We sought to investigate the effects of diabetes and hyaluronidase on the thickness of the endothelial glycocalyx layer in the mouse retina. In our study, the retinal circulation of diabetic Ins2(Akita) mice and their nondiabetic littermates were observed via intravital microscopy. The endothelial glycocalyx thickness was determined from the infusion of two fluorescently labeled plasma markers, one of which was a high molecular weight rhodamine dextran (MW=155,000) excluded from the glycocalyx, and the other a more permeable low molecular weight sodium fluorescein (MW=376). In nondiabetic C57BL/6 mice, the glycocalyx thickness also was evaluated prior to and following infusion of hyaluronidase, an enzyme that can degrade hyaluronic acid on the endothelial surface. A leakage index was used to evaluate the influence of hyaluronidase on the transport of the fluorescent tracers from the plasma into the surrounding tissue, and plasma samples were obtained to measure levels of circulating hyaluronic acid. Both diabetes and hyaluronidase infusion significantly reduced the thickness of the glycocalyx in retinal arterioles (but not in venules), and hyaluronidase increased retinal microvascular leakage of both fluorescent tracers into the surrounding tissue. However, only hyaluronidase infusion (not diabetes) increased circulating plasma levels of hyaluronic acid. In summary, our findings demonstrate that diabetes and hyaluronidase reduce the thickness of the retinal endothelial glycocalyx, in which hyaluronic acid may play a significant role in barrier function.
Purpose Methamphetamine (METH), a highly addictive stimulant of neurotransmitters induces central retinal artery occlusion with retinal atrophy and neovascularization. We previously have identified METH‐induced loss of endothelial surface molecules including platelet and endothelial cell adhesion molecule‐1 (PECAM‐1) and glycocalyx in mice. However, the pathophysiologic pathway of METH‐induced retinal vascular dysfunction has been insufficiently investigated. The purpose of this study is to investigate the METH‐induced alteration of the retinal vascular circulation. Materials and methods C57BL/6J male and female mice were administrated progressively increasing doses of METH (0 to 6 mg/kg) by repetitive intraperitoneal injections (4 times per day) for 4 weeks. Norepinephrine levels in plasma were measured by ELISA. Cross‐sections of the retina were stained with hematoxylin and eosin. Levels of hypoxia in the retina were determined by immunostaining of pimonidazole adducts with Hypoxyprobe. Retinal blood vessels were visualized with fluorescent plasma tracers injected via femoral vein injection for live imaging, and immunostained with griffonia simplicifolia lectin 1 (GSL‐1) for retinal flatmounts. Expression of various proteins was determined by immunoblot. Results The level of pimonidazole adduct was increased in METH‐treated retinas, compared with saline‐treated retinas, indicating METH‐induced retinal hypoxia. Plasma norepinephrine levels, which can promote hypoxia‐inducible factor 1a (HIF‐1a) at the transcriptional level, were elevated by METH, compared with saline treatment at 1 week (2.2±0.9 vs 7.8±1.6 ng/mL, P<0.01), 2 weeks (0.8±0.3 vs 5.5±1.8 ng/mL, P<0.05), and 4 weeks (0.9±0.2 vs 2.0±0.4 ng/mL, P<0.05). Retinal protein expression levels of HIF‐1a and angiogenic proteins, such as vascular endothelial growth factor a (VEGFa), erythroblast transformation‐specific (ETS)‐related gene (ERG), and Protein C‐ets‐1 (ETS‐1) were increased in the METH‐treated retinas. Counts of retinal arterioles and capillaries were higher in METH‐treated mice than in saline‐treated mice. Moreover, protein concentration in the vitreous humor and immunoreactivity of immunoglobulin in retina, which are measures of retinal permeability, were increased by METH treatment. Conclusion Our present data suggest that METH is associated with retinal neovascularization and hyperpermeability with increases in hypoxia, plasma norepinephrine, and HIF‐1a activation. Support or Funding Information This work was supported by funding from the National Institute of Health (NIH) EY025632.
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