Esther Zemel

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.

Retinal toxicity of intravitreal kenalog in albino rabbits.

Purpose: To evaluate possible toxicity of intravitreal Kenalog (commercial triamcinolone acetonide) to the retina of albino rabbits. Methods: Forty-three albino rabbits were injected intravitreally with 0.1 mL of experimental solution to the right eye and 0.1 mL of saline to the left eye (control). Rabbits in Group A (n = 28) were injected with 4 mg/0.1 mL of Kenalog suspension; rabbits in Group B (n = 8) were injected with 0.1 mL of Kenalog vehicle; and rabbits in Group C (n = 7) were injected with 4 mg/0.1 mL of triamcinolone acetonide. Rabbits were examined ophthalmoscopically and by electroretinogram (ERG) recordings before and at different time intervals after injection. At the end of follow-up, animals were killed and the retinas were prepared for light microscopy. Results: Thirty-eight rabbits completed 4 weeks of follow-up. Follow-up for 8 and 17 weeks was completed by 29 and 3 rabbits, respectively. Intravitreal commercial Kenalog or its vehicle alone caused approximately 50% reduction in the ERG b-wave amplitude at the end of follow-up. Pure triamcinolone acetonide caused only mild (up to 14%) reduction of the ERG b-wave amplitude. Histologic examination of retinas exposed to Kenalog or its vehicle showed severe damage to all retinal layers in areas close to the site of Kenalog injection. Conclusions: Intravitreal injection of 4 mg Kenalog suspension is retinotoxic to albino rabbit eyes. The vehicle of Kenalog is probably the main cause of this toxicity.

NADPH diaphorase activity in mammalian retinas is modulated by the state of visual adaptation.

NADPH diaphorase histochemistry is commonly used to identify cells containing nitric oxide synthase (NOS), the enzyme catalyzing the production of nitric oxide from L-arginine. NADPH diaphorase activity and NOS immunostaining was demonstrated in different cells of the vertebrate retina; photoreceptors, horizontal cells, amacrine cells, ganglion cells, and Müller cells. However, the physiological role of nitric oxide (NO) in the retina has yet to be elucidated. In this study, we tested the assumption that NADPH diaphorase activity in the retinas of rabbits and rats depended on the state of visual adaptation. In the rabbit, light adaptation enhanced NADPH diaphorase activity in amacrine cells and practically eliminated it in horizontal cells. Dark adaptation induced the opposite effects; the NADPH diaphorase activity was reduced in amacrine cells and enhanced in horizontal cells. Retinas from eyes that were injected intravitreally with L-glutamate exhibited a pattern of NADPH diaphorase activity that was similar to that seen in dark-adapted retinas. In rats, the NADPH diaphorase activity of amacrine and horizontal cells exhibited adaptation dependency similar to that of the rabbit retina. But, the most pronounced effect of dark adaptation in the rats retina was an enhancement of NADPH diaphorase activity in Müller cells, especially of the endfoot region. Assuming that NADPH diaphorase activity is a marker for NOS, these findings suggest that NO production in the mammalian retina is modulated by the level of ambient illumination and support the notion that NO plays a physiological role in the retina.

Physiological and toxicological effects of cefuroxime on the albino rabbit retina.

PURPOSE Intracameral cefuroxime was found to lower the risk of endophthalmitis after cataract surgery. The purpose of this study was to evaluate the retinal toxicity of cefuroxime in a rabbit model. METHODS Twenty-two albino rabbits were divided into two cefuroxime groups: low-dose (1mg/0.1 mL, n = 9) and high dose (10 mg/0.1 mL, n = 13). The right eye of each rabbit was injected with 0.1 mL cefuroxime solution (experimental eye) and the left eye with 0.1 mL saline (control eye). Electroretinogram (ERG) responses were recorded at 3 hours, 4 days, and 1, 2, and 4 weeks after injection. After 4 weeks, the rabbits were euthanized, the eyes were enucleated, and the retinas were prepared for histologic evaluation and GFAP immunostaining. RESULTS No functional (ERG) or histologic damage was found in rabbits in the low-dose group. In the high-dose group, a significant decrease in the ERG amplitudes of the experimental eyes was seen 3 hours after injection, followed by partial recovery during 4 weeks of follow-up. Retinal histology of experimental eyes revealed marked damage. GFAP immunoreactivity in Müller cells was expressed in rabbits belonging to both groups, although it was more extensive in the high-dose group. CONCLUSIONS ERG and histologic findings indicated that a dose of 1 mg cefuroxime, administered intravitreally, was not toxic to the rabbit retina. A dose of 10 mg, injected intravitreally, induced transient physiological effects, and was toxic to the rabbit retina, as was evident by the permanent reduction in the ERG responses and by the structural damage to the retina with signs of glial activation.

Towards multifocal ultrasonic neural stimulation II: design considerations for an acoustic retinal prosthesis

Ultrasound waves, widely used as a non-invasive diagnostic modality, were recently shown to stimulate neuronal activity. Functionally meaningful stimulation, as is required in order to form a unified percept, requires the dynamic generation of simultaneous stimulation patterns. In this paper, we examine the general feasibility and properties of an acoustic retinal prosthesis, a new vision restoration strategy that will combine ultrasonic neuro-stimulation and ultrasonic field sculpting technology towards non-invasive artificial stimulation of surviving neurons in a degenerating retina. We explain the conceptual framework for such a device, study its feasibility in an in vivo ultrasonic retinal stimulation study and discuss the associated design considerations and tradeoffs. Finally, we simulate and experimentally validate a new holographic method--the angular spectrum-GSW--for efficient generation of uniform and accurate continuous ultrasound patterns. This method provides a powerful, flexible solution to the problem of projecting complex acoustic images onto structures like the retina.

Development of laser-induced retinal damage in the rabbit.

Abstract · Background: Laser lesions may induce retinal damage that is larger than expected from the size of the coagulated area. This study was designed to follow the development of laser-induced reduction in retinal function and to correlate it with structural changes. · Methods: Pigmented rabbits were treated in one eye with 225 argon laser lesions. The ERG responses were recorded at different times after treatment. The effect of the laser treatment upon the functional integrity of the retina was assessed from the ERG responses. Structural damage was examined by light microscopy. · Results: Shortly (1–2 h) after laser treatment, the ERG responses were reduced by about 50%. ERG deficit continued to develop and reached a maximal level about 24 h after treatment. Thereafter, slow recovery was observed but permanent deficit, relative to the initial laser effect, was seen even 30 days after treatment. Histological observations indicated extensive serous retinal detachment between laser lesions that developed within 24 h after treatment. At 30 days post-treatment, lesioned areas were completely destroyed and heavily pigmented. The retina between lesions was attached to the pigment epithelium but exhibited different degrees of structural damage. · Conclusions: The immediate laser damage is confined to the coagulated areas while secondary functional damage develops within 24 h and probably reflects serous retinal detachment between lesions. The serous retinal detachment completely resolves with time but may induce permanent structural abnormalities in non-coagulated retinal areas that is reflected in a functional deficit larger than the initial laser effect.

Safety evaluation of repeated intravitreal injections of bevacizumab and ranibizumab in rabbit eyes.

Purpose: Repeated intravitreal injections of ranibizumab or bevacizumab are a common treatment for several retinal diseases. The aim of the present study was to evaluate the long-term retinal toxicity of repeated injections of ranibizumab and bevacizumab in rabbits. Methods: Albino rabbits were injected intravitreally with ranibizumab (1 mg/0.1 mL) or bevacizumab (2.5 mg/0.1 mL) into the right eye, whereas the left eye of each rabbit was injected with saline. Nine consecutive injections were administered at 14-day intervals. Electroretinographic responses and flash visual-evoked potentials were recorded periodically. After 18 weeks of follow-up, the rabbits were killed, and the retinas were prepared for morphologic examination and for immunocytochemistry for glial fibrillary acidic protein. Results: Electroretinographic and visual-evoked potential responses of the experimental and control eyes were similar in amplitude and pattern throughout the follow-up period. The histopathologic studies yielded similar results. No retinal damage was observed in the experimental and control eyes of all rabbits. Glial fibrillary acidic protein immunoreactivity showed staining only in retinal astrocytes but not in Müller cells in all rabbits. Conclusion: The electrophysiological tests and the morphologic data indicate that the repeated intravitreal injections of ranibizumab or bevacizumab have no cumulative long-term toxic effect on the retina in rabbits.

Retinal toxicity of gentamicin after subconjunctival injection performed adjacent to thinned sclera.

OBJECTIVE To investigate the potential toxicity to the retina of gentamicin injected near surgically thinned scleral areas in a rabbit model. DESIGN Experimental study. METHODS Scleral scraping to half thickness was performed in the superotemporal scleral area in both eyes of adult rabbits (n = 10). Gentamicin sulfate was injected subconjunctivally to the right eye and saline to the left eye, which always served as a control eye. Four weeks after the procedure, electroretinography (ERG) was performed to assess retinal function. Then, the eyes were enucleated and prepared for histologic evaluation of structural damage. In four eyes of two additional rabbits, vitreous gentamicin concentrations were measured using a fluorescence polarization assay. MAIN OUTCOME MEASURES Dark- and light-adapted ERG responses and histopathologic damage. RESULTS Dark- and light-adapted ERG responses in all rabbits were similar in the experimental and control eyes. Gentamicin levels were more than 10 microg/ml after subconjunctival injection of gentamicin with scraping and 0.29 microg/ml after subconjunctival injection of gentamicin with no scraping. Histopathologic examination revealed significant local damage to the photoreceptors adjacent to the area of scraping and subconjunctival injection. A significantly lesser degree of damage was seen if gentamicin was injected in pigmented rabbits or in albino rabbits, but only 4 weeks after scleral scraping. CONCLUSIONS Increased penetration of gentamicin through thinned sclera may lead to toxic levels of the drug in a localized area adjacent to the site of injection. These toxic effects are also influenced by the degree of pigmentation and acute inflammation.

The effects of lidocaine and bupivacaine on the rabbit retina

The toxic action of two commercial anesthetics, lidocaine and bupivacaine, on the functional and morphologic integrity of the retina was investigated in albino and pigmented rabbits. The experimental drug was injected into the vitreous of one eye, while saline solution was injected into the fellow eye. Retinal function was assessed from the electroretinogram and the visual evoked potential. Retinal structure was examined at the light microscopic level. Ten milligrams of lidocaine did not affect the electroretinogram and the visual evoked potential responses, though structural damage could be detected close to the site of injection. A lower dose of 5 mg did not produce any detectable physiologic or morphologic damage. The only dose of bupivacaine used, 0.5 mg, was not toxic to the albino and pigmented rabbit retinas, as assessed by the electroretinogram, visual evoked potential, and light microscopy. The results of this study demonstrate that lidocaine and bupivacaine are nontoxic to the rabbit retina at concentrations that are effective for retrobulbar anesthesia.

Safety of intravitreal bevacizumab in the developing rabbit retina.

Purpose: The purpose of this was to study the long-term effects of a single intravitreal dose of bevacizumab injected at different postnatal age on retinal function and development of retinal blood vessels in the adult albino rabbit. Methods: Bevacizumab was injected into the right eye of newborn rabbits, aged 11 days to 25 days, whereas the left eye of each rabbit was injected with identical volume of saline and served as control. The electroretinogram was recorded 1 week and 10 weeks after injection. Visual evoked potentials were recorded 10 weeks after injection. At termination of the follow-up period, the rabbits were killed and the retinas were prepared for histopathologic studies at the light microscopic level, for glial fibrillary acidic protein immunoreactivity, and for reduced form of nicotinamide adenine dinucleotide phosphate diaphorase histochemistry to assess the integrity of the retinal vascular system. Results: The electroretinogram responses demonstrated normal retinal function in adult rabbits injected at postnatal age of 11 days to 25 days. Mean Vmax and σ values calculated for each group of rabbits, 10 weeks after bevacizumab injection, indicated similar retinal function of the experimental and control eyes. Visual evoked potentials recorded by stimulating each eye separately were also similar. The histopathologic studies yielded similar results; no structural retinal damage was observed in the experimental eyes compared with the control eyes of rabbits from all age groups, and no increased glial fibrillary acidic protein immunoreactivity in Müller cells was observed in the experimental eyes. Staining of blood vessels with reduced form of nicotinamide adenine dinucleotide phosphate diaphorase revealed decreased branching of the capillary network in the experimental eyes compared with the control eyes in all age groups. Conclusion: The electroretinographic and morphologic data showed no deleterious effects of a single intravitreal dose of bevacizumab, injected during the first 30 days postnatally, on the structural and functional integrity of the sensory retina in the adult rabbit. Even partial blockage of vascular endothelial growth factor with bevacizumab applied during retinal development seems to interfere with the development of the retinal vascular system in the albino rabbit. However, extrapolation from rabbits to humans should be made with caution.