Isolation of Retinal Arterioles for <em>Ex Vivo</em> Cell Physiology Studies

Abstract

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Endothelial CYP2J2 overexpression restores the BRB via METTL3‐mediated ANXA1 upregulation

“…In summary, during the early neonatal period, deletion of ANXA1 in ECs had no impact on endothelial proliferation and migration. Retinal arterioles are the main resistance vessels of the retina and play a key role in regulating retinal hemodynamics through changes in luminal diameter 42 . Retinas of P5 Anxa1 ΔEC exhibited narrow precapillary arterioles and postcapillary venules (Figure 6H–J), reflecting poor vascular perfusion and deficient dynamic flow compared with the controls.…”

“…Retinal arterioles are the main resistance vessels of the retina and play a key role in regulating retinal hemodynamics through changes in luminal diameter. 42 Retinas of P5 Anxa1 ΔEC exhibited narrow precapillary arterioles and (Figure 6H-J), reflecting poor vascular perfusion and deficient dynamic flow with the controls. Stimulation of hemodynamic forces protects vascular branches from regression.…”

Loss of TRPV2-mediated blood flow autoregulation recapitulates diabetic retinopathy in rats

“…Retinal arteriole isolation. First-and second-order rat retinal arterioles were isolated in low Ca 2+ (71). In brief, retinas were rapidly removed from euthanized animals, cut into 2 halves using a scalpel blade, and mechanically triturated using a fire-polished Pasteur pipette.…”

“…Arteriolar pressure myography. Pressure-induced myogenic tone was measured in isolated retinal arterioles as previously described (71). After mounting on a custom-built pressure myography system, vessels were pressurized and maintained at 40 mmHg for 15 minutes to enable the development of steady-state myogenic tone.…”

“…First- and second-order rat retinal arterioles were isolated in low Ca 2+ Hank’s solution (in mM: 140 NaCl [Thermo Fisher Scientific; catalog 11904061]; 6 KCl [Melford; catalog P0515]; 5 D-glucose [Merck; catalog G7528-1KG]; 0.1 CaCl2 [Merck; cagtalog C8106-500G]; 1.3 MgCl2 [Merck, catalog M2393-100G]; and 10 HEPES [Melford; catalog B2001-1KG] [pH 7.4] with NaOH [Merck; catalog S5881-1KG-M]) as previously described ( 71 ). In brief, retinas were rapidly removed from euthanized animals, cut into 2 halves using a scalpel blade, and mechanically triturated using a fire-polished Pasteur pipette.…”