Characterization of a new murine retinal cell line (MU-PH1) with glial, progenitor and photoreceptor characteristics

Unlike fish and amphibians, mammals do not regenerate retinal neurons throughout life. However, neurogenic potential may be conserved in adult mammal retina and it is necessary to identify the factors that regulate retinal progenitor cells (RPC) proliferative capacity to scope their therapeutic potential. Müller cells can be progenitors for retinal neuronal cells and can play an essential role in the restoration of visual function after retinal injury. Some members of the Toll-like receptor (TLR) family, TLR2, TLR3 and TLR4, are related to progenitor cells proliferation. Müller cells are important in retinal regeneration and stable cell lines are useful for the study of retinal stem cell biology. Our purpose was to obtain a Müller-derived cell line with progenitor characteristics and potential interest in regeneration processes. We obtained and characterized a murine Müller-derived cell line (MU-PH1), which proliferates indefinitely in vitro. Our results show that (i) MU-PH1 cells expresses the Müller cell markers Vimentin, S-100, glutamine synthetase and the progenitor and stem cell markers Nestin, Abcg2, Ascl1, α-tubulin and β-III-tubulin, whereas lacks the expression of CRALBP, GFAP, Chx10, Pax6 and Notch1 markers; (ii) MU-PH1 cell line stably express the photoreceptor markers recoverin, transducin, rhodopsin, blue and red/green opsins and also melanopsin; (iii) the presence of opsins was confirmed by the recording of intracellular free calcium levels during light stimulation; (iv) MU-PH1 cell line also expresses the melatonin MT1 and MT2 receptors; (v) MU-PH1 cells express TLR1, 2, 4 and 6 mRNA; (vi) MU-PH1 express TLR2 at cell surface level; (vii) Candida albicans increases TLR2 and TLR6 mRNA expression; (viii) C. albicans or TLR selective agonists (Pam(3)CysSK(4), LPS) did not elicit morphological changes nor TNF-α secretion; (ix) C. albicans and Pam(3)CysSK(4) augmented MU-PH1 neurospheres formation in a statistically significant manner. Our results indicate that MU-PH1 cell line could be of great interest both as a photoreceptor model and in retinal regeneration approaches and that TLR2 may also play a role in retinal cell proliferation.

Changes in the Photoreceptor Mosaic of P23H-1 Rats During Retinal Degeneration: Implications for Rod-Cone Dependent Survival

Purpose. To investigate the spatiotemporal relationship between rod and cone degeneration in the P23H-1 rat. Methods. Control Sprague-Dawley (SD) and P23H-1 rats of ages ranging from P30 to P365 were used. Retinas were processed for whole mounts or cross sections and rods and cones were immunodetected. We used newly developed image analysis techniques to quantify the total population of L/M cones (the most abundant cones in the rat) and analyzed the rings of rod-cone degeneration. Results. In P23H-1 rats, rod degeneration occurs rapidly: first the rod outer segment shortens, at P30 there is extensive rod loss, and by P180 rod loss is almost complete except for the most peripheral retina. The numbers of L/M cones are, at all postnatal ages, lower in P23H-1 rats than in control SD rats, and decrease significantly with age (by P180). Rod and cone degeneration is spatiotemporally related and occurs in rings that appear already at P90 and spread throughout the entire retina. At P180, the rings of rod-cone degeneration are more abundant in the equatorial retina and are larger in the dorsal retina. Conclusions. This work describes for the first time that in the P23H-1 rat, rod and cone degeneration is spatiotemporally related and occurs in rings. Cone loss follows rod loss and starts very soon, even before P30, the first age analyzed here. The characteristics of the rings suggest that secondary cone degeneration is influenced by retinal position and/or other intrinsic or extrinsic factors.

Correlation between SD-OCT, immunocytochemistry and functional findings in an animal model of retinal degeneration

Purpose: The P23H rhodopsin mutation is an autosomal dominant cause of retinitis pigmentosa (RP). The degeneration can be tracked using different anatomical and functional methods. In our case, we evaluated the anatomical changes using Spectral-Domain Optical Coherence Tomography (SD-OCT) and correlated the findings with retinal thickness values determined by immunocytochemistry.Methods: Pigmented rats heterozygous for the P23H mutation, with ages between P18 and P180 were studied. Function was assessed by means of optomotor testing and ERGs. Retinal thicknesses measurements, autofluorescence and fluorescein angiography were performed using Spectralis OCT. Retinas were studied by means of immunohistochemistry. Results: Between P30 and P180, visual acuity decreased from 0.500 to 0.182 cycles per degree (cyc/deg) and contrast sensitivity decreased from 54.56 to 2.98 for a spatial frequency of 0.089 cyc/deg. Only cone-driven b-wave responses reached developmental maturity. Flicker fusions were also comparable at P29 (42 Hz). Double flash-isolated rod-driven responses were already affected at P29. Photopic responses revealed deterioration after P29.A reduction in retinal thicknesses and morphological modifications were seen in OCT sections. Statistically significant differences were found in all evaluated thicknesses. Autofluorescence was seen in P23H rats as sparse dots. Immunocytochemistry showed a progressive decrease in the outer nuclear layer (ONL), and morphological changes. Although anatomical thickness measures were significantly lower than OCT values, there was a very strong correlation between the values measured by both techniques.Conclusions: In pigmented P23H rats, a progressive deterioration occurs in both retinal function and anatomy. Anatomical changes can be effectively evaluated using SD-OCT and immunocytochemistry, with a good correlation between their values, thus making SD-OCT an important tool for research in retinal degeneration.