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The purpose of this study was to investigate the adhesion properties and tissue reactions in an in vitro model of nanofabricated membranes emulating the vitreous cortex. Electrospinning was performed for either 5, 10 or 15 min to create various thicknesses of poly(e-caprolactone) (PCL) fibre mats on a poly(glycerol-co-sebacic acid) (PGS) surface. These were fused with adult porcine retinal explants, with the fibre side facing the inner retina, and cultured for 5 days. Adherence was assessed by macroscopic inspection, and morphological and immunohistochemical analysis was performed using haematoxylin and eosin (HE 15 min composite explants adhered only at focal points, were thin and showed extensive degenerative damage. The physical composition of nanofibre meshes is important for adhesion to the inner retina and has a significant impact on neuronal and glial survival in vitro. The results bearing on research involving retinal transplantation are discussed. (Less)

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 0.5 U/ml or 5.0 U/ml for 3 or 24 h. 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.

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.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.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.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.

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. In addition, the culture medium of all explants was assayed for a panel of cytokines at 2 and 5DIV. Compared with in vivo controls, vehicle controls (CT) as well as CLI-095 explants displayed moderate reduction of total thickness and number of retinal neurons with upregulation of glial fibrillary acidic protein (GFAP) throughout the Muller cells. In contrast, LPS and LPS + CLI-095 treated counterparts showed extensive overall thinning with widespread neuronal degeneration but only minimal signs of classical Muller cell gliosis (limited upregulation of GFAP and no downregulation of glutamine synthetase (GS). These specimens also displayed a significantly increased expression of galectin-3 and TGF-beta activated kinase 1 (TAK1). Multiplex proteomic analysis of culture medium at 2DIV revealed elevated levels of IL-1β, IL-6, IL-4 and IL-12 in LPS-treated explants compared to CLI-095 and CT counterparts. LPS stimulation of the isolated adult retina results in substantial neuronal cell death despite only minimal signs of gliosis indicating a retina-intrinsic neuroinflammatory response directly related to the degenerative process. This response is characterized by early upregulation of several inflammatory related cytokines with subsequent upregulation of Galectin-3, TLR4 and TAK1. Pharmacological block of TLR4 does not attenuate neuronal loss indicating that LPS induced retinal degeneration is mediated by TLR4 independent neuroinflammatory pathways.

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 Muller cell endfeet and inner limiting membrane against the membrane. The grafts were analyzed by quantitative polymerase chain reaction (PCR), immunohistochemistry, and transmission electron microscopy (TEM), and culture medium was assayed for the cell damage and oxidative stress markers lactate dehydrogenase and protein carbonyls.RESULTS:In explants cultured with physical support to the inner border, cone photoreceptors were preserved and lactate dehydrogenase levels were reduced, although an initial (2 h), transient, increased oxidative stress was observed. Elevated expression of the antioxidants α1-microglobulin and heme oxygenase-1 was seen in the mitochondria-rich inner segments after 48 h compared to low-support counterparts. Housekeeping gene expression suggested a higher degree of structural integrity of mitochondria in high-support explants, and TEM of inner segments confirmed preservation of a normal mitochondrial morphology.CONCLUSION:Providing retinal explants with inner retinal support leads to mobilization of antioxidant proteins, preservation of mitochondrial function, and increased cell viability. Consequently, the failure of low-support retinal cultures to mobilize an adequate response to the oxidative environment may play a key role in their rapid demise. These findings shed new light on 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 Muller 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.

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

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. Analysis of total protein level, SDS-PAGE, and Western blotting of A1M was also performed. In a separate experiment, mRNA expression of A1M was measured by RT-PCR in rat retina explants. RESULTS: Levels of carbonyl groups and A1M varied widely in RRD vitreous samples, but were significantly higher in samples derived from eyes with large detachment area and macula-off status, while the presence of vitreous hemorrhage did not show any significant correlation. Compared with MH samples, RRD samples displayed significantly higher levels of A1M, whereas changes in total protein levels and carbonyl groups were not significant. Novel forms of A1M, not previously seen in plasma, were found in the vitreous body by Western blotting. Furthermore, A1M expression was seen in rat retina explants and was upregulated after 24 h of culturing. CONCLUSION: Oxidative stress is a prominent feature of human eyes with primary RRD, and is directly related to detachment severity. Affected eyes can launch a protective response in the form of the radical scavenger A1M possibly derived from the retina. The results thus indicate potential therapeutic cell loss prevention in RRD by employing the endogeneous radical scavenger A1M.

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. Seven days postoperatively, a patent separation of the neuroretina and retinal pigment epithelium was found as well as loss of several rows of photoreceptors in combination with massive terminal transferase-mediated dUTP nick-end labeling (TUNEL) staining for apoptosis in the outer nuclear layer. After 28 days, the neuroretina was reattached, the PGS membrane had degraded, and photoreceptors were absent in the implantation area. Activated Müller cells were found in the entire retina in 7-day specimens, and in the implantation area after 28 days. AII amacrine and rod bipolar cell morphology was not affected, except for disrupted dendritic branching, which was present in rod bipolar cells in 28-day specimens. We conclude that retinal detachment induced by the biodegradable PGS membrane creates a permissive environment in which graft–host neuronal connections may be facilitated in future retinal transplantation experiments.

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. Here, we report subretinal transplantation of these composites into adult porcine eyes. In hematoxylin and eosin stained sections of composite explants after 5–7 days in vitro, excellent fusion of retinas and biomaterial membranes was noted, with the immature retinal components showing laminated as well as folded and rosetted areas. The composite grafts could be transplanted in all cases and, 3 months after surgery, eyes displayed clear media, attached retinas and the grafts located subretinally. Histological examination revealed that the biomaterial membrane had degraded without any signs of inflammation. Transplanted retinas displayed areas of rosettes as well as normal lamination. In most cases inner retinal layers were present in the grafts. Laminated areas displayed well-developed photoreceptors adjacent to an intact host retinal pigment epithelium and degeneration of the host outer nuclear layer (ONL) was often observed together with occasional fusion of graft and host inner layers.

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. PGS membranes were chemically modified with peptides containing an arginine-glycine-aspartic acid (RGD) extracellular matrix ligand sequence. PGS membranes were also coated with electrospun nanofiber meshes, containing laminin and poly(epsilon-caprolactone) (PCL). Following in vitro co-culture of biomaterial membranes with isolated embryonic retinal tissue, composites were tested for surgical handling and examined with hematoxylin and eosin staining and immunohistochemical markers. Electrospun nanofibers composed of laminin and PCL promoted sufficient cell adhesion for simultaneous transplantation of isolated photoreceptor layers and PGS membranes. Composites developed large populations of recoverin and rhodopsin labeled photoreceptors. Furthermore, ganglion cells, rod bipolar cells and AII amacrine cells were absent in co-cultured retinas as observed by neurofilament, PKC and parvalbumin labeling respectively. These results facilitate retinal transplantation experiments in which a composite graft composed of a biodegradable membrane adhered to an immature retina dominated by photoreceptor cells may be delivered in a single surgery, with the possibility of improving graft-host neuronal connections.

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. 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). 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. Muller 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. 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.

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. 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. In vitro detachment resulted in gliosis and severe progressive loss of retinal neurons. In contrast, if the explant was reattached, gliosis and overall cell death was attenuated, ganglion cell death was arrested, and the number of transducin-expressing cone photoreceptors increased. These results support the hypothesis that removal of the elastic retina from its normal physical environment results in degenerative damage, and, if restored, rescues retinal neurons. Our study reinforces the notion of a strong relationship between the biomechanical environment and homeostasis within the retina, which has significant bearing on pathologic events related to RRD, and may also have impact on other regions within the CNS under biomechanical influence.

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.

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.

Adult and embryonic rabbit retinal sheets were transplanted into the subretinal space of adult rabbits. The transplants were either full-thickness with intact layering, or gelatin embedded and vibratome sectioned with the inner retina removed. The full-thickness grafts were positioned subretinally by means of a glass capillary in which they were partially folded. The vibratome sectioned ones were placed using a plastic injector in which the gelatin embedded graft was flat. The embryonic full-thickness grafts were followed clinically up to 3 months, and the other 3 transplant types up to 1 month postoperatively, after which the retina was sectioned and stained for light microscopy. Surgical complications were more common in eyes receiving vibratome sectioned grafts with 10 out of 34 eyes displaying blood in the vitreous. Four of these eyes also developed total retinal detachment. Out of 17 eyes receiving full-thickness grafts, only one displayed these complications. Histologically, 11 out of 13 embryonic full-thickness transplants revealed straight, laminated transplants with correct polarity, and with all normal retinal layers present. In these transplants, fusion with the host increased in time. Of the adult full-thickness transplants, only 1 out of 4 survived, and this graft showed signs of degeneration. The vibratome sectioned adult transplants in a few cases survived the first two postoperative weeks. In these grafts, both inner and outer retina were present, indicating an incomplete vibratome sectioning. With longer postoperative times, the number of surviving transplants in this group diminished considerably. All vibratome sectioned embryonic transplants developed into rosettes and sometimes also into laminated sections with reversed polarity. It can be concluded that in rabbits, the surgical technique used for vibratome sectioned transplants requires a larger sclerotomy and retinotomy, since they have to be kept flat in the transplanting instrument due to the surrounding gelatin. This technique is associated with a higher frequency of complications than the one used for full-thickness grafts which are more flexible and can be transplanted with a smaller instrument. Vibratome sectioning of embryonic grafts results in abnormal morphology and their adult counterparts only survive if the sectioning is incomplete. Adult full-thickness grafts show poor survival. Embryonic full-thickness transplants in the majority of cases develop into laminated retinas with layers parallel to the host retinal pigment epithelium. They also survive and integrate well with the host retina.

PURPOSE: To develop an improved surgical technique making full-thickness retinal transplant possible, thereby achieving a normal laminated transplant with minimal rosette formation.METHODS: A total of 23 rabbits underwent vitrectomy, retinotomy, and subsequent subretinal transplant of a complete embryonic neuroretina using a specially crafted glass cannula. Of the 23 animals, 15 received a prenatal day 16 or 19 (E16 or E19) retina; the remaining eight received an E15 retina. The animals were followed from 10 to 35 days, and after this period, the transplants were sectioned and stained for light microscopy.RESULTS: In 11 of the 15 transplants with E16 or E19 donors, histology showed regions up to 1.8 mm of straight, correctly positioned transplants with layering corresponding to their age. The eight animals kept alive longest postoperatively, 31 or 35 days, all showed normal retinal layers, including photoreceptor outer segments appositioned against the host retinal pigment epithelium. Tissue from the youngest donors (E15) yielded less well-organized transplants, indicating a critical stage in retinal embryogenesis before which transplant in this respect is less favorable.CONCLUSIONS: Our procedure makes it possible to transplant embryonic retina to the appropriate position adjacent to the host retinal pigment epithelium, keeping the transplant architecture intact. The transplants show good layering and well-developed photoreceptors abutting the retinal pigment epithelium. (Less)

PURPOSE: The purpose of this study is 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 to standard (no stretch) and free-floating cultured explants. Cell survival was analysed using immunohistochemistry, and retinal architecture using hematoxylin and eosin staining. RESULTS: Compared to unstretched specimens, which at 10 DIV degenerate into a gliotic cell mass, stretched retinas display 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 pathological responses within the CNS.

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.

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 (PBS) as artificial vitreous substitutes. A 20 gauge pars plana vitrectomy and posterior vitreous detachment were performed in the right eye of 8 pigmented rabbits. Approximately 1 mL of the viscoelastic PEG sols were then injected into the vitreous space of 6 eyes. PEG with an average molecular weight of 300,000 g mol(-1) and 400,000 g mol(-1) was used in 2 and 4 eyes respectively. Two eyes received balanced salt solution (BSS) and served as controls. Full-field electroretinography (ERG) and intraocular pressure (IOP, palpation) was measured pre-and post-operatively at regular intervals up to 41 days. The rabbits were sacrificed and the eyes were examined by retinal photography, gross macroscopic examination and histology. The viscoelastic sols were successfully injected and remained translucent throughout the postoperative period, with some inferior formation of precipitates. None of the eyes displayed IOP elevation postoperatively, but in 3 of the PEG sol injected eyes, transient hypotony was noted. One eye sustained a retinal detachment during surgery and another 2 in the postoperative period. ERG recordings confirmed preservation of retinal function in 3 out of 4 eyes injected with 400,000 g mol(-1) PEG. Histological examination revealed upregulation of glial acidic fibrillary protein (GFAP) in Müller cells in PEG sol injected eyes, but normal overall morphology in eyes with attached retinas. The viscosity of the sol was not retained throughout the postoperative period, indicating the demand for polymer crosslinking to increase residence time. The results provide promising preliminary results of the use of PEG hydrogels as a vitreous substitute.

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.

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 Muller 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.

Purpose. In previous studies of subretinal transplantation in rabbits, the host photoreceptor layer seemed to prevent the bridging of neuronal fibers between the graft and the host retina. The current study was undertaken to determine whether the same phenomenon occurs in transplants to the subretinal space of the vascularized retina of rats. Bridging of fibers was examined in transplants to animals of different genetic backgrounds (normal versus dystrophic rats), of different ages, and after different survival times. Methods. Sprague-Dawley (SD) rat retinal tissue from embryonic day (E)18 was subretinally grafted to adult (60-day-old) normal SD rats, to RCS rats (32 and 73 days old), and to adult (60-day-old) transgenic P23H rats. After various survival times (28–183 days), transplanted retinas were processed for routine histology and immunocytochemistry. Antibodies against calbindin, neuronal nitric oxide synthase (NOS), and protein kinase C (PKC) were used to identify specific retinal cell types and their processes. Results. The shape and position of the immunoreactive cell bodies indicated that the expected neuronal populations were labeled within the grafts and in the host retina. Labeled neuronal processes were also observed. In each case, NOS-, calbindin-, and PKC-immunolabeled fibers formed bridges between the graft and the host tissues. However, regardless of the extent of host photoreceptor cell loss, the age of the recipient, or the genetic background, bridging fibers were observed only in areas where the host photoreceptor layer was discontinuous or completely missing. Conclusions. The present study demonstrates that the host photoreceptor layer plays a role in limiting graft–host anatomical integration. (Less)

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. (Less)

The evoked release of purines from rabbit retinae preloaded with [(3)H]adenosine was studied in vitro. Potassium (8.6-43.6 mM) and ouabain (1 or 10 microM) increased the release of radioactivity in a concentration-dependent manner. The K(+)-evoked release was significantly reduced when the superfusion was carried out at 2-4 degrees C. The effect of K(+) (8.6, 13.6 and 23.6 mM) and of ouabain (1 microM) were completely abolished when the retinae were superfused with a Ca(2+)-free medium containing 0.1 mM EGTA. Calcium removal only partially reduced the effect of higher K(+) and ouabain concentrations (43.6 mM and 10 microM, respectively). Further, the effect of K(+) was found to be independent of extracellular Ca(2+) when retinae were pretreated with ouabain for 30 min. Stimulation of the retina with light flashes induced a small, persistent increase in the release of radioactivity observable for several minutes after the end of stimulation. The superfusate contained mainly hypoxanthine and inosine. There were no significant changes in the relative proportions of the different purine compounds released before or in response to either K(+) (23.6 mM) or ouabain (10 microM) stimulation. Potassium stimulation significantly increased the release of adenosine, inosine and hypoxanthine. Addition of the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), significantly increased the relative proportions of released endogenous adenosine and inosine. The results indicate that K(+) stimulation induces the release of purines from the rabbit retina by a Ca(2+)- and energy-dependent process. Light flashes also induce a purine release. The results suggest an active role for adenosine in retinal neurotransmission.

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. Therefore, we wanted to explore the contribution of Galectin-3 (Gal-3) to hypoperfusion-induced retinal degeneration in mice. Methods Gal-3 knockout (Gal-3 KO) and wildtype (WT) C57BL/6 mice were subjected to chronic cerebral hypoperfusion by bilateral narrowing of the common carotid arteries using metal coils resulting in a 30% reduction of blood flow. Sham operated mice served as controls. After 17 weeks, the mice were sacrificed and the eyes were analyzed for retinal architecture, neuronal cell survival, and glial reactivity using morphological staining and immunohistochemistry. Results Hypoperfusion caused a strong increase in Gal-3 expression and microglial activation in WT mice, coupled with severe degenerative damage to all retinal neuronal subtypes, remodeling of the retinal lamination and Müller cell gliosis. In contrast, hypoperfused Gal-3 KO mice displayed a retained laminar architecture, a significant preservation of photoreceptors and ganglion cell neurons, and an attenuation of microglial and Müller cell activation. Conclusion Moderate cerebral blood flow reduction in the mouse results in severe retinal degenerative damage. In mice lacking Gal-3 expression, pathological changes are significantly attenuated. Gal-3 is thereby a potential target for treatment and prevention of hypoperfusion-induced retinal degeneration and a strong candidate for further research as a factor behind retinal degenerative disease. Electronic supplementary material The online version of this article (doi:10.1186/s12974-015-0312-x) contains supplementary material, which is available to authorized users.