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

Cell Death by SecTRAPs: Thioredoxin Reductase as a Prooxidant Killer of Cells

BACKGROUND: SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins) can be formed from the selenoprotein thioredoxin reductase (TrxR) by targeting of its selenocysteine (Sec) residue with electrophiles, or by its removal through C-terminal truncation. SecTRAPs are devoid of thioredoxin reductase activity but can induce rapid cell death in cultured cancer cell lines by a gain of function. PRINCIPAL FINDINGS: Both human and rat SecTRAPs killed human A549 and HeLa cells. The cell death displayed both apoptotic and necrotic features. It did not require novel protein synthesis nor did it show extensive nuclear fragmentation, but it was attenuated by use of caspase inhibitors. The redox active disulfide/dithiol motif in the N-terminal domain of TrxR had to be maintained for manifestation of SecTRAP cytotoxicity. Stopped-flow kinetics showed that NADPH can reduce the FAD moiety in SecTRAPs at similar rates as in native TrxR and purified SecTRAPs could maintain NADPH oxidase activity, which was accelerated by low molecular weight substrates such as juglone. In a cellular context, SecTRAPs triggered extensive formation of reactive oxygen species (ROS) and consequently antioxidants could protect against the cell killing by SecTRAPs. CONCLUSIONS: We conclude that formation of SecTRAPs could contribute to the cytotoxicity seen upon exposure of cells to electrophilic agents targeting TrxR. SecTRAPs are prooxidant killers of cells, triggering mechanisms beyond those of a mere loss of thioredoxin reductase activity

Cell Type Differentiation Dynamics in the Developing Porcine Retina.

The dynamics of retinal embryogenesis have been well characterized previously in terms of cell proliferation, genesis and migration, whereas overall cell type differentiation within the retinal layers has been less thoroughly explored. In the present study, phenotypical differentiation of all 7 major retinal cell types was examined in the developing porcine retina using one cell-specific immunohistochemical marker per cell type. At the end of the first trimester at E39 (39 days after gestation), neurofilament labeled ganglion cells, recoverin labeled photoreceptors, vimentin labeled Müller cells and synaptophysin labeled presynaptic vesicles were found. Rhodopsin labeled rod photoreceptors were present at E60, whereas cone transducin labeled cone photoreceptors were not seen until E99. Differentiation of inner nuclear cells coincided with the appearance of the retinal layers at E70-E99 with the presence of parvalbumin labeled amacrine cells, calbindin labeled horizontal cells and PKC labeled rod bipolar cells. At postnatal day 4, all retinal subtypes except for cone photoreceptors displayed a labeling pattern corresponding to the one found in the adult porcine retina. The immunohistochemical labeling pattern suggests that phenotypic differentiation of the 7 principal retinal cell types in the porcine retina follows a central-to-peripheral spatio-temporal gradient similar to the one reported for cell proliferation and genesis. Differentiation of the non-laminated retinal cell mass appears to be initiated at its outer and inner margins and progresses inwards, a process which ends in the formation of the characteristic plexiform and nuclear layers. The dynamics of retinal cell type differentiation are of interest from a biological standpoint and are also important for therapeutical strategies in retinal degenerative disease

Transplantation of full-thickness retina in the normal porcine eye: surgical and morphologic aspects.

PURPOSE: To report a surgical technique for transplantation of full-thickness neuroretinal sheets into the subretinal space of a large animal with a vascularized retina and to establish the light microscopic morphology of such specimens. METHODS: Twelve normal pigs underwent transplantation of a neuroretinal sheet from a neonatal donor into the subretinal space by means of a vitrectomy-based technique. After a survival of 33 to 72 days, eye specimens were studied with a light microscope. RESULTS: In most eyes, the transplants displayed a laminated morphology, with photoreceptor outer segments facing the host retinal pigment epithelium. These grafts had normal outer retinal layers, while the inner layers were less developed. The host retina straddling the graft showed evidence of photoreceptor degeneration, but the inner layers were well preserved. CONCLUSION: Full-thickness neuroretinal sheets can be transplanted to the subretinal space of a large animal eye with a vascularized retina. The grafts survive well and display mostly photoreceptors, which in combination with the well-preserved host inner retina may be of importance in attempts at reconstructing the retina in photoreceptor degenerative disease

Neuroretinal xenotransplantation to immunocompetent hosts in a discordant species combination.

In spite of its immune privileged state, xenotransplantation within the CNS is associated with rapid graft destruction in immunocompetent hosts. Efforts to enhance graft survival have mostly focused on host immune response, whereas relatively little attention has been paid to donor tissue characteristics. In the present paper, we explore long-term survival of xenogeneic full-thickness neuroretinal transplants in immunocompetent hosts and investigate the significance of tissue integrity in relation to graft survival. Adult rabbits receiving no immunosuppression were used as hosts and fetal Sprague-Dawley rat neuroretina as donors. Using vitreoretinal surgical techniques, rabbits received either a full thickness or a fragmented neuroretinal graft to the subretinal space of one eye. Eyes receiving full-thickness grafts were examined morphologically after 91 days and fragmented grafts after 7-14 days. Surviving full thickness grafts were found in six of eight eyes, four of which displayed the normal laminated appearance. Major histocompatibility complex (MHC) up-regulation in surviving grafts was minimal and they contained a well-organized photoreceptor layer, protein kinase C (PKC) labeled rod bipolar cells, parvalbumin labeled AII amacrine cells and glial fibrillary acidic protein (GFAP) labeled Müller cells. Fragmented grafts (n=6) were all destroyed or showed severe signs of rejection. A mass of inflammatory cells derived from the choroid was evident in these specimens, and no labeling of retina-specific cells was seen. We conclude that full-thickness rat neuroretina can survive for several months after subretinal transplantation to the subretinal space of immunocompetent rabbits, while fragmented counterparts are rapidly rejected. Surviving full-thickness grafts can develop many of the normal retinal morphological characteristics, indicating a thriving relationship between the initially immature donor tissue and its foreign host. Our results strongly indicate that donor tissue integrity is a crucial factor for graft survival in CNS xenotransplantation

Immune privilege of allogeneic neuroretinal transplants in the subconjunctival space.

BACKGROUND: The extent of site and tissue-associated immune privilege is of great interest in transplantation experiments involving the CNS. In the present paper we have explored neuroretinal immune privilege by transplantation to a non-immune privileged site. METHODS: Fetal and adult full-thickness rabbit neuroretinal grafts were placed in the subconjunctival space of immunocompetent rabbit hosts. Morphological examination was performed after 2-31 days (fetal grafts, n = 46), and after 8 days (adult grafts, n = 4). RESULTS: Hematoxylin and eosin-stained sections and immunohistochemistry directed against microtubule-associated protein 2 (MAP2) revealed surviving grafts containing retinal neurons in the majority of eyes with fetal grafts. In all specimens, a mild inflammatory reaction was evident as seen with major histocompatibility complex class II (MHC-II) labeling. Short-term grafts survived well and displayed lamination and rosette formation whereas older grafts appeared more disorganized and were more often rejected. Müller cell fibers labeled with glial fibrillary acidic protein (GFAP) were present in grafts from 15 days and onwards. Adult grafts were destroyed after 8 days. CONCLUSIONS: Allogeneic fetal full-thickness neuroretinal transplants can survive for several weeks in a non-immune privileged environment in which adult grafts are rapidly rejected. Fetal grafts gradually shrink, lose their architecture and go through a glial transformation accompanied by low-grade inflammation. The rabbit neuroretina thus appears to enjoy partial immune privilege, the extent of which depends on the development state of the tissue. The characterization of neuroretinal immune privilege will hopefully influence future clinical trials of retinal transplantation

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

Isolation of photoreceptors in the cultured full-thickness fetal rat retina.

Purpose. To create a retina consisting mainly of photoreceptors for future use as donor tissue in retinal transplantation. Methods. Fetal full-thickness neuroretinas from Sprague Dawley rats 17 (E17) or 20 (E20) days post conception were placed in culture for 7 or 14 days. Explants and age-matched control retinas were examined by light microscopy and with a panel of immunohistochemical markers labeling all seven of the major retinal cell types. Results. E17 and E20 control retinas displayed vimentin labeled Muller cells, NF160 labeled ganglion cells and synaptic vesicles labeled with synaptophysin. The remaining cell types were found in control specimens of postnatal age 2 days and older. After 7 or 14 days in culture, all explants were significantly thinner than their aged-matched controls, and displayed multiple rows of cells organized in a single layer. Within this layer, they contained rhodopsin labeled rod photoreceptors, presynaptic vesicles and vertically arranged Muller cells. Transducin labeled cone photoreceptors were found in all but the youngest explants. Scattered PKC labeled rod bipolar cells and calbindin labeled horizontal cells were found in the inner part of most explants whereas beta-III-tubulin labeled ganglion cells and parvalbumin labeled amacrine cells were seen only sporadically. No NF160 labeled ganglion cells were found. Conclusions. Fetal full-thickness rat retina in vitro develops into a retina consisting of predominantly synapse containing cone and rod photoreceptors embedded in a scaffold of well organized Muller cells. These explant retina characteristics are well adapted for use as donor tissue in future retinal transplantation experiments