Providing physical support to the inner but not the outer retina appears to prevent the tissue collapse resulting from perturbation of the normal biomechanical milieu in the isolated retinal sheet. Using this novel paradigm, gliotic reactions are attenuated and metabolic processes vital for tissue health are preserved, which significantly increases neuronal cell survival. This finding opens up new avenues of adult retinal tissue culture research and increases our understanding of pathological reactions in biomechanically related conditions in vivo.

Signaling through the polymodal cation channel Transient Receptor Potential Vanilloid 4 (TRPV4) has been implicated in retinal neuronal degeneration. To further outline the involvement of this channel in this process, we here explore modulation of Transient Receptor Potential Vanilloid 4 (TRPV4) activity on neuronal health and glial activation in an in vitro model of retinal degeneration. For this purpose, adult porcine retinal explants were cultured using a previously established standard protocol for up to 5 days with specific TRPV4 agonist GSK1016790A (GSK), or specific antagonist RN-1734, or culture medium only. Glial and neuronal cell health were evaluated by a battery of immunohistochemical markers, as well as morphological staining. Specific inhibition of TRPV4 by RN-1734 significantly enhanced ganglion cell survival, improved the maintenance of the retinal laminar architecture, reduced apoptotic cell death and attenuated the gliotic response as well as preserved the expression of TRPV4 in the plexiform layers and ganglion cells. In contrast, culture controls, as well as specimens treated with GSK, displayed rapid remodeling and neurodegeneration as well as a downregulation of TRPV4 and the Müller cell homeostatic mediator glutamine synthetase. Our results indicate that TRPV4 signaling is an important contributor to the retinal degeneration in this model, affecting neuronal cell health and glial homeostasis. The finding that pharmacological inhibition of the receptor significantly attenuates neuronal degeneration and gliosis in vitro, suggests that TRPV4 signaling may be an interesting pharmaceutical target to explore for treatment of retinal degenerative disease.

PURPOSE.To explore the effect of lateral tension as a survival factor for retinal explants in vitro. The central nervous system (CNS) resides in a highly mechanical milieu. However, the importance of biomechanical homeostasis for normal CNS function has not been extensively explored. Diseases in which normal mechanical forces are disrupted, such as retinal detachment of the eye, are highly debilitating and the mechanisms underlying disease progression are not fully understood.METHODS. Using a porcine animal model, we developed a novel technique of culturing adult retinal explants under stretch for up to 10 days in vitro (DIV). These were compared with standard (no stretch) and free-floating cultured explants. Cell survival was analyzed using immunohistochemistry, and retinal architecture using hematoxylin and eosin staining.RESULTS. Compared with unstretched specimens, which at 10 DIV degenerated into a gliotic cell mass, stretched retinas displayed a profound preservation of the laminar retinal architecture as well as significantly increased neuronal cell survival, with no signs of impending gliosis. CONCLUSIONS.The results confirm that biomechanical tension is a vital factor in the maintenance of retinal tissue integrity, and suggest that mechanical cues are important components of pathologic responses within the CNS. (Invest Ophthalmol Vis

Abbreviations: AMD, age-related macular degeneration; BSA, bovine serum albumin; CHOP, C/EBP homology protein; CtBP2, C-terminal binding protein 2; ER, endoplasmic reticulum; GRP78/BiP, glucoseregulated protein 78kDa/Binding immunoglobulin protein, INL, inner nuclear layer; IPL, inner plexiform layer; LC3B, microtubule-associated protein light chain 3B; mTOR, mammalian target of rapamycin; ONL, outer nuclear layer; OPL, outer nuclear layer; PBS, phosphate buffered saline; PNA, peanut agglutinin; p62/SQSTM1; nucleoporin p62/sequestosom 1; PSD-95, postsynaptic density protein 95; rd1, retinal degeneration 1; RPE, retinal pigment epithelium; UPS, ubiquitin-proteasome system; 2 AbstractThe aim of this study was to investigate rod and cone photoreceptor degeneration in organotypic cultures of adult porcine retina. Our hypothesis was that the photoreceptors accumulate opsins, which, together with exposure to cyclic dim light illumination, induce autophagy and endoplasmic reticulum stress (ER-stress) to overcome damaging protein overload. For this purpose, retinas were cultured for 48 h and 72 h during which they were illuminated with dim light for 8 h/day; specimens were analyzed by means of immunohistochemistry, western blot and transmission electron microscopy. ER-stress and photoreceptor degeneration was observed in conventionally cultured retinas. The additional stress in the form of dim light illumination for 8 h/day resulted in increased levels of the ER-stress markers GRP78/BiP and CHOP, as well as increased level of active caspase-12. Increased autophagic processes in cone and rod photoreceptors were detected by LC3B-II increases and occurrence of autophagosomes at the ultrastructural level. Illumination also resulted in altered protein expression for autophagy inducers such as p62 and Beclin-1. Moreover, there was a decrease in phosphorylated mammalian target of rapamycin (mTOR), which further indicate an increase of autophagy. Rod and cone photoreceptors in retinas from a diurnal animal that were exposed to dim light illumination in vitro displayed autophagy and ER-stress processes. In particular, these processes resulted in decreased protein levels for rhodopsin.3

The procedure of culturing retina involves several steps causing severe traumatic effects on the tissue, such as ganglion cell axotomy, interruption of the blood flow as well as separation from the retinal pigment epithelium (RPE). In this paper, we have shown that addition of GDNF in the culture medium attenuates the effect of these steps, resulting in enhanced preservation of several retinal neuronal subtypes. The results may be of importance for research in retinal transplantation where storage time of the donor tissue prior to transplantation is a critical issue.