ATF3 Regulates the Expression of AChE During Stress.

Acetylcholinesterase (AChE) expresses in non-cholinergic cells, but its role(s) there remain unknown. We have previously attributed a pro-apoptotic role for AChE in stressed retinal photoreceptors, though by unknown mechanism. Here, we examined its promoter only to find that it includes a binding sequence for the activating transcription factor 3 (ATF3); a prototypical mediator of apoptosis. This suggests that expression of AChE could be regulated by ATF3 in the retina. Indeed, ATF3 binds the AChE-promoter to down-regulate its expressions in vitro. Strikingly, retinas of "blinded" mice display hallmarks of apoptosis, almost exclusively in the outer nuclear layer (ONL); coinciding with elevated levels of AChE and absence of ATF3. A mirror image is observed in the inner nuclear layer (INL), namely prominent levels of ATF3 and lack of AChE as well as lack of apoptosis. We conclude that segregated patterns of expressions of ATF3 reflect its ability to repress apoptosis in different layers of the retina-a novel mechanism behind apoptosis.

Infliximab exerts a dose-dependent effect on retinal safety in the albino rabbit.

PURPOSE: To assess the retinal toxicity of an intravitreal injection of infliximab, a monoclonal antibody to tumor necrosis factor α, in a rabbit model. MATERIALS AND METHODS: Two groups of adult albino rabbits (n = 5) received intravitreal injections of infliximab (0.1 ml) in the study eye and balanced salt solution (BSS, 0.1 ml) in the control eye at baseline. Group 1 was administered with 1.5 mg/0.1 ml, and group 2 was injected with 7.5 mg/0.1 ml of infliximab solution. Electroretinography (ERG) was performed at baseline and at 1, 7, 30, and 45 days after the injection. Visual evoked potentials (VEPs) were recorded at 7 and 45 days after the injection. After the last electrophysiological assessment, the rabbits were euthanized and retinal histopathology and immunhistochemistry for glial fibrillary acidic protein (GFAP) were performed. RESULTS: ERG responses demonstrated no significant deficit in retinal function in eyes injected with infliximab. Mean dark-adapted a-wave and b-wave maximal amplitude and semi-saturation constant values at baseline and throughout the 45 days of follow-up after the injection indicated no remarkable difference in outer retinal function between the control and experimental eyes. VEP responses were similar at each time point (7 and 45 days). No difference was seen in retinal histopathology and immunocytochemistry sections in eyes receiving the 1.5 mg/0.1 ml dose compared to the control eyes. However, increased GFAP labeling in retinal Müller cells was detected in rabbit eyes treated with the 7.5 mg/0.1 ml dose. CONCLUSIONS: Intravitreal injection of 1.5 mg/0.1 ml infliximab dose has no toxic effect on the integrity (functional or structural) of the retina in rabbits. A higher dose of 7.5 mg/0.1 ml may be slightly toxic as suggested by positive Müller cell GFAP expression. Additional studies of retinal toxicity at higher doses and after multiple injections are needed to establish the retinal safety of intravitreal infliximab therapy in humans.

Light Modulates Ocular Complications in an Albino Rat Model of Type 1 Diabetes Mellitus.

PURPOSE: The purpose of the study was to assess potential interactions of light exposure and hyperglycemia upon ocular complications in diabetic rats. METHODS: Streptozotocin-induced (STZ-induced) diabetic rats (N = 39) and non-diabetic rats (N = 9) were distributed into eight groups according to the irradiance and color of the light phase during the 12/12-hour light/dark regime. Follow-up lasted 90 days and included assessment of cataract development and electroretinogram (ERG) recordings. Stress to the retina was also assessed by glial fibrillary acidic protein immunocytochemistry. RESULTS: Cataract development was fast in diabetic rats that were exposed to unattenuated white light or to bright colored lights during the light phase. Diabetic rats that were kept under attenuated brown or yellow light during the light phase exhibited slower rate of cataract development. Electroretinogram responses indicated very severe retinal damage in diabetic rats kept under bright colored lights in the blue-yellow range or bright white light during the light phase. Electroretinogram damage was milder in rats kept under bright red light or attenuated yellow or brown light during the light phase. Glial fibrillary acidic protein expression in retinal Müller cells was consistent with ERG assessment of retinal damage. CONCLUSIONS: Attenuating white light and filtering out short wavelengths have a protective effect on the eyes of diabetic rats as evident by slower rate of cataract formation and a smaller degree of retinal damage. TRANSLATIONAL RELEVANCE: Our findings suggest that special glasses attenuating light exposure and filtering out short wavelengths (400-530 nm) may be beneficial for diabetic patients.

Retinal toxicity of intravitreal rituximab in albino rabbits.

PURPOSE: To evaluate retinal toxicity of intravitreal rituximab. METHODS: Twelve albino rabbits were included in the study. 1 mg/0.1 mL of rituximab was injected to the right (experimental) eye of each rabbit, and 0.1 mL of saline was injected into the left (control) eye. Electroretinogram was recorded before injection and 3 hours, 4 days, 1 week, 2 weeks and 4 weeks after injection (12 rabbits), and visual evoked potential was recorded before injection and 4 weeks after injection (10 rabbits). Histology and glial fibrillary acidic protein immunocytochemistry (12 rabbits) were performed at 4 weeks after injection. Clinical examination was conducted at all time points in all rabbits. RESULTS: The average dark-adapted electroretinogram b-wave Vmax ratios, and the average light-adapted b-wave amplitude ratios were approximately 1, and the average log σ difference was around zero throughout the follow-up period. The average visual evoked potential amplitude ratio and the average visual evoked potential implicit time difference were approximately 1 and 0, respectively. No histologic damage was seen, but glial fibrillary acidic protein was mildly expressed in 6 of 12 experimental eyes. Results of clinical examination were normal in all the eyes. CONCLUSION: Intravitreal injection of 1 mg rituximab does not cause functional or histologic signs of retinal toxicity in albino rabbits. Mild glial fibrillary acidic protein expression in Müller cells probably indicates a mild degree of retinal stress.

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.

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.

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.

Normal physiological and pathophysiological effects of trypan blue on the retinas of albino rabbits.

PURPOSE: To determine whether intravitreal injection of trypan blue is toxic to the retina of the albino rabbit. METHODS: Sixteen albino rabbits were studied for the effects of intravitreal trypan blue (eight with 0.06% solution and eight with 0.15% solution). Saline was injected into the fellow control eye of all rabbits. The electroretinogram and visual evoked potentials were recorded from each rabbit at different time intervals after injection. The rabbits were killed at the termination of the follow-up period, and their retinas were prepared for histologic examination under light microscopy. RESULTS: In all rabbits, short-term follow-up showed significant reduction of ERG responses in the experimental eye, with the b-wave more affected than the a-wave. Partial to complete recovery was observed during follow-up. After 4 weeks, negligible ERG deficit was observed in the rabbits treated with 0.06% trypan blue, whereas significant ERG deficit was measured in rabbits tested by the 0.15% trypan blue. No differences in flash VEP responses between experimental and control rabbit eyes were found. Light microscopy showed no significant histologic effects in the retinas exposed to the 0.06% solution. Marked disorganization of all retinal layers was observed in areas close to the site of injection in the rabbits injected with the 0.15% solution. CONCLUSIONS: Trypan blue exerts transient physiological effects on the distal retina of the rabbit, but in concentrations of 0.15% it can induce permanent toxic effects. Therefore, caution should be used when using this dye in vitreoretinal surgery.

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.

The developing mammalian retina is partially protected from gentamicin toxicity.

Gentamicin retinal toxicity was tested in numerous studies on adult animal models, but not in the developing retina. Since differentiating cells and developing tissues may exhibit different degrees of sensitivity to toxic drugs, we aimed here to test the susceptibility of the developing mammalian retina to the toxic action of gentamicin. Gentamicin was injected in the right eye of newborn rabbits, aged 11-38 days. The left eye of each rabbit was injected with saline. Eight weeks after injection, electroretinographic (ERG) responses were recorded in the dark- and light-adapted states. The rabbits were then sacrificed, and the retinas were prepared for morphological examination and immunostaining for Glial Fibrillary Acidic Protein (GFAP) and Protein Kinase C (PKC). The ERG responses demonstrated practically no functional damage to the retina of rabbits injected at postnatal age of 11 and 13 days, and a gradually increasing severity of functional damage with increasing postnatal age of intravitreal gentamicin injection. The histopathological studies yielded similar results. GFAP immunoreactivity showed staining in Müller cells only in retinas that exhibited functional and structural damage. PKC immunoreactivity indicated lesser damage to the rod-bipolar cells compared to the photoreceptors. The ERG data and morphological observations suggest that processes involved in development of the rabbit retina, such as outer segment elongation may provide partial or even complete protection to the retina from toxic insults such as a single dose of gentamicin.

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.

A novel isoform of acetylcholinesterase exacerbates photoreceptors death after photic stress.

PURPOSE: To study the involvement of stress-induced acetylcholinesterase (AChE) expression in light-induced retinal damage in albino rats. METHODS: Adult albino rats were exposed for 24 hours to bright, damaging light. AChE expression was monitored by in situ hybridization, by histochemistry for AChE activity, and by immunocytochemistry. An orphan antisense agent (Monarsen; Ester Neurosciences, Ltd., Herzlia Pituach, Israel) was administered intraperitoneally to minimize light-induced AChE expression. The electroretinogram (ERG) was recorded to assess retinal function. RESULTS: Twenty-four-hour exposure to bright light caused severe reduction in the ERG responses and augmented expression of mRNA for the "read-through" variant of AChE (AChE-R) in photoreceptor inner segments (IS), bipolar cells, and ganglion cells. AChE activity increased in IS. The expressed AChE protein was a novel variant, characterized by an extended N terminus (N-AChE). Systemic administration of the orphan antisense agent, Monarsen, reduced the photic induction of mRNA for AChE-R, and of the N-AChE protein. Rats exposed to bright, damaging light and treated daily with Monarsen exhibited larger ERG responses, relatively thicker outer nuclear layer (ONL), and more ONL nuclei than did rats exposed to the same damaging light but treated daily with saline. CONCLUSIONS: The findings indicate that the photic-induced novel variant of AChE (N-AChE-R) may be causally involved with retinal light damage and suggest the use of RNA targeting for limiting such damage.

Retinal toxicity of indocyanine green in albino rabbits.

PURPOSE: Intravitreal indocyanine green (ICG) is commonly used in vitreoretinal surgery. The purpose of this study was to evaluate possible toxicity of ICG in the retina of albino rabbits. METHODS: Twenty-two albino rabbits were injected intravitreally with 0.1 mL ICG solution in one eye, and three rabbits were studied for the effects of 0.1 mL distilled water. All rabbits were injected intravitreally with 0.1 mL saline into the fellow eye, which served as the control. The electroretinogram (ERG) and visual evoked potential (VEP) were recorded from each rabbit at different time intervals after injection. The rabbits were killed at the termination of the follow-up periods and their retinas prepared for histologic examination at the light microscopic level. RESULTS: Three hours after injection, the ERG responses were reduced in amplitude in all ICG-injected eyes, and the VEPs were of abnormal pattern (reduced amplitude and delayed). Partial dose-dependent recovery was observed during 4 weeks of follow-up. Light microscopy of the retinas of the experimental eyes exhibited considerable damage to all retinal layers in all eyes studied that received the highest ICG dose. CONCLUSIONS: ICG is potentially toxic to all retinal layers of the albino rabbit. Although it is difficult to extrapolate these findings directly to human eyes, caution should be exercised when using ICG intravitreally.

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.

NADPH diaphorase activity in the rat retina during the early stages of experimental diabetes.

BACKGROUND: Nitric oxide (NO) plays an important physiological role in inter-cellular communication, but when produced in excess it can become toxic. Our goal was to evaluate possible involvement of NO in the development of retinopathy in diabetic rats. METHODS: Diabetes was induced in male albino rats by intravenous injection of streptozotocin. Some of the normal and diabetic rats were raised with added L-arginine to increase in vivo NO synthesis, or with added L-NAME to inhibit the rate of in vivo NO synthesis. NADPH diaphorase histochemistry was conducted on retinal whole mounts and transverse sections at different time intervals after induction of diabetes. The electroretinogram (ERG) was recorded to assess retinal function. RESULTS: After 6 weeks of diabetes, NADPH diaphorase amacrine cells in the diabetic retinas appeared abnormal in their morphology and the degree of staining was decreased in their processes. In contrast, NADPH diaphorase activity was augmented in Müller cells. Supplementing the rats' diet with L-arginine for 10 weeks slightly reduced NADPH diaphorase activity in amacrine cell in normal rats but had no effect on the diabetic rats. Adding L-NAME for 10 weeks did not alter NADPH diaphorase histochemistry in either normal or diabetic rats. The ERG responses were reduced by L-arginine supplementation in normal and diabetic rats, and were unaffected by adding L-NAME to the drinking water. CONCLUSIONS: Our findings are consistent with the hypothesis that high glucose levels are deleterious to the rat retina and that excessive synthesis of NO may contribute to the development of diabetic retinopathy.