Specific inhibition of TRPV4 enhances retinal ganglion cell survival in adult porcine retinal explants

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

In vitro biomechanical modulation-retinal detachment in a box.

To illustrate the importance of biomechanical impact on tissue health within the central nervous system (CNS), we herein describe an in vitro model of rhegmatogenous retinal detachment (RRD) in which disruption and restoration of physical tissue support can be studied in isolation

Autophagy and ER-stress contribute to photoreceptor degeneration in cultured adult porcine retina.

The 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 48h and 72h during which they were illuminated with dim light for 8h/day; specimens were analyzed by means of immunohistochemistry, Western blot, real-time polymerase chain reaction (PCR) 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 8h/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. As no alteration of rhodopsin mRNA was observed, autophagy and ER-stress are suggested to decrease rhodopsin protein at the posttranscriptional level

Biochemical and biomechanical modulation of the in vitro retina

The porcine retinal explant culture paradigm offers the possibility to study retinal health, disease and development independently from the surrounding tissues of the eye. Retina-intrinsic effects of molecular and mechanical cues on the isolated retinal sheet can thus be studied with great precision. For the purpose of creating a donor tissue optimized for transplantation, we used this system to explore the impact of biochemical modulation on cell survival and embryogenesis. Further, biomechanical modulation and its significance in retinal homeostasis was investigated. Biochemical modulation of the embryonic retina using glutamate revealed an accelerated photoreceptor maturation coupled with increased developmental apoptosis in the inner layers. In adult tissue, glial cell-derived neurotrophic factor (GDNF) treatment was found to increase photoreceptor cell survival and attenuate gliosis. These results demonstrate the potential of biochemical modulation in the creation of a donor tissue well adapted for retinal transplantation. Biomechanical modulation through emulation of the tissue tension and inner retinal support found in the living eye was found to significantly increase neuronal survival and preserve glial structure and function in adult explants. The results demonstrate the importance of the biomechanical milieu for retinal homeostasis and provide an important link between glial cell health and neuronal preservation. In conclusion, we have shown that biochemical and biomechanical modulation of the porcine retina in vitro can be used to modify development and enhance cell survival. The findings provide further understanding of mechanisms behind retinal disease and reveals new avenues in the search for treatment of degenerative retinal disease

N-methyl-N-nitrosourea-induced neuronal cell death in a large animal model of retinal degeneration in vitro

N-methyl-N-nitrosourea (MNU) has been reported to induce photoreceptor-specific degeneration with minimal inner retinal impact in small animals in vivo. Pending its use within a retinal transplantation paradigm, we here explore the effects of MNU on outer and inner retinal neurons and glia in an in vitro large animal model of retinal degeneration. The previously described degenerative culture explant model of adult porcine retina was used and compared with explants receiving 10 or 100 μg/ml MNU (MNU10 and MNU100) supplementation. All explants were kept for 5 days in vitro, and examined for morphology as well as for glial and neuronal immunohistochemical markers. Rhodopsin-labeled photoreceptors were present in all explants. The number of cone photoreceptors (transducin), rod bipolar cells (PKC) and horizontal cells (calbindin) was significantly lower in MNU treated explants (p <0.001). Gliosis was attenuated in MNU10 treated explants, with expression of vimentin, glial fibrillary protein (GFAP), glutamine synthetase (GS), and bFGF comparable to in vivo controls. In corresponding MNU100 counterparts, the expression of Müller cell proteins was almost extinguished. We here show that MNU causes degeneration of outer and inner retinal neurons and glia in the adult porcine retina in vitro. MNU10 explants display attenuation of gliosis, despite decreased neuronal survival compared with untreated controls. Our results have impact on the use of MNU as a large animal photoreceptor degeneration model, on tissue engineering related to retinal transplantation, and on our understanding of gliosis related neuronal degenerative cell death

First Responders: Dynamics of Pre-Gliotic Müller Cell Responses in The Isolated Adult Rat Retina.

Abstract Purpose: To explore the early reactions of the retinal Müller glia in response to retinal insult prior to gliotic remodeling and the sustained upregulation of intermediate filament glial fibrillary acidic protein (GFAP), which has traditionally been considered the most sensitive early indicator of reactive gliosis. Methods: To study pre-gliotic events, we used a model of adult rat retinal explants and related the dynamic expression of GFAP as well as apoptosis, to four key regulators of retinal homeostasis (glutamine synthetase (GS), cellular retinaldehyde binding protein (CRALBP), basic fibroblast growth factor (bFGF), carbonic anhydrase II (CAII)) using immunohistochemistry. Results: We found that a sustained GFAP upregulation couple with gliotic remodeling occurred comparatively late and that this phenomenon was preceded by an initial upregulation followed by depletion of GS, CRALBP, bFGF and CAII in retinal Müller cells. The initial increase of the regulatory proteins, seen after 1-12 h, preceded a first phase of moderate apoptosis, and their depletion after 48 h was followed by massive apoptosis and widespread GFAP upregulation in the Müller cells at 5 days. Conclusion: We conclude that, in the explant model, changes in the expression of the four homeostatic regulatory proteins as well as apoptotic cell death precedes sustained GFAP upregulation and reactive gliosis. Müller cell reactivity has been linked to several retinal conditions, and the herein provided novel information on the dynamics of pre-gliotic events in the lesioned retina may help us understand important pathological mechanisms crucial for future therapeutic intervention

Feet on the ground: Physical support of the inner retina is a strong determinant for cell survival and structural preservation in vitro.

Purpose: The purpose of this study was to explore the importance of local physical tissue support for homeostasis in the isolated retina. Methods: Full-thickness retinal sheets were isolated from adult porcine eyes. Retinas were cultured for 5 or 10 days using the previously established explant protocol with photoreceptors positioned against the culture membrane (porous polycarbonate) or the Müller cell endfeet and inner limiting membrane (ILM) apposed against the membrane. The explants were analyzed morphologically using hematoxylin and eosin staining, immunohistochemistry, TUNEL labeling, and transmission electron microscopy (TEM). Results: Standard cultures displayed a progressive loss of retinal lamination and extensive cell death, with activated, hypertrophic Müller cells. In contrast, explants cultured with the ILM facing the membrane displayed a maintenance of the retinal laminar architecture, and a statistically significant attenuation of photoreceptor and ganglion cell death. TEM revealed intact synapses as well as preservation of normal cellular membrane structures. Immunohistochemistry showed no signs of Müller cell activation (GFAP), with maintained expression of important metabolic markers (GS, bFGF). Conclusion: 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

Exogenous Glutamate Modulates Porcine Retinal Development in vitro.

Embryogenesis of the retina is a complex event orchestrated by a multitude of physical and biochemical signals. To study the impact of intrinsic developmental cues, the retinal tissue can be isolated in culture which also enables modulation of normal development for other purposes, i.e. transplantation of specific neuronal cell types. In the present experiment, cell type development of immature porcine retinal tissue kept in culture was explored using specific immunohistochemical markers. Retinal explants were either kept under standard culture conditions or supplemented with glutamate and their morphology was compared with in vivo controls of corresponding age. After 15 days in vitro (DIV), E45 retinal explants displayed several signs of atypical development when compared with E60 in vivo controls. First, an accelerated photoreceptor differentiation was evident, seen in sections labeled with antibodies directed against recoverin, rhodopsin and synaptophysin. Second, apoptotic cells in the inner retina were more prevalent in the cultured retinas (TUNEL). Rod photoreceptor differentiation as well as inner retinal apoptosis was even more pronounced in glutamate-supplemented specimens in which they occurred already at 8 DIV. Müller cell, vimentin and GFAP expression was not affected in any of the cultured retinas. These results suggest that normal retinal embryogenesis is more dependent on tissue extrinsic factors than what has been deduced from previous small animal experiments. Glutamate, which has been identified as an important regulator of cell cycle exit, may also be important for photoreceptor differentiation and induction of developmental apoptosis. Insights into retinal cell type differentiation under in vitro conditions is of interest from a biological standpoint, and the possibility of modulation of this process is valuable for research directed towards cell replacement in retinal degenerative disease

Scaffolding the retina: The interstitial extracellular matrix during rat retinal development.

PURPOSE: To examine the expression of interstitial extracellular matrix components and their role during retinal development. MATERIAL AND METHODS: Fibronectin (FN), collagen IV (Coll IV) and laminin 5 (Lam 5) expression in rat retinas from developmental stages E17 to adult were studied. In addition, PN5 full-thickness retinas were cultured for 7 days with dispase, which selectively cleaves FN and Coll IV, at either 0.5U/ml or 5.0U/ml for 3 or 24h. Eyecups and retinal cultures were examined morphologically using hematoxylin and eosin staining and immunohistochemistry. RESULTS: Coll IV, Lam 5 and FN were all transiently expressed in the interstitial matrix of the retinal layers during development. The retinal layers in dispase treated explants was severely disturbed in a dose and time dependent manner. CONCLUSIONS: FN, Lam 5 and Coll IV, are present in the interstitial extracellular matrix during rat retinal development. Enzymatic cleavage of FN and Coll IV early in the lamination process disrupts the retinal layers implicating their pivotal role in this process