For eyes perfused in euthanatized mice, Fu was reduced in OS (0.0215 ± 0.0101 μL/min) compared to OD (0.1543 ± 0.0241 μL/min, P = 0.010). For eyes perfused ex-vivo while submerged in PBS, there was no difference in Fu between OS (0.0222 ± 0.0065 μL/min) and OD (0.0137 ± 0.0078 μL/min, P = 0.175). For eyes perfused ex-vivo while exposed to air, Fu was reduced in OS (0.0702 ± 0.0087 μL/min) compared to OD (0.1377 ± 0.0106 μL/min, P = 0.008). Fin showed a trend towards a reduction in the eyes in which TGFβ2 was over-expressed, but this effect did not reach statistical significance. There was a significant increase in Pe in eyes in which TGFβ2 was expressed (8.6 ± 0.7 mmHg in OS to 6.4 ± 0.2 mmHg in OD, P = 0.015). Given these results, the present study further quantifies the effect of TGFβ2 in POAG, providing more insight into its mechanism of action in this disease.