Steven K. Fisher

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.

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.

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.

Synaptic Connexions Made by Horizontal Cells within the Outer Plexiform Layer of the Retina of the Cat and the Rabbit

Two ultrastructurally distinctive types of horizontal cells are described in the retinae of the cat and the rabbit. Evidence is presented that they have different synaptic connexions in the outer plexiform layer. The majority of the presynaptic structures identified in the outer plexiform layer of the rabbit (as defined on page 320) belong to a neurofilamentous type of horizontal cell. It is suggested that the cat may be the same. No synapses have been identified on to, or from, the second, predominantly neurotubular, type of horizontal cell. No chemical synapses on to, or between, horizontal cells have been found. Thus input of this kind to both types of horizontal cells is as yet only known to be from the photoreceptors. All positively identified postsynaptic processes were the dendrites or perikarya of bipolar cells. Other cell types that are possibly pre- or postsynaptic in the outer plexiform layer are discussed.

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.

Interplexiform Cells of the Mammalian Retina and their Comparison with Catecholamine-Containing Retinal Cells

Retinal interplexiform cells have processes that branch within both the inner and outer plexiform layers. Their morphology is described from Golgi-preparations of cat, rhesus macaque and squirrel monkey retinae. Comparisons are made with similar cells, known to be catecholamine-containing, which have been observed histofluorometrically in the teleost fish and New World monkeys. It is concluded that there may be more than one pharmacological type of interplexiform cell. In addition an inner nuclear layer plexus of fibres is described for the first time from Golgi-material of the squirrel monkey’s retina. Electron microscopy reveals that this plexus synapses within the inner nuclear layer on to bipolar and amacrine cells. It is compared with the catecholamine-containing inner nuclear layer plexus of New World monkeys.