Inducible nitric oxide synthase and vascular endothelial growth factor are colocalized in the retinas of human subjects with diabetes

PURPOSE: Nitric oxide (NO) mediates vascular endothelial growth factor (VEGF)-induced angiogenesis and vascular hyperpermeability. This study was undertaken to study the cellular distribution of inducible nitric oxide synthase (iNOS) and VEGF in the retinas from human subjects with diabetes mellitus. In addition, glial reactivity and peroxynitrite generation were detected by immunolocalization of glial fibrillary acidic protein (GFAP) and nitrotyrosine, respectively. METHODS: Eight post-mortem eyes from four consecutive subjects with diabetes mellitus and eight eyes from four subjects without diabetes and without known ocular disease were prospectively collected and examined. We used immunohistochemical techniques and antibodies directed against iNOS, VEGF, GFAP, and nitrotyrosine. RESULTS: In retinas from all subjects without diabetes, weak GFAP immunoreactivity was confined to nerve fibre and ganglion cell layers. There was no immunoreactivity for iNOS, nitrotyrosine, and VEGF. All diabetic retinas showed GFAP induction in Müller cells and GFAP upregulation in nerve fibre and ganglion cell layers. All diabetic retinas showed cytoplasmic immunoreactivity for iNOS, and VEGF in ganglion cells, cells in the inner nuclear layer, and glial cells. In serial sections, ganglion cells and cells in the inner nuclear layer expressing VEGF were localized in the same area of iNOS-expressing ganglion cells and cells in the inner nuclear layer. Six retinas from three subjects with diabetes showed immunoreactivity for nitrotyrosine in vascular endothelial cells in inner retinal layer. CONCLUSIONS: iNOS and VEGF are colocalized in diabetic retinas. Increased GFAP immunoreactivity is a pathological event in the retina during diabetes.     

Expression of apoptosis markers in the retinas of human subjects with diabetes

PURPOSE: To investigate the expression of the apoptotic mediators in the retinas from human subjects with diabetes mellitus. METHODS: Ten donor eyes from five subjects with diabetes mellitus, and eight eyes from four nondiabetic subjects without known ocular disease serving as control subjects were examined. Immunohistochemical techniques were used with antibodies directed against glial fibrillary acidic protein (GFAP), caspase-3, Fas, Fas ligand (FasL), Bax, Bcl-2, survivin, p53, extracellular signal-regulated kinases (ERK1/2), and p38. RESULTS: In retinas from all subjects without diabetes, weak Bcl-2 immunoreactivity was confined to GFAP-positive glial cells in the nerve fiber layer. Weak immunoreactivity for ERK1/2 was noted in a few nuclei in the inner nuclear layer and in a few Müller cell processes. Cytoplasmic immunostaining for survivin was noted in the retinal pigment epithelial cells. There was no immunoreactivity for the other antibodies tested. All diabetic retinas showed cytoplasmic immunoreactivity for caspase-3, Fas, and Bax in ganglion cells. FasL immunoreactivity was detected in GFAP-positive cells. Upregulation of Bcl-2 immunoreactivity was noted in GFAP-positive cells in nerve fiber and ganglion cell layers, and Bcl-2 induction was noted in Müller cell processes. Strong immunoreactivity for ERK1/2 was observed in many nuclei in the inner nuclear layer in GFAP-positive cells in the nerve fiber and ganglion cell layers and numerous Müller cell processes. Survivin immunoreactivity was not altered in the diabetic retinas. There was no immunoreactivity for p53 and p38. CONCLUSIONS: Ganglion cells in diabetic retinas express several proapoptosis molecules, suggesting that these cells are the most vulnerable population. Glial cells in diabetic retinas are activated and express several antiapoptosis molecules in addition to the cytotoxic effector molecule FasL, suggesting a possible role of glial cells in induction of apoptosis in ganglion cells.     

Long-term glial reactivity in rat retinas ipsilateral and contralateral to experimental glaucoma

Although glaucoma is known to alter glial reactivity, the long-term effect of elevated intraocular pressure (IOP) on glial change has not been fully elucidated. This study aimed to examine how chronically elevated IOP induced by episcleral vein cauterization (EVC) in unilateral eyes affect reactivities of astrocytes and Müller cells of rats in the treated as well as contralateral eyes over time. EVC in unilateral eyes of Sprague-Dawley rats were performed to produce chronically elevated IOP. Flat mounted retina preparations were made at several points until 6 months, which were subjected to immunostaining for glial fibrillary acidic protein (GFAP). Retinal homogenates were one- or two-dimensionally electrophoresed, followed by GFAP immunoblotting. EVC significantly increased IOPs up to 27.8 from 13.1 mmHg, which gradually decreased over time. In flat mounted retinas, astrocytes lost but Müller cells gained GFAP immunoreactivity at 3 days after cauterization. The glial changes were partially reversed over time but last even after IOP normalization. In the contralateral eyes, similar glial changes gradually appeared at 1 month after EVC and thereafter. Immunoblotting demonstrated not only molecular size shifts but also alteration of isoelectric focusing of GFAP both in treated and contralateral retina as compared with age-matched control retina. EVC led to opposite reactions in astrocytes and Müller cells in terms of GFAP immunoreactivity. Late-onset glial reactivity also occurred in the contralateral retina.     

Altered expression of retinal occludin and glial fibrillary acidic protein in experimental diabetes. The Penn State Retina Research Group

PURPOSE: To investigate how diabetes alters vascular endothelial cell tight junction protein and glial cell morphology at the blood-retinal barrier (BRB). METHODS: The distribution of the glial marker, glial fibrillary acidic protein (GFAP), and the endothelial cell tight junction protein occludin were explored by immunofluorescence histochemistry in flatmounted retinas of streptozotocin (STZ)-diabetic and age-matched control rats, and in BB/Wor diabetes-prone and age-matched diabetes-resistant rats. RESULTS: GFAP immunoreactivity was limited to astrocytes in control retinas. Two months of STZ-diabetes reduced GFAP immunoreactivity in astrocytes and increased GFAP immunoreactivity in small groups of Müller cells. After 4 months of STZ-induced diabetes, all Müller cells had intense GFAP immunoreactivity, whereas there was virtually none in the astrocytes. BB/Wor diabetic rats had similar changes in GFAP immunoreactivity. Occludin immunoreactivity in normal rats was greatest in the capillary bed of the outer plexiform layer and arterioles of the inner retina but much less intense in the postcapillary venules. Diabetes reduced occludin immunoreactivity in the capillaries and induced redistribution from continuous cell border to interrupted, punctate immunoreactivity in the arterioles. Forty-eight hours of insulin treatment reversed the pattern of GFAP and occludin immunoreactivity in the STZ-diabetic rats. CONCLUSIONS: Diabetes alters GFAP expression in retinal glial cells, accompanied by reduction and redistribution of occludin in endothelial cells. These changes are consistent with the concept that altered glial-endothelial cell interactions at the BRB contribute to diabetic retinopathy.     

Early retinal damage in experimental diabetes: electroretinographical and morphological observations

A growing body of evidence indicates that impairment of retinal function precedes the earliest signs of vascular complications. The aim of this study was to follow the development of retinopathy both functionally and morphologically in a rat model of diabetes mellitus. Diabetes was induced in rats by intravenous injection of streptozotocin (STZ). Age-matched rats raised under similar conditions served as control. The electroretinogram (ERG) was recorded in order to assess retinal function. The expression of glial fibrillary acidic protein (GFAP) in Müller cells was used as a cellular marker for retinal damage. The ERG responses of the diabetic rats were reduced in amplitude compared to the responses recorded from the control rats as early as 2 weeks after onset of diabetes. The b-wave was more affected than the a-wave. GFAP expression in the diabetic retina did not differ from that in the control retina during the first 5 weeks of diabetes. GFAP was demonstrated only in astrocytes in the vitreo-retinal border. After 6-7 weeks of diabetes, GFAP expression in the retinas of the diabetic rats was also detected in the endfeet of the Müller cells. With the progression of diabetes, GFAP expression spreads throughout the entire length of the Müller cells. In the retinas from control rats, GFAP expression was limited to astrocytes and was not detected in Müller cells even at 40 weeks of follow-up. The observations indicate that the functional integrity of retinal cells is compromised already at short time intervals after onset of experimental diabetes in rats.     

Induction of nestin, Ki-67, and cyclin D1 expression in Müller cells after laser injury in adult rat retina

Purpose The purpose was to examine the expression of nestin, Ki-67, and cyclin D1 in Müller cells after laser injury in adult rat retina. Methods The right eyes of adult Brown Norway rats were treated with laser photocoagulation. The eyes were removed 3, 7, and 14 days after laser treatment. The retinas were investigated immunocytochemically by confocal microscopy. Agarose-embedded sections were immunostained with antibodies to nestin, vimentin, glial fibrillary acidic protein (GFAP), glutamate-aspartate transporter (GLAST), rhodopsin, Ki-67, and cyclin D1. Cell death was examined using terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) assay on agarose sections. Results Nestin expression was induced in Müller cells following laser injury. In addition, Ki-67 and cyclin D1 expression was found in the nuclei of Müller cells after the treatment. TUNEL assay demonstrated that Müller cells were not labeled; hence these cells were not apoptotic. Conclusions These results suggest that dedifferentiation and proliferation of Müller cells can be induced by laser injury in adult rat retina.     

Oxidative stress induces heme oxygenase-1 immunoreactivity in Müller cells of mouse retina in organ culture

PURPOSE. Heme oxygenase (HO)-1 immunoreactivity (IR) was examined in normal untreated retina and in retinal explants after in vitro treatment with stress agents. METHODS. Enucleated eyes from young adult C3H mice were immediately fixed and cryosectioned and the retina sections processed for immunocytochemistry with antibodies against HO-1 and glial fibrillary acidic protein (GFAP). From other eyes retinas were isolated and maintained in organ culture, either untreated for 4 days maximum or for 21 hours during which the explants were treated the first 3 hours with selected doses of sodium arsenate or hydrogen peroxide. Thereafter, the explants were processed identically with the normal tissue. RESULTS. In the normal retina, HO-1 and GFAP IR was very low. The culturing itself resulted in an increase in both HO-1 and GFAP immunolabeling in Müller cells of explanted retinas. Both sodium arsenate and hydrogen peroxide further induced strong HO-1 IR in Müller cells but not in other retinal cells. In contrast to HO-1, GFAP staining in Müller cells was not altered as a result of treatment, either by sodium arsenate or hydrogen peroxide at any concentration used. CONCLUSIONS. The results show for the first time that HO-1 can be induced in the retina in vitro by conditions of oxidative stress and that enzyme expression is confined exclusively to Müller cells.     

Müller glial cells of the goldfish retina are phagocytic in vitro but not in vivo.

The role of Müller glial cells in the process of degeneration and regeneration of the goldfish retina is poorly understood. One potential role is phagocytosis of neuronal debris in degenerating retinas. We investigated the phagocytic capacity of Müller glial cells of the goldfish retina both in vitro and in vivo. Müller glial cells from primary or first passage cultures were incubated with latex beads to assess their phagocytic ability, and acridine orange staining was used to identify phagolysosomes in living Müller glial cells. These experiments showed that Müller glial cells are phagocytic in culture. Cell identity was verified with an antibody raised against glial fibrillary acidic protein (GFAP). For the in vivo experiments fluorescent latex beads alone or in combination with the metabolic poison ouabain were injected into the posterior chamber. At various intervals (4 days to 8 weeks) after injection the retinas were prepared for immunocytochemistry. Polyclonal anti-GFAP and NN-1, a monoclonal antibody which recognizes macrophages and microglia within the goldfish retina, were used to identify the phagocytic cells. When the beads were injected into the eye, they were phagocytosed by macrophages/microglia cells but not by Müller cells.     

Glial cell reactivity in a porcine model of retinal detachment

PURPOSE: Detachment of the neural retina from the pigment epithelium causes, in addition to photoreceptor deconstruction and neuronal cell remodeling, an activation of glial cells. It has been suggested that gliosis contributes to the impaired recovery of vision after reattachment surgery that may involve both formerly detached and nondetached retinal areas. Müller and microglial cell reactivity was monitored in a porcine model of rhegmatogenous retinal detachment, to determine whether gliosis is present in detached and nondetached retinal areas. METHODS: Local detachment was created in the eyes of adult pigs by subretinal application of hyaluronate. Retinal slices were immunostained against glial intermediate filaments and K+ and water channel proteins (aquaporin-4, Kir4.1, Kir2.1), and P2Y receptor proteins. In retinal wholemounts, adenosine 5'-triphosphate (ATP)-induced intracellular Ca2+ responses of Müller cells were recorded, and microglial and immune cells were labeled with Griffonia simplicifolia agglutinin isolectin I-B4. K+ currents were recorded from isolated Müller cells. RESULTS: At 3 and 7 days after surgery, Müller cells in detached retinas showed a pronounced gliosis, as revealed by the increased expression of the intermediate filaments glial fibrillary acidic protein and vimentin, by the decrease of Kir4.1 immunoreactivity and of the whole-cell K+ currents, and by the increased incidence of cells that showed Ca2+ responses on stimulation of purinergic (P)2 receptors by ATP. By contrast, the immunohistochemical expression of Kir2.1 and aquaporin-4 were not altered after detachment. The increase in the expression of intermediate filaments, the decrease of the whole-cell K+ currents and of the Kir4.1 immunolabeling, and the increase in the Ca2+ responsiveness of Müller cells were also observed in attached retinal areas surrounding the focal detachment. The density of microglial-immune cells at the inner surface of the retinas increased in both detached and nondetached retinal areas. The immunoreactivities for P2Y1 and P2Y2 receptor proteins apparently increased only in detached areas. CONCLUSIONS: Reactive responses of Müller and microglial cells are not restricted to detached retinal areas but are also observed in nondetached regions of the porcine retina. The gliosis in the nondetached retina may reflect, or may contribute to, neuronal degeneration that may explain the impaired recovery of vision observed in human subjects after retinal reattachment surgery.     

Proliferative gliosis causes mislocation and inactivation of inwardly rectifying K(+) (Kir) channels in rabbit retinal glial cells

Retinal glial (Müller) cells are proposed to mediate retinal potassium homeostasis predominantly by potassium transport through inwardly rectifying K(+) (Kir) channels. Retinal gliosis is often associated with a decrease in glial potassium conductance. To determine whether this decrease is caused by a downregulation of glial Kir channels, we investigated a rabbit model of proliferative vitreoretinopathy (PVR) which is known to be associated with proliferative gliosis. The membrane conductance of control Müller cells is characterized by large Kir currents whereas Müller cells of PVR retinas displayed an almost total absence of Kir currents. In control tissues, Kir2.1 immunoreactivity is localized in the inner stem processes and endfeet of Müller cells whereas Kir4.1 immunoreactivity is largely confined to the Müller cell endfeet. In PVR retinas, there is a mislocation of Kir channel proteins, with Kir4.1 immunoreactivity detectable in Müller cell fibers throughout the whole retina, and a decrease of immunoreactivity in the cellular endfeet. Real-time PCR analysis revealed no alteration of the Kir4.1 mRNA levels in PVR retinas as compared to the controls but a slight decrease in Kir2.1 mRNA. Western blotting showed no difference in the Kir4.1 protein content between control and PVR retinas. The data suggest that proliferative gliosis in the retina is associated with a functional inactivation of glial Kir channels that is not caused by a downregulation of the channel proteins but is associated with their mislocation in the cell membrane.     

GFAP promoter drives Müller cell-specific expression in transgenic mice

PURPOSE: In an attempt to identify Müller cell-specific promoters and to better understand the gene regulatory mechanisms in retinal glial cells, the expression of the glial fibrillary acidic protein (GFAP) gene was studied in Müller cell cultures and in GFAP-enhanced green fluorescent protein (EGFP) transgenic mice. METHODS: A transfection assay of GFAP-luciferase constructs carrying a series of nested deletions was performed in an established Müller cell line. For in vivo analysis, transgenic mice were generated by injecting a construct carrying a 2.5-kb, 5' fragment of the mouse GFAP gene linked to the EGFP gene. Isolated retinas from transgenic mice were screened for GFP expression. Subsequently, the identity of the GFP-expressing cells was established by immunostaining cryostat sections of the retina with antibodies against Müller cell antigenic markers. Induction of the transgene and the endogenous GFAP gene was examined by injecting ciliary neurotrophic factor (CNTF) into the eye. RESULTS: The DNA transfection data suggested that proximal 5' sequences of the GFAP gene are sufficient to direct high-level reporter expression in Müller cell cultures. In transgenic mice, GFP fluorescence appeared in radially oriented processes that spanned almost the entire thickness of the retina. Immunostaining with antibodies to cellular retinaldehyde-binding protein (CRALBP) and glutamine synthetase showed that the GFP-expressing cells were Müller cells. GFP-expressing Müller cells were observed in the retinas of both albino and pigmented transgenic mice. In eyes injected with CNTF, both GFP and GFAP levels were highly elevated. These observations suggest that the 2.5-kb, 5' GFAP sequence can direct inducible reporter gene expression in Müller cells. In addition to Müller cells, a few GFP-labeled astrocytes were present in the adult retina. In the developing retina, GFP-expressing astrocytes were first present at the optic nerve head, and as development progressed, the cells gradually moved toward the periphery of the retina and acquired their adult, stellate morphology. CONCLUSIONS: The present study shows that the 2.5-kb, 5' flanking region of the mouse GFAP gene can be used to express GFP, and possibly other genes, specifically in Müller cells in the mouse retina. Furthermore, expression of the transgene can be upregulated by intravitreal injection of CNTF.     

Nitric oxide synthase expression in ischemic rat retinas

PURPOSE: To investigate the expression of nitric oxide synthase (NOS) in the ischemic retina. METHODS: Retinal ischemia was induced in rats by bilateral common carotid artery occlusion (BCCAO) for various lengths of time. Using the retina after BCCAO, expression of neuronal NOS (nNOS) and inducible NOS (iNOS) and identification of their positive cells were studied by histological and immunohistochemical examinations. RESULTS: Histological examinations revealed significant reduction in the thickness of the inner plexiform layer and the outer plexiform layer of the retina. Expression of nNOS was detected in retinal ganglion cells, amacrine cells, and Müller cells after BCCAO. The expression of nNOS and iNOS detected in Müller cells became stronger and persisted long after BCCAO. CONCLUSIONS: In the ischemic retina, Müller cells and retinal ganglion cells expressed nNOS and iNOS. These phenomena may be involved in the ischemic damage to the retina.     

Experimental dispase-induced retinopathy causes up-regulation of P2Y receptor-mediated calcium responses in Müller glial cells

During proliferative vitreoretinopathy (PVR) Müller glial cells show an up-regulation of their responsiveness to extracellular adenosine 5'-triphosphate (ATP). In the present study, we investigated if such a glial cell response is also a feature for other retinopathies besides PVR. To this aim, the proteolytic enzyme, dispase (0.1 U), was injected into the vitreous of rabbit eyes. After 3 weeks, a distinct retinopathy had developed which showed no signs of PVR. The retinopathy was characterized by strong alterations of the retinal vasculature in the medullary rays, by photoreceptor degeneration, retinal atrophy, and activation of microglial cells. Müller cells became reactive, as indicated by up-regulation of glial fibrillary acidic protein immunoreactivity and by hypertrophy involving subretinal fibrosis. Müller cell reactivity was also evidenced electrophysiologically by a down-regulation of their inwardly rectifying potassium currents and by an up-regulation of their responsiveness to extracellular ATP. Significantly more Müller cells from dispase-treated eyes showed ATP-evoked calcium (83%) and current responses (69%) when compared with cells from control eyes (13 and 9%, respectively). The results indicate that increased responsiveness to extracellular ATP may be a more general feature of Müller cell gliosis, and is also observed in retinopathies besides PVR.     

Glial fibrillary acidic protein increases in Müller cells after retinal detachment

Retinal detachment, separation of the neural retina from the retinal pigment epithelium (RPE), initiates a series of changes in the eye which result in loss of vision if the retina is not rapidly reattached to the RPE. Many of the complex effects of this separation on the cell biology of the retina have yet to be determined. We report here a change in the amount and location of a specific cytoskeletal protein, glial fibrillary acidic protein (GFAP), within Müller cells after retinal detachment. Cat neural retina and RPE were separated by injecting fluid into the extracellular space between the retina and RPE. Normal retinas and retinas detached for 30 days were fixed and embedded for conventional electron microscopy or immunocytochemistry, or homogenized and processed by SDS-PAGE for immunoblot analysis with anti-GFAP. In normal retinas and in attached retinal regions of eyes with retinal detachment, GFAP was detected only in the end feet of the Müller cells as 10 nm diameter filaments and as a diffuse component over the cytoplasm. By contrast, in regions where the retina was detached from the RPE, GFAP was localized throughout the Müller cells in both of these forms. Immunoblots revealed a significant increase in anti-GFAP labeling of a 51,000 MW band from the detached retina.     

Endothelial nitric oxide synthase (eNOS) is localized to Müller cells in all vertebrate retinas.

The distribution of endothelial nitric oxide synthase immunoreactivity (eNOS-IR) was investigated in the retinas of all phylogenetic vertebrate classes by using a monoclonal eNOS antibody. Confocal light microscopy showed immunoreactive labeling in Müller cells of fish, frog, salamander, turtle, chicken, rat, ground squirrel, and monkey retina. In vascularized retinas (rat, monkey), astrocytes and some blood vessels were also stained. Furthermore, eNOS-IR was localized to axon terminals of turtle and fish horizontal cells. These observations are the first to show the presence of eNOS-IR in Muller glia and horizontal cell structures of the vertebrate retina.     

A mouse model of proliferative vitreoretinopathy induced by dispase

A proliferative vitreoretinopathy-like condition induced by intravitreal dispase injection in C57BL/6J mice was studied using ophthalmoscopic and histochemical procedures. The frequency of intravitreal hemorrhage, intravitreal spots, retinal folds and epiretinal membranes was scored by ophthalmoscopic examination at 1, 2, 4, 6 and 8 weeks after the injection. Intravitreal spots corresponded to free cells exhibiting F4/80 immunoreactivity, a macrophage/microglial marker. Retinal folds always appeared before an epiretinal membrane could be observed. Dispase-injected eyes always showed a much higher frequency of folds and membranes than saline-injected eyes. Folds and membranes appeared earlier and were more extensive in the presence of intravitreal hemorrhage than in its absence. Müller retinal cells exhibited significant changes in glial fibrillary acidic protein-immunoreactivity. This was absent in normal Müller cells but, in dispase-injected animals, it was expressed in radial processes at the site of retinal folds, later extending to the whole retina. Both epi- and subretinal membranes contained cells probably derived from Müller cells, since they exhibited co-localization of glial fibrillary acidic protein- and glutamine synthase immunoreactivities. F4/80 was also present in numerous cells within the retina, epi- and subretinal membranes. By contrast, the retinal pigment epithelium cell marker RPE65 was restricted to subretinal membranes. It can be concluded that dispase induced a proliferative vitreoretinopathy-like condition in mice, with a strong contribution of macrophage- and glial-derived cells.     

Temporary impairment of Müller cell metabolism in the rat retina by intravitreal injection of fluorocitrate

Fluorocitrate was injected in the vitreum of rats in order to define the experimental conditions for a temporary impairment of Müller cell metabolism in the retina. Injection of 16 nmol of fluorocitrate appeared to fulfil this requirement since this dose resulted in a large decrease in retinal endogenous glutamine and a smaller decrease in glutamate within 6 hr of administration. The reversible nature of the effect was attested by a substantial recovery of the retinal levels of the two amino acids within 24 hr of injection. In vitro experiments of carbon incorporation from different substrates, carried out with retinas dissected from eyes previously injected with fluorocitrate, were consistent with a metabolic impairment of glial cells, since carbon incorporation from [14C]acetate into glutamine was almost completely abolished in the fluorocitrate-treated retinas. Electron microscopic examination in fluorocitrate-poisoned retinas demonstrated essentially selective ultrastructural alterations of Müller cells at times corresponding to their maximal metabolic impairment. Since Müller cells are by far the largest glial population of the rat retina, common astrocytes being only scattered in the nerve fibre layer, the present experimental model may be used to study the role of Müller cells in the metabolism of retinal neurotransmitters.     

Reduction of ocular blood flow results in glial fibrillary acidic protein (GFAP) expression in rat retinal Müller cells.

Reduction of blood flow to the rat retina was achieved by either clamping both carotid arteries briefly (24 min) or combining clamping of the carotid arteries with permanent occlusion of the vertebral arteries. Analysis of retinas 6 days after operations showed that GFAP immunoreactivity is expressed throughout the retinal Müller cells, although this was variable in retinas from animals where only the carotid arteries were clamped. GFAP immunoreactivity was not associated with retinal Müller cells from control animals and no obvious neuronal damage was observed in retinas from operated animals. These data suggest that Müller-cell GFAP expression may be used as an index to follow possible processes leading to an ischemic insult.     

Changes in intermediate filament immunolabeling occur in response to retinal detachment and reattachment in primates

The immunolabeling patterns for vimentin and glial fibrillary acidic protein (GFAP) were studied in five rhesus monkeys that had undergone retinal detachment or detachment and reattachment. Anti-vimentin and anti-GFAP labeling intensity increased in Müller cells after 2 days of detachment. Weak anti-vimentin labeling of the basal RPE cytoplasm, which was absent in control tissue, was detected 2 days after detachment. After detachment for 7 days and reattachment for 7 or 14 days, the pattern and extent of intermediate filament (IF) labeling changed. In Müller cells, the labeling, which in controls was restricted to processes near the vitreal border of the retina, was present in Müller processes spanning the entire retina. In retinal pigment epithelium (RPE) cells, prominent anti-vimentin labeling was identified in the basal and basolateral cytoplasm. The extent of RPE and Müller cell IF labeling in two animals whose retinas had been detached and then reattached for 150 days was different from that found at either the 7- or 14-day reattachment time points. This suggests that the abnormal IF distribution triggered by detachment may be attenuated after a lengthy period of reattachment.     

Expression and localisation of BDNF, NT4 and TrkB in proliferative vitreoretinopathy

Exogenous brain derived neurotrophic factor (BDNF) is known to rescue ganglion cell death after optic nerve injury. Its mechanism of action is believed to be indirect via glial cells in the retina. In this study we investigated the changes in expression and localisation of BDNF, neurotrophin-4 (NT4) and their common receptor (TrkB) in retinectomy sections of patients with proliferative vitreoretinopathy (PVR). Nine full-thickness retinectomy specimens obtained at retinal reattachment surgery for PVR were fixed in 4% paraformaldehyde immediately after excision and compared to similarly processed normal donor retinas (4 eyes). Agarose-embedded sections (100 microm thick) were double labelled for immunohistochemistry by confocal microscopy, with antibodies against BDNF, NT4, TrkB, rod opsin, glial fibrillary acidic protein (GFAP), cellular retinaldehyde binding protein (CRALBP) and Brn3. This study demonstrates expression of NT4 by ganglion cells and shows expression of BDNF and NT4 in the outer photoreceptor segments is downregulated during PVR, whilst NT4 is markedly upregulated throughout the retina during this condition. The findings here suggest that NT4 may play a neural protective role during the development of PVR. It also shows that upregulation of NT4 in PVR is localised to Müller glial cells, indicating either over-expression of this factor by Müller cells or that Müller cells internalise NT4 for trafficking across the retina. TrkB expression was not observed in PVR retina. The observations that Müller glia demonstrate upregulation of NT4 suggests that retinal injury may lead to activation of this neurotrophin by Müller cells as part of their neuroprotective functions.