Retinal mRNA Levels Research Articles

Photoreceptors are preferentially affected in the rat retina following permanent occlusion of the carotid arteries

Carotid artery occlusion (two vessel occlusion; 2-VO) for 3 or 9 months causes a suppression of the electroretinogram. However, after 3 months the retinal morphology appears unaffected judging from the localisation of GABA, ChAT, αPKC, Thy-1 and GFAP immunoreactivities. Moreover, no difference in NMDA-R1, opsin or Thy-1 mRNA levels were detected. In contrast, after 9 months 2-VO photoreceptor degeneration occurred as indicated by thinning of the outer nuclear layer and reduced Ret-P1 immunoreactivity. All other immunoreactivities appeared normal. These findings were supported by analysis of retinal mRNA levels. We conclude that the major effect of prolonged 2-VO is photoreceptor degeneration.

Ocular Neovascularization: Clarifying Complex Interactions

Neovascularization within the eye contributes to visual loss in several ocular diseases, the most common of which are proliferative diabetic retinopathy, neovascular age-related macular degeneration, and retinopathy of prematurity. Together, these three diseases afflict persons in all stages of life from birth through late adulthood and account for most instances of legal blindness. Retinopathy of prematurity (ROP) occurs in premature neonates. Normally, the retina becomes completely vascularized at full term. In the premature baby, the retina remains incompletely vascularized at the time of birth. Rather than continuing in a normal fashion, vasculogenesis in the premature neonatal retina becomes disrupted. Abnormal new proliferating vessels develop at the juncture of vascularized and avascular retina. These abnormal new vessels grow from the retina into the vitreous, resulting in hemorrhage and tractional detachment of the retina. Although laser ablation of avascular peripheral retina may halt the neovascular process if delivered in a timely and sufficient manner, some premature babies nevertheless go on to develop retinal detachment. Surgical methods for treating ROP-related retinal detachments in neonates have limited success at this time because of unique problems associated with this surgery, such as the small size of the eyes and the extremely firm vitreoretinal attachments in neonates. Diabetic retinopathy is the leading cause of blindness in adults of working age. In persons with diabetes mellitus, retinal capillary occlusions develop, creating areas of ischemic retina. Retinal ischemia serves as a stimulus for neovascular proliferations that originate from pre-existing retinal venules at the optic disk or elsewhere in the retina posterior to the equator. Severe visual loss in proliferative diabetic retinopathy (PDR) results from vitreous hemorrhage and tractional retinal detachment. Again, laser treatment (panretinal photocoagulation to ischemic retina) may arrest the progression of neovascular proliferations in this disease but only if delivered in a timely and sufficiently intense manner. Some diabetic patients, either from lack of ophthalmic care or despite adequate laser treatment, go on to sustain severe visual loss secondary to PDR. Vitrectomy surgery can reduce but not eliminate severe visual loss in this disease. Age-related macular degeneration is the leading cause of severe visual loss in persons over 65 years old. In contrast to ROP and PDR, in which neovascularization emanates from the retinal vasculature and extends into the vitreous cavity, AMD is associated with neovascularization originating from the choroidal vasculature and extending into the subretinal space. Choroidal neovascularization causes severe visual loss in AMD patients because it occurs in the macula, the area of retina responsible for central vision. The stimuli which lead to choroidal neovascularization are not understood. Laser ablation of the choroidal neovascularization may stabilize vision in selected patients. However, only 10% to 15% of patients with neovascular AMD have lesions judged to be appropriate for laser photocoagulation according to current criteria. Retinopathy of prematurity, proliferative diabetic retinopathy, and neovascular age-related macular degeneration are but three of the ocular diseases which can produce visual loss secondary to neovascularization. Others include sickle cell retinopathy, retinal vein occlusion, and certain inflammatory diseases of the eye. These, however, account for a much smaller proportion of visual loss caused by ocular neovascularization. Additional treatments beyond laser photocoagulation and vitrectomy surgery are needed to improve outcomes in these patients. Pharmacological antiangiogenic therapy can potentially assist in prevention of the onset or progression of ocular neovascularization and is a current goal of many research laboratories and pharmaceutical companies.

Upregulation of Retinal Vascular Endothelial Growth Factor mRNAs in Spontaneously Diabetic Rats without Ophthalmoscopic Retinopathy

Vascular endothelial growth factor (VEGF) has recently been shown to be involved in the pathogenesis of proliferative diabetic retinopathy. However, its involvement in the development of the early phase of diabetic retinopathy is not fully understood. In this study we investigated the retinal VEGF mRNA level in spontaneously diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats, a model of non-insulin-dependent diabetes, without overt retinopathy, using quantitative reverse-transcription polymerase chain reaction. The retinal VEGF mRNA level was 2.2 times higher (p < 0.0005) in OLETF rats than in control rats at the age of 60 weeks. Moreover, their retinal mRNA level was positively correlated with serum concentration of advanced glycation end products (AGEs) but not to serum glucose concentration. Furthermore, the peak latency of the oscillatory potentials in the electroretinogram, one of the most sensitive markers for the early phase of diabetic retinopathy, was significantly prolonged in OLETF rats (p < 0.05), being also correlated with the serum AGE concentration. The results thus suggest that AGEs, which are formed acceleratedly in diabetic conditions, are involved in the development of the early phase of diabetic retinopathy probably through the induction of retinal VEGF mRNAs.

Localization of a 42-kDa inositol 1,3,4,5-tetrakisphosphate receptor protein in retina and change in expression after optic nerve injury.

The mRNA and protein expression of a 42-kDa inositol 1,3,4,5-tetrakisphosphate receptor (InsP4R) was investigated in cryostat and paraffin sections from rat, porcine and bovine retina. InsP4R mRNA was localized by in situ hybridization in the ganglion cell layer, the inner nuclear cell layer and the outermost part of the outer nuclear cell layer. For immunocytochemistry, we used an antibody raised against a 19-amino-acid peptide (peptide-3) derived from previous microsequencing of proteolytic fragments of the porcine InsP4R (Stricker et al., FEBS Lett., 370 (1995) 236). The distribution of immunoreactivity was similar in all species investigated. Two cell types, most likely wide-field amacrine and retinal ganglion cells, were intensely stained. Prominent immunoreactivity in the on/off sublaminae of the inner plexiform layer and in the optic nerve layer indicates a pre- and/or post-synaptic localization of the protein. Moreover, significant InsP4R protein expression in the inner segment of photoreceptors points to a putative role of the second messenger InsP4 in signaling processes related to phototransduction. However, also the endfeet of Müller glia cells in the optic nerve layer were intensely stained. Optic nerve crush caused only minor changes in retinal InsP4R mRNA levels whereas InsP4R immunoreactivity was attenuated for more than 4 weeks in the photoreceptor inner segments, wide-field amacrine cells, and in retinal ganglion cells. The immunopositive sublaminae of the inner plexiform layer appeared to have shrunken. However, the signal intensity gradually recovered after 10 weeks. Since in parallel sections stained with a monoclonal antibody directed against the vesicular protein synaptophysin no changes were found, the alterations in InsP4R immunoreactivity induced by nerve injury are not due to a general decline in the expression of pre-synaptic proteins. We, therefore, hypothesize that the InsP4R might be linked to altered intracellular Ca2+ signaling after neuronal injury.

Reactive oxygen intermediates increase vascular endothelial growth factor expression in vitro and in vivo.

Elevated vascular endothelial growth factor (VEGF) levels are required for ocular and tumor angiogenesis in animal models. Ischemic hypoxia is strongly correlated with increased VEGF expression in these systems and is considered a physiologically relevant stimulus. Because ischemic hypoxia is often followed by reperfusion and reactive oxygen intermediate (ROI) generation, we examined the potential role of ROI in the control of VEGF gene expression. Human retinal pigment epithelial cells exposed to superoxide or hydrogen peroxide rapidly increased VEGF mRNA levels. Superoxide-associated mRNA increases were dose dependent, blocked by antioxidants, and associated with elevated VEGF protein levels in conditioned media. Increases in VEGF mRNA levels were also observed in cultured human melanoma and rat glioblastoma cells with superoxide or hydrogen peroxide. Cycloheximide prevented the ROI-associated increases in VEGF mRNA. Transcriptional inhibition with actinomycin D revealed an inducible increase in VEGF mRNA half-life, but nuclear run-on experiments showed no increase in VEGF transcriptional rate. Reoxygenation of human retinal pigment epithelial cells in vitro and ocular reperfusion in vivo increased retinal VEGF mRNA levels. Antioxidants prevented the reperfusion-associated VEGF mRNA increases in retina. We conclude that ROIs increase VEGF gene expression in vitro and during the reperfusion of ischemic retina in vivo. The ROI-associated increases are mediated largely through increases in VEGF mRNA stability.

Light and dark cause a shift in the spatial expression of a neuropeptide-processing enzyme in the rat retina

Carboxypeptidase E (CPE, EC 3.4.17.10) is an essential enzyme in the post-translational processing of most neuroactive peptides. We have studied how the expression of CPE in the rat retina is modified in response to the light/dark cycle. Retinal CPE mRNA levels increase immediately after a change in lighting condition. After prolonged exposure to the dark or the light, however, CPE mRNA levels drop to comparable amounts. The increase CPE mRNA is most dramatic during the dark-to-light transition. Interestingly, during the same interval, CPE protein levels transiently decrease. This apparently contradictory change in mRNA and protein levels is attributable to a change in the spatial pattern of CPE expression in the retina in response to light and dark. Both in situ hybridization and indirect immunofluorescence studies indicate that CPE is expressed in photoreceptors in the dark. During light onset, CPE expression is rapidly induced in retinal ganglion cells, whereas expression in photoreceptors is reduced. This pattern is reversed when the animals are exposed to the dark. In contrast, CPE is apparently constitutively expressed in a subpopulation of cells in the inner nuclear layer in both the light and the dark. These changes in CPE expression are similar to light-induced changes in c-fos expression, suggesting that CPE may be a downstream target for Fos activation.

Effects of long-term diabetes and galactosaemia upon lens and retinal mRNA levels in the rat

The levels of mRNAs encoding the α1 chain of collagen IV and the B1 chain of laminin were assayed in the lenses and retinas of long-term (28-week) diabetic and galactosaemic rats in order to gain some insight into the effects on basement membrane (BM) synthesis in these tissues. mRNAs coding for β-actin, glucose transporter protein and the α2 catalytic subunit of Na +,K +-ATPase were also assayed to determine whether any effects on BM-coding mRNA levels were specific. Long-term diabetes had no significant effect on the levels of α1(IV) collagen mRNA but caused a significant reduction in the laminin B1 message in the lens. In the same samples, the level of the glucose transporter protein mRNA was found to be elevated significantly in the diabetic tissue, whereas the mRNAsen coding β-actin and α2 Na +,K +-ATPase were unaffected in comparison with age-matched controls. Long-term galactosaemia resulted in significant increases in the levels of all mRNAs assayed when expressed per μg total RNA used for each analysis. However, this effect appeared to be due to a specific loss of ribosomal RNA from these severely cataractous lenses. When related to the β-actin mRNA internal control, the levels of mRNA in the galactosaemic lenses were very similar to that found in the diabetics. Laminin B1 mRNA levels were decreased significantly. Diabetes had little influence on any of the mRNAs assayed in the retina. mRNAs coding for the glucose transporter protein and α2 Na +,K +-ATPase increased slightly but these levels were not of statistical significance. In the galactosaemic rat retinas, no significant differences were detected in the levels of mRNAs coding for α1(IV) collagen, laminin B1, β-actin or glucose transporter protein in comparison with age-matched controls. However, the α2 Na +,K +-ATPase mRNA was increased 2·3-fold in comparison with controls. The data demonstrate that whereas long-term diabetes and galactosaemia induce specific increases in certain mRNA levels, neither condition affects the levels of mRNAs coding for the BM components examined. These results, therefore, provide no support for the hypothesis that the thickening of BMs apparent in diabetes and galactosaemia is the result of increased synthesis of the extracellular matrix components studied.