Geoffrey P. Lewis

Electrophysiologic and retinal penetration studies following intravitreal injection of bevacizumab (Avastin).

Purpose: Intravitreal bevacizumab (Avastin; Genentech Inc., San Francisco, CA) is a new treatment for age-related macular degeneration. The aim of this study was to evaluate retinal penetration and toxicity of bevacizumab. Methods: Ten albino rabbits were injected intravitreally with 0.1 mL (2.5 mg) of Avastin into one eye and 0.1 mL saline into the fellow eye. The electroretinogram (ERG) was recorded after 3 hours, 3 days, and 1, 2, and 4 weeks. The visual evoked potential (VEP) was recorded after 4 weeks. Confocal immunohistochemistry was used to assess retinal penetration. Results: The ERG responses of the control and experimental eyes were similar in amplitude and pattern throughout the follow-up period. The flash VEP responses of the experimental eyes were of normal pattern and amplitude and did not differ from those recorded by stimulation of the control eye alone. Full thickness retinal penetration was present at 24 hours and was essentially absent at 4 weeks. Conclusions: Bevacizumab was found to be nontoxic to the retina of rabbits based on electrophysiologic studies. Full thickness retinal penetration may explain observed clinical effects of intravitreal bevacizumab. Although it is difficult to directly extrapolate to humans, our study supports the safe use of intravitreal bevacizumab injection.

Cellular remodeling in mammalian retina: results from studies of experimental retinal detachment.

Retinal detachment, the separation of the neural retina from the retinal pigmented epithelium, starts a cascade of events that results in cellular changes throughout the retina. While the degeneration of the light sensitive photoreceptor outer segments is clearly an important event, there are many other cellular changes that have the potential to significantly effect the return of vision after successful reattachment. Using animal models of detachment and reattachment we have identified many cellular changes that result in significant remodeling of the retinal tissue. These changes range from the retraction of axons by rod photoreceptors to the growth of neurites into the subretinal space and vitreous by horizontal and ganglion cells. Some neurite outgrowths, as in the case of rod bipolar cells, appear to be directed towards their normal presynaptic target. Horizontal cells may produce some directed neurites as well as extensive outgrowths that have no apparent target. A subset of reactive ganglion cells all fall into the latter category. Muller cells, the radial glia of the retina, undergo numerous changes ranging from proliferation to a wholesale structural reorganization as they grow into the subretinal space (after detachment) or vitreous after reattachment. In a few cases have we been able to identify molecular changes that correlate with the structural remodeling. Similar changes to those observed in the animal models have now been observed in human tissue samples, leading us to conclude that this research may help us understand the imperfect return of vision occurring after successful reattachment surgery. The mammalian retina clearly has a vast repertoire of cellular responses to injury, understanding these may help us improve upon current therapies or devise new therapies for blinding conditions.

Müller cell and neuronal remodeling in retinal detachment and reattachment and their potential consequences for visual recovery: a review and reconsideration of recent data

Recent evidence suggests that the adult mammalian retina is far more plastic than was previously thought. Retinal detachment induces changes beyond the degeneration of outer segments (OS). Changes in photoreceptor synapses, second- and even third-order neurons may all contribute to imperfect visual recovery that can occur after successful reattachment. Changes that occur in Müller cells have obvious effects through subretinal fibrosis and proliferative vitreoretinopathy, but other unidentified effects seem likely as well. Reattachment of the retina induces its own set of responses aside from OS re-growth. Reattachment halts the growth of Müller cell processes into the subretinal space, but induces their growth on the vitreal surface. It also induces the outgrowth of rod axons into the inner retina.

Limiting photoreceptor death and deconstruction during experimental retinal detachment: the value of oxygen supplementation ☆

PURPOSE To assess the role of hypoxia in causing the death and deconstruction of photoreceptors in detached retinas and the effectiveness of supplemental oxygen in limiting such damage. METHODS Retinal detachment was induced surgically in the right eye of each of 10 cats. The cats were allowed to survive surgery for 3 days. Two were kept for these 3 days in normoxia (room air, 21% oxygen) and eight in hyperoxia (70% oxygen). The retinas were examined for cell death by use of labels for normal and fragmenting DNA, with antibodies and a cone sheath-specific lectin to demonstrate the status of their inner and outer segments, the synaptic structures of the outer plexiform layer, and the distribution of basic fibroblast growth factor (bFGF) and with in situ hybridization to demonstrate bFGF mRNA. RESULTS Retinal detachment without oxygen supplementation caused the death of some photoreceptors; the loss of cytochrome oxidase from the inner segments and the collapse of the outer segments of surviving photoreceptors; the loss of synaptophysin profiles from the outer plexiform layer; and the loss of bFGF protein from retinal neurons and neuroglia but not from retinal vessels. Oxygen supplementation (hyperoxia) during detachment mitigated all these changes, reducing photoreceptor death, maintaining the specialized structures of surviving photoreceptors, and stabilizing the bFGF within the retina. CONCLUSIONS In experimental retinal detachment, hypoxia caused by the separation of outer retina from its normal source of nutrients is a factor in inducing the death and deconstruction of photoreceptors as well as in the loss of bFGF from the detached retina. Hyperoxia offered to human patients between diagnosis of retinal detachment and surgery may enhance the function of the reattached 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.

Rapid changes in the expression of glial cell proteins caused by experimental retinal detachment

We examined the expression of several proteins normally present in Müllers glia after the production of experimental retinal detachment in adult cats. Retinas were detached for one-half to seven days, after which the tissue was processed for correlative immunocytochemistry and biochemistry. Previous studies demonstrated that the intermediate filament proteins glial fibrillary acidic protein and vimentin, increase after long-term retinal detachment (30 to 60 days), whereas glutamine synthetase, carbonic anhydrase C, and cellular retinaldehyde-binding protein all decrease to barely detectable levels. Alterations in Müller cell protein expression are rapid and specific events that can be detected as early as two days after retinal detachment. By seven days, levels of protein expression are similar to those in the long-term retinal detachments. Within the first week after injury the Müller cell processes hypertrophy and begin forming glial scars, which indicates that early intervention may be required to halt or reverse the effects of detachment.

Limiting the proliferation and reactivity of retinal Müller cells during experimental retinal detachment : The value of oxygen supplementation

PURPOSE To assess the role of hypoxia in inducing the proliferation, hypertrophy, and dysfunction of Muller cells in detached retina and the effectiveness of supplemental oxygen in limiting these reactions. METHODS Retinal detachments were produced in the right eye of each of 13 cats; the cats survived surgery for 3 days, during which six were kept in normoxia (room air, 21%) and seven in hyperoxia (70% oxygen). Retinas were labeled for proliferation with an antibody (MIB-1) to a cell cycle protein (Ki-67), for evidence of hypertrophy employing antibodies to the intermediate filament protein glial fibrillary acidic protein (GFAP) and to beta-tubulin and for disturbance of glutamate neurochemistry employing antibodies to glutamate to a glutamate receptor (GluR-2) and to glutamine synthetase. RESULTS Results from the two animals kept in normoxia after retinal detachment confirmed previous reports that detachment caused the proliferation of Muller cells, the hypertrophy of Muller cell processes, and the disruption of glutamate recycling by Muller cells. Oxygen supplementation during detachment reduced Muller cell proliferation and hypertrophy and reduced the abnormalities in the distributions of glutamate, GluR-2, and glutamine synthetase. CONCLUSIONS Oxygen supplementation reduced the reaction of retinal Muller cells to retinal detachment, limiting their proliferation and helping to maintain their normal structure and function. In the clinical setting, oxygen supplementation between diagnosis and reattachment surgery may reduce the incidence and severity of glial-based complications, such as proliferative vitreoretinopathy.

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.