Eye/Vision
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21759
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Browsing Eye/Vision by Author "Krishnamoorthy, Raghu R."
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Item CHANGES IN ENDOTHELIN RECEPTOR A EXPRESSION IN A RAT MODEL OF OCULAR HYPERTENSION(2014-03) McGrady, Nolan R.; Minton, Alena Z.; Krishnamoorthy, Raghu R.Glaucoma is the leading cause of blindness in developed countries and is the second leading cause of blindness worldwide. The most common and currently only treatable symptom associated with glaucoma is an increase in intraocular pressure (pressure inside the eye). Rodent models have been routinely used to understand the effects elevated pressure has on the eye, and this study focuses on the changes in expression of a protein molecule (endothelin A receptor) within the retina due to elevated intraocular pressure in a rat model of elevated intraocular pressure. Purpose (a): The endothelin system of peptides and their receptors have been implicated for their neurodegenerative role in glaucoma. The purpose of this study was to determine changes in ETA receptor expression within the retina in the Morrison’s elevated IOP model of glaucoma in rats. Methods (b): IOP was elevated in the left eye of adult male retired breeder Brown Norway rats using the Morrison’s model of glaucoma (by injection of hypertonic saline through episcleral veins) while the contralateral eye served as the control. The rats were maintained for two to four weeks following IOP elevation and sacrificed. Retinal sections were obtained from both control and IOP-elevated eyes, and analyzed for changes in ETA receptor expression using immunohistochemistry. ETA receptor immunostaining was co-localized with β-III-Tubulin, which is selectively expressed in retinal ganglion cells. Results (c): After two weeks, rat eyes with IOP elevation showed an increase in immunostaining for ETA receptors in several retinal layers including the inner and outer plexiform layers with a modest increase in the retinal ganglion cell layer. Following four weeks of IOP elevation, ETA receptor expression was modestly increased in the inner and outer plexiform layers of the retina, compared to that in the corresponding contralateral eyes. Conclusions (d): Elevated intraocular pressure results in a time-dependent change in ETA receptor expression. Increased ETA receptor expression is associated with neurodegenerative changes in glaucoma.Item ENDOTHELIN B (ETB) RECEPTORS CONTRIBUTE TO NEURODEGENERATION IN A RODENT MODEL OF GLAUCOMA VIA UPREGULATION OF C-JUN AND BAX(2014-03) Minton, Alena Z.; He, Shaoqing; Ma, Hai-Ying; Krishnamoorthy, Raghu R.Glaucoma is a group of eye conditions that, if left untreated, can result in blindness. It is commonly associated with an increased pressure inside the eye, known as intraocular pressure or simply IOP. As the pressure builds up inside the eye, it causes damage to the optic nerve, which in turn results in the death of retinal ganglion cells (RGCs). Studies from our lab and others have shown that endothelin 1 (ET-1), the potent vasoactive peptide, contributes to glaucoma. Currently, our lab is interested in understanding the role of the endothelin receptor B (ETB) in glaucoma. We are using rats that do not have ETB receptor (ETB KO rats) and those that have the receptor (WT rats). To mimic glaucoma, the high salt solution is injected into the special vein in the eye (episcleral vein). This causes the build up of pressure inside the eye within 7 to 10 days. This model of glaucoma is called the Morrison’s ocular hypertension model. Previously, we found that IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in ETB KO rats. In addition, pathological changes in the optic nerve were greatly reduced in ETB KO rats, as compared to those in WT rats. To find out the molecular mechanisms responsible for the death of RGCs, we elevated the pressure inside one eye of adult WT and ETB KO rats, while the contralateral eye served as control. After 2 weeks of IOP elevation, retinal sections were obtained and stained with specific antibodies to detect the levels of c-Jun (the member of the activator protein-1 (AP-1) family) and Bax (protein involved in cell death). We found that WT rats have higher levels of c-Jun and Bax in the retina (especially in the ganglion cell layer), as compared to ETB KO rats. Interestingly, using the Promo 3 software, we found 15 binding sites for members of the AP-1 family of proteins on the rat 1.95 kb upstream promoter region of Bax. Therefore, the transcription factor c-Jun may be an upstream regulator of Bax. In conclusion, transcription factor AP-1 could be involved in the elevation of the ETB receptor levels in the Morrison's model of glaucoma. Conversely, deletion of the ETB receptor results in the lower expression of c-Jun. Taken together, there may be a reciprocal relationship between the AP-1 and ETB receptors. Purpose (a): Previously, our lab has demonstrated that increased levels of ETB receptors contribute to the death of retinal ganglion cells (RGCs) and degeneration of optic nerve axons in the Morrison's elevated intraocular pressure (IOP) model of glaucoma in rats. Moreover, these pathological changes were greatly attenuated in ETB receptor-deficient transgenic Wistar Kyoto rats. Interestingly, an increase in ETB receptor levels in RGCs, following 2 weeks of IOP elevation in Brown Norway rats, was shown to be associated with increased expression of c-Jun, a member of the activator protein-1 (AP-1) family. The current study was aimed at investigating whether the increased expression of c-Jun observed in wild type rats is reduced in ETBreceptor-deficient Wistar Kyoto rats subjected to the Morrison’s model of glaucoma. The status of another apoptotic protein, Bax, was also assessed in these rats. Methods (b): IOP was elevated in one eye of adult wild type and ETB receptor-deficient transgenic Wistar Kyoto rats using the Morrison’s method (injection of hypertonic saline through episcleral veins), while the contralateral eye served as control. After IOP was elevated, rats were maintained for 2 weeks and sacrificed. Retinal sections were obtained and stained with specific antibodies to detect the expression of c-Jun and Bax by immunohistochemistry. In addition, retinal sections were immunostained using an antibody to βIII-tubulin, which is selectively expressed by RGCs in the retina. Images were taken using Zeiss LSM-510 confocal microscope with Z-scan. Results (c): Immunohistochemical analysis showed that IOP elevation for 2 weeks caused increased expression of c-Jun and Bax mainly in the ganglion cell layer (GCL) of wild type transgenic Wistar Kyoto rats as compared to ETB receptor-deficient transgenic Wistar Kyoto rats. Interestingly, using the Promo 3 software, we found 15 binding sites for members of the AP-1 family of proteins on the rat 1.95 kb upstream promoter region of Bax. Therefore, the transcription factor c-Jun may be an upstream regulator of Bax (pro-apoptotic factor). Conclusions (d): Transcription factor AP-1 could be involved in the elevation of the ETB receptor levels in the Morrison's model of glaucoma. Conversely, deletion of the ETB receptor results in the downregulation of c-Jun. Taken together, there may be a reciprocal feedback loop between the AP-1 and ETB receptors.