Inflammatory responses after vitrectomy with vitreous substitutes in a rabbit model.

Purpose: To investigate the inflammatory response of current and future potential vitreous substitutes in an experimental in vivo vitrectomy model.

Methods: Twenty-five gauge pars plana vitrectomy was performed in the right eye of 60 pigmented rabbits, with subsequent injection of 0.5-1.

Retinal neuroinflammatory induced neuronal degeneration - Role of toll-like receptor-4 and relationship with gliosis.

The purpose of this study was to explore retina-intrinsic neuroinflammatory reactions, effects on neuronal survival, relationship with classic gliosis, and possible role of the toll-like receptor 4 (TLR4). To isolate the adult retina from the systemic immune system, a previously described large animal explant culture model was used in which full-thickness porcine retinal sheets can be kept in vitro for extended time periods. Explants were kept for 5 days in vitro (DIV) and were treated with either; lipopolysaccharide (LPS), a Toll-like receptor-4 (TLR4) inhibitor (CLI-095), LPS + CLI-095, or solvent vehicle throughout the culture period after which retinal sections were examined with hematoxylin and eosin staining and extensive immunohistochemistry.

The Role of Mitochondria, Oxidative Stress, and the Radical-binding Protein A1M in Cultured Porcine Retina.

Purpose: The purpose of this study was to explore the relationship between oxidative stress, antioxidant defense, mitochondrial structure, and biomechanical tissue support in the isolated porcine retina.

Methods: Full-thickness retinal sheets were isolated from adult porcine eyes. Retinas were cultured for 2 or 48 h using (1) a previously established low-support explant protocol with photoreceptors positioned against the culture membrane (porous polycarbonate) or (2) a high-support procedure developed by our group, apposing the Müller cell endfeet and inner limiting membrane against the membrane.

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.

Complimentary action: C1q increases ganglion cell survival in an in vitro model of retinal degeneration.

Using a previously described retinal explant culture system as an acute injury model, we here explore the role of C1q, the initiator of the classical complement pathway, in neuronal cell survival and retinal homeostasis. Full-thickness adult rat retinal explants were divided into four groups, receiving the following supplementation: C1q (50nM), C1-inhibitor (C1-inh; Berinert; 500mg/l), C1q+C1-inh, and no supplementation (culture controls). Explants were kept for 12h or 2days after which they were examined morphologically and with a panel of immunohistochemical markers.

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.

In vitro biomechanical modulation--retinal detachment in a box.

Background: 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.

Methods: Adult retinal porcine explants were kept in culture for 3 or 12 hours without any tissue support, simulating clinical RRD, after which they were either maintained in this state or reattached to the culture membrane for an additional 48 hours.

Results: In vitro detachment resulted in gliosis and severe progressive loss of retinal neurons.

Who let the dogs out?: detrimental role of Galectin-3 in hypoperfusion-induced retinal degeneration.

Background: Retinal ischemia results in a progressive degeneration of neurons and a pathological activation of glial cells, resulting in vision loss. In the brain, progressive damage after ischemic insult has been correlated to neuroinflammatory processes involving microglia. Galectin-3 has been shown to mediate microglial responses to ischemic injury in the brain.

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.

First responders: dynamics of pre-gliotic Müller cell responses in the isolated adult rat retina.

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.

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.

Stretch to see: lateral tension strongly determines cell survival in long-term cultures of adult porcine retina.

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.

Effects of glial cell line-derived neurotrophic factor on the cultured adult full-thickness porcine retina.

Background: The tissue culture system offers a possibility to study factors involved in neuronal survival which may be important in a transplantation paradigm. The use of adult tissue in this setting poses specific challenges since traditionally mature neurons survive poorly in vitro. In the current paper, we have explored effects of glial cell line-derived neurotrophic factor (GDNF) on cultures of adult porcine retina.

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.

Vitreous levels of oxidative stress biomarkers and the radical-scavenger α1-microglobulin/A1M in human rhegmatogenous retinal detachment.

Purpose: To explore oxidative stress and the radical scavenger α(1)-microglobulin (A1M) in the vitreous body of human eyes with primary rhegmatogenous retinal detachment (RRD).

Methods: Levels of carbonyl groups, a marker of oxidative stress, and A1M were measured by ELISA and RIA in 14 vitreous samples derived from patients suffering from RRD, and compared with 14 samples from macula hole (MH) patients. Carbonyl group and A1M levels in RRD samples were statistically related to detachment characteristics.

Evaluation of viscoelastic poly(ethylene glycol) sols as vitreous substitutes in an experimental vitrectomy model in rabbits.

The aim of this study was to employ an experimental protocol for in vivo evaluation of sols of 5 wt.% poly(ethylene glycol) (PEG) in phosphate-buffered saline as artificial vitreous substitutes. A 20 gauge pars plana vitrectomy and posterior vitreous detachment were performed in the right eye of eight pigmented rabbits.

Retinal transplantation using surface modified poly(glycerol-co-sebacic acid) membranes.

In retinal transplantation experiments it is hypothesized that remaining diseased photoreceptor cells in the host retina and inner retinal cells in transplants physically obstruct the development of graft-host neuronal contacts which are required for vision. Recently, we developed methods for the isolation of donor photoreceptor layers in vitro, and the selective removal of host photoreceptors in vivo using biodegradable elastomeric membranes composed of poly(glycerol-co-sebacic acid) (PGS). We also coated PGS membranes with electrospun nanofibers, composed of laminin and poly(epsilon-caprolactone) (PCL), to promote attachment of embryonic retinal explants, allowing the resulting composites to be handled surgically as a single entity.

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.

The use of surface modified poly(glycerol-co-sebacic acid) in retinal transplantation.

Retinal transplantation experiments have advanced considerably during recent years, but remaining diseased photoreceptor cells in the host retina and inner retinal cells in the transplant physically obstruct the development of graft-host neuronal contacts which are required for vision. Recently, we developed methods for the isolation of donor photoreceptor layers in vitro, and the selective removal of host photoreceptors in vivo using biodegradable elastomeric membranes composed of poly(glycerol-co-sebacic acid) (PGS). Here, we report the surface modification of PGS membranes to promote the attachment of photoreceptor layers, allowing the resulting composite to be handled surgically as a single entity.

Selective removal of photoreceptor cells in vivo using the biodegradable elastomer poly(glycerol sebacate).

Retinal transplantation experiments have advanced considerably during recent years, but remaining diseased photoreceptor cells in the host retina physically obstruct the development of graft-host neuronal contacts that are required for vision. We here report selective removal of photoreceptors using the biodegradable elastomer poly(glycerol sebacate) (PGS). A 1 × 3 mm PGS membrane was implanted in the subretinal space of normal rabbit eyes, and morphologic specimens were examined with hematoxylin and eosin staining and a panel of immunohistochemical markers.

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 embryonic day (E) 17 or E20 rats 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.

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.