Structural and functional changes in retinal vasculature induced by retinal ischemia-reperfusion in rats
Introduction
Retinal ischemia is a major contributor to tissue damage in diseases such as acute angle-closure glaucoma, retinal vascular occlusions, diabetic retinopathy, and retinopathy of prematurity (Bek, 2009). An important aspect of this injury is the ischemia–reperfusion (I/R) condition, in which blood flow returns to a tissue after a period of ischemia. The absence of oxygen and nutrients during ischemia creates a condition in which restoration of circulation results in the generation of reactive oxygen species (Szabo et al., 1991), leading to inflammation (Tsujikawa et al., 1999).
The mechanisms of neuronal degeneration induced by I/R are not completely understood, but it has been recognized that excessive activation of N-methyl-d-aspartate (NMDA) receptor plays an important role (Lam et al., 1997, Lagrèze et al., 1998). Indeed, intravitreal injection of high doses of NMDA induces similar retinal neuronal degeneration, as indicated by retinal ganglion cell apoptosis and thinning of the inner retina (Lam et al., 1999, Manabe and Lipton, 2003). In NMDA-induced neuronal injury or death, excessive Ca2+ influx and subsequent activation of Ca2+-dependent responses, including formation of nitric oxide (NO) via neuronal NO synthase (NOS) (Morizane et al., 1997, Vorwerk et al., 1997), are thought to be important events. In addition, upregulation of inflammatory proteins and recruitment of leukocytes into the retina are involved (Nakazawa et al., 2007, Al-Gayyar et al., 2011). Furthermore, reductions in retinal blood flow may be an additional cause of progression of the ischemic retinal damage, because damage to the retinal microvasculature was observed in these experimental models of retinal neurodegeneration (Zheng et al., 2007, Chen et al., 2009).
Several similarities in the altered retinal microvasculature were found between I/R- or NMDA-induced injury and diabetic retinopathy models, e.g., pericyte loss and elevation of inducible NOS expression (Zheng et al., 2007, Al-Gayyar et al., 2010). Therefore, these retinal degeneration models would be useful for investigating pathological processes of neuronal and capillary degeneration and for testing novel interventions for treating such retinal diseases through protection against neuronal/vascular injury.
In the present study, we aimed to provide a detailed description of the structural and functional changes in the retinal vasculature induced by I/R to clarify the implications of retinal neuronal degeneration in capillary degeneration. We also examined the distribution and expression of vascular endothelial growth factor (VEGF) in the retina. Although VEGF is an important factor for physiological vascular development (Ozaki et al., 2000, Gerhardt et al., 2003) and pathological neovascularization (Aiello et al., 1995) in the retina and the choroid, increasing evidence has revealed its important role in the trophic maintenance of neurons (Nishijima et al., 2007, Foxton et al., 2013).
Section snippets
Animals
Male Sprague–Dawley rats weighing 220–240 g were maintained in a room at a constant temperature (22 ± 2 °C) and at constant humidity (55 ± 5%) under a 12-h light/dark cycle, and allowed free access to regular food and tap water.
All animal procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement on the Use of Animals in Ophthalmic and Vision Research, and the Regulations for the Care and Use of Laboratory Animals in Kitasato University adopted
Morphological changes in overall retinal architecture and retinal capillary network
We first determined the time-dependent morphological changes in overall retinal architecture and retinal capillary network in paraffin sections (Fig. 1A–D) and whole-mounts of retina stained with the antibody to RECA, an endothelial cell marker (Fig. 1E–H). Loss of retinal ganglion cells and thinning of the inner plexiform layer (IPL) were detected 2 days after I/R, and these histological changes progressed in a time-dependent manner (Fig. 1B–D). Marked thinning of inner retinal layers and mild
Discussion
The present study demonstrates that a time-dependent degeneration of capillaries occurs in the retina after a high pressure-induced retinal I/R. Histological changes in the inner retina, such as loss of retinal ganglion cells and thinning of the IPL, preceded the capillary degeneration. The results also indicated that loss of retinal ganglion cells, which are a source of VEGF, is likely to become one cause of the reduction in VEGF in the inner retina, and subsequently induce retinal capillary
Acknowledgments
This study was supported by Grants-in-Aid for Scientific Research on Innovative Areas (Nos. 23122517, 25122712, T.N.) and Grants-in-Aid for Scientific Research (C) (Nos. 20590090, 23590112, T.N.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, Suzuken Memorial Foundation (T.N.) and by Kitasato University Research Grant for Young Researchers (T.N.).
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