ABSTRACT
Purpose. To determine the neuroprotective properties of a latanoprost acid derivative (ACS67) that donates the gas hydrogen sulfide (H(2)S). Methods. Ischemia to the rat retina was induced by elevation of intraocular pressure. Electroretinograms (ERGs) were recorded and the retinas analyzed 2 days later by immunohistochemistry, Western blot analysis, and RT-PCR. Hydrogen peroxide (H(2)O(2)) was used to impose an insult on RGC-5 cells in culture. The nature of the insult to cultures was quantified by the resazurin-reduction assay procedure, staining for reactive oxygen species (ROS) and for apoptosis. ACS67, its sulfurated moiety (ACS1), and latanoprost were tested for both their toxicity and ability to blunt the negative effect of H(2)O(2) on RGC-5 cells. In addition, an assay was used to see whether any of the substances influenced glutathione (GSH) levels in RGC-5 cells. Results. Partial damage to the retina in situ after ischemia was characterized by an alteration of the ERG, a reduction in the retinal localization of specific antigens and a reduction and elevation of defined retinal proteins and mRNAs. Optic nerve axonal proteins were also drastically reduced by ischemia. Most of these changes were significantly blunted by an intravitreal injection of ACS67 directly after ischemia. ACS67, ACS1, and the antioxidant epigallocatechin gallate (EGCG) all stimulated GSH levels and significantly attenuated H(2)O(2)-induced toxicity to RGC-5 cells, whereas latanoprost did not. Conclusions. ACS67 acts as an H(2)S donor through its donating moiety ACS1 and as a consequence is able to act as a neuroprotectant.
Subject(s)
Hydrogen Sulfide/metabolism , Oxidative Stress/drug effects , Prostaglandins F, Synthetic/pharmacology , Reperfusion Injury/prevention & control , Retinal Diseases/prevention & control , Retinal Ganglion Cells/drug effects , Thioctic Acid/analogs & derivatives , Animals , Blotting, Western , Caspases/genetics , Cells, Cultured , Disease Models, Animal , Electrophoresis, Polyacrylamide Gel , Electroretinography , Fluorescent Antibody Technique, Indirect , Glial Fibrillary Acidic Protein/genetics , Glial Fibrillary Acidic Protein/metabolism , Glutathione/biosynthesis , Hydrogen Peroxide/toxicity , Latanoprost , Nitric Oxide Synthase Type I/metabolism , Prostaglandins F, Synthetic/chemistry , RNA, Messenger/metabolism , Rats , Reperfusion Injury/metabolism , Reperfusion Injury/pathology , Retinal Diseases/metabolism , Retinal Diseases/pathology , Retinal Ganglion Cells/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Thioctic Acid/chemistry , Thioctic Acid/pharmacology , Thy-1 Antigens/metabolismABSTRACT
PURPOSE: To localize different prostaglandin E(2) receptors in rat retinas of varying age, deduce how they are affected by acute stress insult, and determine whether the negative effect of ischemia/reperfusion is attenuated by the EP2 agonist butaprost. METHODS: Ischemia was induced by the elevation of intraocular pressure. Butaprost was injected intravitreally immediately after ischemia. Standard methods were used for recording of electroretinograms (ERGs) and processing of immunohistochemistry. Extracts of whole retinas were analyzed for specific proteins by Western blotting or by RT-PCR for defined mRNAs. RESULTS: The localization of different EP receptor types is similar in retinas of all aged rats. However, differences exist in the monomer/dimer ratios in retinas of different age. Acute stress insult (48 hours after ischemia) affects the ratio of monomer/dimer of all EP receptor types and increases EP2 and EP3 immunoreactivities in Müller cells of the adult retina. Ischemia and 5 to 7 days of reperfusion to the retina caused the normal ERG and the localization of nNOS and ChAT immunoreactivities to be affected. Certain proteins and mRNAs were lowered in content, whereas other proteins and mRNAs were upregulated. In addition, specific optic nerve proteins were drastically reduced. Most of these changes induced by ischemia/reperfusion were significantly blunted by butaprost. CONCLUSIONS: All subtypes of EP receptors exist primarily in the inner retina at different ages, but their monomer/dimer ratios vary. Stress affects the monomer/dimer ratio and EP2 and EP3 immunoreactivities in Müller cells. Butaprost injected intravitreally significantly blunts the detrimental influence of ischemia/reperfusion to the retina.
Subject(s)
Aging/physiology , Alprostadil/analogs & derivatives , Oxidative Stress , Receptors, Prostaglandin E/metabolism , Reperfusion Injury/prevention & control , Retinal Diseases/prevention & control , Alprostadil/therapeutic use , Animals , Blotting, Western , DNA Primers/chemistry , Electroretinography , Immunohistochemistry , Injections , Nerve Tissue Proteins/genetics , Nitric Oxide Synthase Type I/metabolism , Prostaglandins E, Synthetic/therapeutic use , RNA, Messenger/metabolism , Rats , Rats, Wistar , Receptors, Prostaglandin E/agonists , Receptors, Prostaglandin E, EP2 Subtype , Reperfusion Injury/metabolism , Reperfusion Injury/physiopathology , Retina/metabolism , Retina/physiopathology , Retinal Diseases/metabolism , Retinal Diseases/physiopathology , Reverse Transcriptase Polymerase Chain Reaction , Vitreous BodyABSTRACT
EGCG, a major component of green tea, has a number of properties which includes it being a powerful antioxidant. The purpose of this investigation was to deduce whether inclusion of EGCG in the drinking water of albino rats attenuates the effect of a light insult (2200lx, for 24h) to the retina. TUNEL-positive cells were detected in the outer nuclear layer of the retina, indicating the efficacy of the light insult in inducing photoreceptor degeneration. Moreover, Ret-P1 and the mRNA for rhodopsin located at photoreceptors were also significantly reduced as well as the amplitude of both the a- and b-waves of the electroretinogram was also reduced showing that photoreceptors in particular are affected by light. An increase in protein/mRNA of GFAP located primarily to Müller cells caused by light shows that other retinal components are also influenced by the light insult. However, antigens associated with bipolar (alpha-PKC), ganglion (Thy-1) and amacrine (GABA) cells, in contrast, appeared unaffected. The light insult also caused a change in the content of various proteins (caspase-3, caspase-8, PARP, Bad, and Bcl-2) involved in apoptosis. A number of the changes to the retina caused by a light insult were significantly attenuated when EGCG was in the drinking water. The reduction of the a- and b-waves and photoreceptor specific mRNAs/protein caused by light were significantly less. In addition, EGCG attenuated the changes caused by light to certain apoptotic proteins (especially at after 2 days) but did not appear to significantly influence the light-induced up-regulation of GFAP protein/mRNA. It is concluded that orally administered EGCG blunts the detrimental effect of light to the retina of albino rats where the photoreceptors are primarily affected.
