Loss of retinal capillary vasoconstrictor response to Endothelin-1 following pressure increments in living isolated rat retinas
Introduction
Elevated intraocular pressure (IOP) is a major risk factor for glaucoma, but how pressure exerts its deleterious effects is still debated. Damage is likely to be multi-factorial, the main hypotheses being that increased IOP may directly damage retinal axons mechanically, but also indirectly affect neurons by triggering neurotoxic effects and altering retinal circulation (see for reviews (Burgoyne et al., 2005, Flammer et al., 2002, Libby et al., 2005, Neufeld and Liu, 2003, Quigley, 1999)). In favor of a vascular component in the progression of glaucoma, reduced ocular blood flow (reviewed in (Flammer et al., 2002)), and a narrowing of retinal arterioles (Mitchell et al., 2005, Wang et al., 2007) have been observed in glaucoma patients, but whether these are causally related to neuronal damage is still debated (see for example (Ikram et al., 2005)).
Retinal ganglion cells (RGCs), the neurons most affected in glaucoma (Quigley, 1999), are nourished by the retinal vessels, which branch from the ophthalmic artery to form two dense capillary plexuses (Flammer et al., 2002, Osborne et al., 2004). It is at the level of capillaries that most metabolic exchange occurs (reviewed in Bechmann et al., 2007). Furthermore, although capillaries lack smooth muscle, retinal pericytes regulate capillary caliber in response to local stimuli, indicating that capillaries have an active role in local blood flow control (Metea and Newman, 2007, Peppiatt et al., 2006). Therefore, an important issue is whether IOP elevation leads to capillary alterations in the retina that may adversely affect neurons.
Current techniques to image retinal vasculature in vivo, such as fundus photography, fluorescent angiography and Laser Doppler velocimetry, although valuable in identifying gross changes in retinal vessels, lack the resolution necessary to visualize the smallest capillaries (Ritter et al., 2005), calling for different experimental approaches to address this question. Here, we imaged capillaries in living explanted rat retinas, where capillary viability and responsiveness are preserved (Metea et al., 2007, Metea and Newman, 2006a, Metea and Newman, 2006b, Metea and Newman, 2007, Peppiatt et al., 2006), and tested whether pressure increments alter capillary viability, diameter and/or responses to vasoactive stimuli. Our results show that capillary viability, diameter and response to vasodilator stimulation are not affected after pressure increments; however, a lack of capillary response to the vasoconstrictor Endothelin-1 (Et-1) is observed. Since Et-1 is a major component of the endogenous mechanisms controlling retinal blood flow (Haefliger et al., 2001, Polak et al., 2003), we speculate that, in case of prolonged/repeated IOP elevation, abnormal capillary regulation would ensue that could contribute to neuronal damage over time.
Section snippets
Animal and tissue handling
Experiments were performed on 3–4 week old Long Evans hooded rats, in accordance to the national and the ARVO regulation on animal experimentation in ophthalmic research. Animals were killed with an overdose of anesthetic (Avertine, 1 cc/hg b.w. i.e. 29 mg tribromoethanol/hg b.w.), retinas quickly dissected in oxygenated artificial cerebrospinal fluid (ACSF) containing (in mM) NaCl (119), KCl (2.5), MgCl2 (1.3), CaCl2 (2.5), NaH2PO4 (1), glucose (11), and HEPES (20) (described in Kettunen
Testing the model system
In a series of preliminary experiments we tested whether retinal vessels were functional once the retina was isolated and normal blood perfusion lost. In agreement with previous studies on this model system (Metea et al., 2007, Metea and Newman, 2006a, Metea and Newman, 2006b Metea and Newman, 2007, Peppiatt et al., 2006), we found that, for at least 4 h after retinal isolation, erythrocyte movements could be observed within retinal capillaries (Supplemental movie 1), capillaries responded to
Discussion
In the present study we have explored the effects of pressure increments on retinal capillaries, focusing on changes in capillary caliber, viability and response to vasoactive stimuli in isolated retinas. We found that capillary viability, caliber and response to Fsk vasodilator stimulation are not affected after pressure increments; yet, a prolonged lack of capillary response to the vasoconstrictor Endothelin-1 (Et-1) is observed. Here, we will first discuss the potential limitations deriving
Acknowledgments
We thank Giulio Cappagli for invaluable technical assistance, Giovanni Vozzi and Arti Ahluwalia for help with the bioreactor, Enrica Strettoi and M. Cristina Cenni for reading the manuscript, and C. Marini for help with the Bosentan experiments. Supported by grants from the Institute fur Paraplegia and ASI to L.G.R.
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