Many disease processes in the back of the eye result in pathologic changes occurring that affect the physiology and function of the RPE/retina interface and can lead to new blood vessel formation (neovascularization or angiogenesis), retinal edema, and subsequent retinal detachment retinal detachments;age-related macular degeneration (AMD) in the elderly (60 yrs or older);retinopathy of prematurity (infants);diabetic retinopathy (in adults). Use of animal models helps understand the role of retinal pigment epithelium (RPE) cells in visual physiology and pathology, and to develop intervention strategies of degenerative eye diseases. In pre-clinical experiments trials, we are using a rat model of retinal reattachment that allows us to directly measure RPE function as well as determine efficacy and dosing requirements for subsequent clinical trials. In these experiments we use pharmacological interventions (vitreous or SRS) to induce the RPE to up-regulate fluid transport from the retinal side of the epithelium to the choroidal blood supply (Maminishkis et al., IOVS, 2003). For these in vivo measurements of fluid transport we use a novel Polarization Optical Coherence Tomography (POCT) technique. This non-invasive technique allows us to record anatomical changes inside the living animals eye without interfering with the physiological processes of any ocular (or other) tissues. Using POCT techniques over the past three years we have been able to measure, for the first time, rates of fluid absorption from the subretinal space in artificially created retinal detachments. Using this model we have obtained the first quantative data for fluid absorption modulation by Sandostatin, ATP, INS37217 (hydrolysis resistant P2Y2 receptor agonist), and pH by retinal pigment epithelium (RPE) in vivo. In addition, this model provided crucial data for ongoing clinical trial (phase I) aimed to reduce retinal edema using IFNg. In another set of animal experiments using KO mice models we study the fine tuning of RPE physiology by microRNA (miRNA) and its network interaction effects on RPE function. From in vitro studies we know that miRNAs regulate a significant portion of the transcriptome. The miRNAs are highly conserved across many species, indicating their functional importance. Several miRNAs were identified whose expression is relative high in hfRPE compared to its adjacent tissues, including retina and choroid. These hfRPE-enriched miRNAs are miR-184, miR-187, miR-200, miR-221/222, miR-204, and miR-211, and several of them were found to be critical to the total tissue resistance of the hfRPE, suggesting their importance in RPE barrier function. To carry out this study, we produced a miR-204 knockout (KO) mouse line by a gene-targeting approach. The miR-204 gene was completely ablated and replaced with a neomycin resistant gene expression cassette via homologous recombination. Loss of miR-204 expression was determined in tissues normally enriched in miR-204, such as eye and brain, by Northern hybridization. The knockout mice are viable and dont have gross developmental defects. However, OCT analysis of adult eye showed an irregular vasculature in retina and an extra mass protruding from the lens epithelium in 2/3 of the knockout mice. Their physiological function in eye is likely compromised as ERG obtained from these mice showed an a-wave and b-wave amplitude response that was diminished in rod photoreceptors compared to control (n = 3-5). Additional functional studies (OCT, ERG) will be required to characterize the retinal defects and future studies will determine the possible structural alteration in eyes of miR-204 KO mice and identity the miR-204 target genes responsible for the physiological changes. In other sets of experiments we studied miR-155, an inflammatory miRNA, which was found to be enriched in choroid compared to RPE and retina. Its expression in RPE can be increased by pro-inflammatory cytokine cocktail. Its physiological significance has been extensively studied in monocytes, macrophages, and dendritic cells where pro-inflammatory stimuli, such as bacterial lipopolysaccharide, polyinosinic:polycytidylic acid, or IFN- increased miR-155 expression and miR-155 was shown to play an important role in inflammation. We propose to perform dark and light adapted ERGs on 3, 6, 9, 12, and 24 month old miR-204 and mir-155 KO mice to understand the role of these miRNAs in rod and cone photoreceptor function, during the progression of age. Additionally, dc-ERGs will be performed on these KO mice. dc-ERGs are a very powerful tool to visualize RPE function through monitoring of electrophysiological responses. dc-ERG consists of three parts, a positive c-wave comprised of RPE apical membrane hyperpolarization due to increased K+ conductance and slow PIII response of Mller cells, a negative fast oscillation (FO) comprised of basal membrane hyperpolarization due to reduced conductance of CFTR-mediated Cl channels, and positive light peak (LP) comprised of basal membrane depolarization by increased activity of Ca2+-activated Cl- channels. Thus, dc-ERG will enable us to isolate the electrical responses of the RPE from the rod and cone photoreceptors, which will primarily be studied using normal ERG. The nm3342 mouse may be the first clinically appropriate animal model for studying serous detachment and CSR. An early goal of our study will be to determine if there are gene differences in the RPE and choroidal between mutant and wild type mice that may disrupt regulation and allow for fluid leakage. In these mutants, the choroidal or RPE cells may be abnormal, tight junctions that seal the extracellular space between cells may be affected, or there may be RPE cell death. We will also determine if the mutation itself causes abnormalities in the neural retina or if abnormalities only arise after retinal detachment. Rhegmatogenous detachment (where fluid flows through a hole or break in the retina) creates many rapid cellular changes in the retina. Whether serous detachments cause similar changes is unknown. Current observations in human patients with CSR suggest degenerative changes in the photoreceptor layer, but their nature is unknown. We will determine if the photoreceptor degeneration and cell death occurs in the mice and if there is neuronal or glial remodeling that may predict mechanisms for visual imperfections in human CSR patients. Comparisons to gene expression in rhegmatogenous detachments of the same duration will be important as well. We will obtain time-line ocular structural changes in nm3342 mice by high-resolution OCT, a powerful non-invasive technique that will provide additional structural data and help guide the cellular characterization of nm3342. We will do pilot studies to determine if previously tested small molecules resolve detachment in the nm3342 mice, and begin exploring methods for primary cultures of nm3342 RPE cells. The proposed experiments should help us understand the special characteristics of serous retinal detachments, an important first step to understanding CSR in humans.
|Oh, Hyun-Mee; Yu, Cheng-Rong; Lee, Yongjun et al. (2011) Autoreactive memory CD4+ T lymphocytes that mediate chronic uveitis reside in the bone marrow through STAT3-dependent mechanisms. J Immunol 187:3338-46|
|Maminishkis, Arvydas; Miller, Sheldon S (2010) Experimental models for study of retinal pigment epithelial physiology and pathophysiology. J Vis Exp :|