Page A. Erickson

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.

Changes in the expression of specific Müller cell proteins during long-term retinal detachment.

Retinal detachments were produced in domestic cats by injecting fluid between the retinal pigment epithelium and neural retina. Retinas were allowed to remain detached for 30 or 60 days at which time the animals were killed. Tissue areas from detached and attached retinal regions from the same eye were processed for correlative biochemical and structural analysis, i.e. SDS-PAGE and Western blots of tissue homogenates were correlated with tissue processed for postembedding immunoelectron microscopy. Antibodies to six proteins were used as probes. Glial fibrillary acidic protein in Müller cells has previously been shown to increase after retinal detachment; here we show that vimentin, another intermediate filament protein present in Müller cells, also increases after detachment. In contrast, cellular retinaldehyde binding protein, cellular retinol binding protein, glutamine synthetase, and carbonic anhydrase C--all normally found in Müller cells--decrease after detachment. The results of this study indicate that retinal Müller cells dramatically alter their expression of proteins in response to retinal detachment.

Basic fibroblast growth factor: a potential regulator of proliferation and intermediate filament expression in the retina

Proliferation of astrocytes, and a concomitant increase of intermediate filaments in astrocytes are two fundamental responses of the CNS to injury. We have previously identified these two events in the retinas response to detachment of the neural retina from the adjoining monolayer of retinal pigmented epithelium. In order to analyze the potential role of basic fibroblast growth factor (bFGF) in these responses, we studied cellular proliferation and intermediate filament protein expression in the retinas of cats and rabbits 4 d and 4 weeks after a single intravitreal injection of 1 microgram of bFGF. Our results show that bFGF stimulates both of these processes in an otherwise normal eye. The eyes that received bFGF had significantly elevated numbers of 3H-thymidine-labeled Muller cells, astrocytes, vascular cells, retinal pigmented epithelial cells, microglia, and macrophages by comparison to control eyes. This proliferation was apparent at 4 d after the injection of bFGF but not after 4 weeks. In control eyes, antibodies to glial fibrillary acidic protein and vimentin labeled intermediate filaments only in the inner (vitread) portion of the Muller cells, the specialized radial astrocytes that span the width of the retina. In eyes that had been injected with bFGF, almost the entire Muller cell cytoplasm was labeled at 4 d after injection; after 4 weeks, the cytoplasmic labeling intensity had increased significantly. Release or activation of endogenous stores of bFGF after injury or disease may be involved in the control of cellular proliferation and intermediate filament expression in the retina and elsewhere in the CNS.

Fluorouracil therapy for proliferative vitreoretinopathy after vitrectomy.

Fluorouracil effectively inhibits epiretinal membrane formation and traction retinal detachment after vitrectomy surgery. When 0.5 mg of fluorouracil was administered intraocularly every 24 hours for seven days, traction retinal detachment two weeks after the intraocular injection of 200,000 cultured retinal pigment epithelial cells occurred in 12 of 12 control eyes but in only six of 14 eyes treated with fluorouracil (P less than .001). Four weeks after cell injection, eight of 12 eyes treated with fluorouracil had traction retinal detachments whereas 12 of 12 control eyes did (P less than .001). The height of the traction retinal detachment four weeks after intraocular injection of 200,000 cultured retinal pigment epithelial cells was reduced 50% in eyes treated with 0.5 mg of fluorouracil every 24 hours for seven days compared to control eyes (P less than .001). When the number of injected retinal pigment epithelial cells was increased to 400,000 cells and 1.25 mg of fluorouracil was administered intraocularly every 24 hours for seven days, traction retinal detachment two weeks after injection occurred in 15 of 15 eyes in the control group but in none of ten eyes in the treated group. Four weeks after cell injection, eight of eight eyes in the control group and five of five eyes in the fluorouracil-treated group had detachments and the mean height of the detachments in the two groups was equal. Autoradiography of the epiretinal membranes in eyes injected with 200,000 cultured retinal pigment epithelial cells and labeled for two hours with tritiated thymidine showed that 0.8% of the epiretinal cell nuclei were labeled two weeks after cell injection but that no labeled cells were present in the fluorouracil-treated eyes. Tritiated thymidine labeling of epiretinal cells in the fluorouracil-treated eyes was first noted three weeks after the cell injection. The presence of tritiated thymidine labeling in the fluorouracil-treated eyes correlated with an increase in the number of epiretinal cells and an increase in the incidence of traction retinal detachment.

Ocular Toxicity of Fluorouracil after Vitrectomy

The retinal and corneal toxicity of fluorouracil in the rabbit eye after lensectomy and vitrectomy depended on both the dosage and the frequency of intraocular injection and was reversible at certain dosages. All eyes in Group 1 (1.25 mg of fluorouracil every 12 hours for four days and then every 24 hours for three days) had opaque corneas by three days; these did not clear for four weeks. Histologic studies showed loss of photoreceptor outer segments and loss of ribosomes in all the retinal cells examined. The electroretinographic b-wave decreased to 0% of the baseline value (no b-wave), and did not recover after three weeks. In Group 2 eyes (1.25 mg of fluorouracil every 24 hours for seven days), corneal opacification increased to a maximum after two weeks and gradually decreased by four weeks. The electroretinographic b-wave diminished to 9.6% of the baseline value at two weeks but later recovered to 62.5% of the baseline value at three weeks. Histologic studies showed loss of photoreceptor outer segments and ribosomes at nine days; both returned to near normal after five weeks. Clinical, electrophysiologic, and histologic studies showed no toxicity in Group 3 eyes (0.5 mg of fluorouracil every 24 hours for seven days). This dosage of fluorouracil exerts a significant antiproliferative effect on injected retinal pigment epithelial cells and is well tolerated by the rabbit eye.

An immunocytochemical comparison of Müller cells and astrocytes in the cat retina.

Immunocytochemical localization, at the light and electron microscopic levels, of five different known glial proteins was used to compare Müller cells with astrocytes in the adult cat retina. Retina from two different areas of the eye was examined. A region of retina on the border of the optic nerve was used because of its large population of astrocytes, and a region away from the optic nerve was used to examine Müller cells (astrocytes are sparse in this region). Antibodies to cellular retinaldehyde binding protein and glutamine synthetase labeled the Müller cells but not the astrocytes, while labeling with anti-carbonic anhydrase C, anti-alpha crystallin and anti-glial fibrillary acidic protein was found in both Müller cells and astrocytes.

Rod and cone disc shedding in the rhesus monkey retina: a quantitative study.

Abstract Four Rhesus monkeys were maintained for several months on a daily lighting schedule of 12 hr light and 12 hr darkness (12L:12D). The animals were then fixed by intracardiac perfusion at 1 and 5 hr following the onset of light, and at 1 and 5 hr after the offset of light. The number of phagosomes/mm of retinal pigment epithelium (RPE) was counted in the light microscope for both foveal and extrafoveal locations. At extrafoveal sites, phagosomes within the RPE cell bodies and those within the ensheathing processes bordering cone outer segments were counted separately. Counts at foveal and extrafoveal locations suggest that the rate of disc shedding, and therefore the rate of membrane replacement, is probably less for foveal cones than it is for rods. The number of phagosomes above the rod tips 1 hr following light onset and their diminution several hours later suggests that an increase in rod shedding, similar to the “burst” of rod shedding reported for other species, also exists in the monkey retina. The number and location of phagosomes 5 hr after the offset of light indicates that there could be another phase of increased rod shedding somewhere around the middle of the dark period. Phagosome counts in the foveal RPE were highest 5 hr after light offset, however the counts at 1 and 5 hr following light onset were nearly as high. The number of RPE phagosomes at both foveal and extrafoveal locations was lowest 1 hr after light offset. Counts of phagosomes within the sheaths of extrafoveal cones showed that about 20% contained one or more phagosomes within their sheaths. Of those sheaths that did contain phagosomes, approximately 25% had multiple phagosomes. There appears to be a considerable period of time during which extrafoveal cone phagosomes remain within the ensheathing processes, moving from the cone tips toward the apical RPE surface. Rod and cone disc shedding in the monkey retina are probably cyclic processes—although the temporal pattern of shedding may be different than that reported for other species.

Opsin distribution and protein incorporation in photoreceptors after experimental retinal detachment

The distribution of opsin was examined immunocytochemically after experimental retinal detachment in adult cats. Retinal detachments were produced by injecting fluid between the retinal pigment epithelium and neural retina. One to 60 days later the animals were killed. Tissue areas from detached and attached retinal regions from the eye with the detached retina, as well as normal (control) retinas, were processed for post-embedding light and electron microscopic immunocytochemistry. In normal and attached retinal regions, anti-opsin labeled the outer segments and Golgi apparatus most heavily, although the entire photoreceptor plasma membrane was labeled at a low level. Beginning at 2 days after retinal detachment, immunolabeling increased in the photoreceptor inner segment, cell body and synaptic terminal plasma membranes. This pattern of anti-opsin labeling continued at all intervals up through the 60-day detachment time-point. Injection of radiolabeled amino acid in detachments from 1 to 30 days show that radiolabeled protein is still transported to the truncated outer segments of the photoreceptor cells. In addition, these outer segment disks label with anti-opsin. These data imply that opsin continues to be transported and incorporated into the outer segments of photoreceptors showing severe degeneration as a result of long-term detachment from the RPE.

Glial fibrillary acidic protein and its mRNA: Ultrastructural detection and determination of changes after CNS injury☆

We have previously demonstrated that glial fibrillary acidic protein (GFAP) containing intermediate filaments in retinal Müller cells undergo both quantitative induction and subcellular reorganization as a response to long-term retinal detachment (an induced CNS degeneration wherein the Müller cells form a multicellular scar). This study demonstrates by RNA blotting analysis that normal retina expresses a low basal level of GFAP mRNA, which is induced approximately 500% within 3 days of retinal detachment. At the cellular level, electron microscopic in situ hybridization analysis readily detects GFAP mRNA in Müller cells of detached retinas, but not in normal retinas. On the other hand, GFAP mRNA was readily detected in retinal astrocytes (which appear to express GFAP mRNA at high, constitutive levels). In both cell types, the ultrastructural localization of GFAP mRNA was the same. In the nuclei, the GFAP mRNA was associated with amorphous, electron-dense regions within the euchromatin. In the cytoplasm, the GFAP mRNA was associated with intermediate filaments near the nuclear pores, along the filaments when no other structures were apparent, and when the filaments appeared to be associated with ribosomes and polysomes. The ultrastructural location of the GFAP mRNA (especially along the intermediate filaments) may be unique to this mRNA or may represent a more generalized mRNA phenomenon.

Glial fibrillary acidic protein (GFAP) immunoreactivity in rabbit retina: Effect of fixation

The binding of monoclonal and polyclonal antibodies to glial fibrillary acidic protein (GFAP) antigenic sites in the rabbit retina was shown to be sensitive to aldehyde fixation. In chemically unfixed retina, the polyclonal anti-GFAP labeled Müller cells, astrocytes, and unidentified profiles in the outer plexiform layer; the monoclonal anti-GFAP labeled Müller cell endfeet and astrocytes only. The outer plexiform layer label with the polyclonal antibody was lost after fixation for 1 hr in 1% paraformaldehyde; elsewhere, the label was reduced. Fixation also reduced labeling by the monoclonal antibody. Such fixation sensitivity may underlie the different patterns reported for retinal GFAP immunoreactivity in the literature.