Ketogenic diet attenuates NMDA-induced damage to rat's retinal ganglion cells in an age-dependent manner.

OBJECTIVE: This study was conducted to investigate neuroprotective effects of a high fat/low carbohydrate and protein diet (ketogenic diet, KD) in a model of N-methyl D-aspartate (NMDA)-induced retinal ganglion cell (RGC) damage in juvenile and young adult rats. METHODS: Juvenile (30-35 days old) and young adult (56-70 days old) female Brown Norway rats were fed the KD for 21 days; rats exposed to a standard rodent diet (SRD) served as controls. The main constituents of the KD used in the present study were approximately 80% fats, 8% proteins, and less than 1% carbohydrates. On day 14 of exposure to the KD (or the SRD in the control group), each rat received a single intravitreal injection of NMDA; RGCs were then retrogradely labelled by hydroxystilbamidine on day 19 and collected on day 21 to assess the degree of damage induced by NMDA. Blood biomarkers to confirm the expected metabolic response to the KD (i.e. ketosis and hypoglycaemia) were also assessed. RESULTS: Although both the juvenile and young adult rats developed comparable ketosis and hypoglycaemia when fed the KD, NMDA-induced loss in RGCs was significantly attenuated only in juvenile rats exposed to the KD in comparison with those fed the SRD; exposure to the KD had no protective effect in young adult rats. In summary, exposure to the KD had a neuroprotective effect in NMDA-induced RGC damage in juvenile rats, but not in young adult rats. CONCLUSION: These results support further exploration of metabolic interventions to treat optic neuropathies associated with neurodegeneration.

Caspase inhibitors protect against NMDA-mediated retinal ganglion cell death.

BACKGROUND: Apoptosis is a major mechanism of cell death in glutamate-induced excitotoxicity and caspases as the executors of apoptosis play an important role in the development of various central nervous system and eye diseases. We studied the involvement of certain caspases in excitotoxic retinal ganglion cell death, which was experimentally induced in Brown Norway Rats by application of the glutamate receptor agonist N-methyl-D-aspartate (NMDA). METHODS: Animals were injected intravitreally with one of six caspase inhibitors (against caspases 1, 3, 4, 6, 8 and 9). Seven hours later, NMDA or phosphate-buffered saline as a control was injected intravitreally into the respective eyes. The neuroprotective potential against NMDA toxicity was assessed by retinal ganglion cell quantification. Additionally, wholemount TUNEL was performed. RESULTS: Statistical analysis revealed significant neuroprotective effects for the inhibitors of caspases 3, 6, 8 and 9, but not for those of caspases 1 and 4. The inhibitors of caspases 6 and 9 showed greater neuroprotective potential than those of caspases 3 and 8, although cell death was not entirely averted in any case. Results of ganglion cell counts were confirmed for the most pronounced treatment groups using wholemount TUNEL. CONCLUSION: Excitotoxic retinal ganglion cell death after NMDA injection is mediated mainly through apoptosis, whereby extrinsic as well as intrinsic pathways of caspase activation play a role.

Neuroprotection by acetoacetate and ß-hydroxybutyrate against NMDA-induced RGC damage in rat--possible involvement of kynurenic acid.

PURPOSE: This study investigated the effects of systemically administered lithium acetoacetate (ACA) and sodium ß-hydroxybutyrate (BHB) in a rat model of N-methyl-D-aspartate (NMDA)-induced damage of retinal ganglion cells (RGC). Additionally, the influence of ACA and BHB on kynurenic acid (KYNA) production was assessed in vitro in bovine retinal slices. METHODS: Female adult Brown-Norway rats in groups of 5-8 animals were used. ACA and BHB were administered intraperitoneally once a day for 21 consecutive days, and phosphate buffered saline (PBS) was administered to control animals. After 2 weeks, the animals received intraocular NMDA (2 µl of a 10 mM solution in PBS) or intraocular PBS as a control. On day 19, retinal ganglion cells were labeled retrogradely with hydroxystilbamidine. Two days later, RGC density (cells per mm(2)) was assessed on retinal flatmounts. Additionaly, bovine retinal slices were incubated with NMDA and ACA or BHB at concentrations of 1.0 mM and 3.0 mM, and de novo KYNA production was measured using HPLC. RESULTS: Intraperitoneal ACA (250 mg/kg) or BHB (291.2 mg/kg) significantly protected RGC against NMDA-induced neurodegeneration. De novo KYNA production in bovine retinal slices was lowered by NMDA. Both ACA and BHB at a concentration of 3.0 mM significantly reduced the effects of NMDA. CONCLUSIONS: ACA and BHB had a significant dose-dependent neuroprotective effect on RGC in a rat model of NMDA-induced RGC damage. Both ketone bodies also significantly attenuated NMDA-induced reduction of retinal KYNA production in vitro, suggesting that this mechanism may be essential for the neuroprotective effects of ACA and BHB in vivo. Our results imply that ketone bodies may represent an additional treatment option in chronic neurodegenerative disorders of the eye.

Toxicity testing of the VEGF inhibitors bevacizumab, ranibizumab and pegaptanib in rats both with and without prior retinal ganglion cell damage.

PURPOSE: To evaluate the effects of intravitreally introduced vascular endothelial growth factor (VEGF) inhibitors in rat eyes with healthy retinal ganglion cells (RGC) and into others with N-methyl-D-aspartate (NMDA)-induced RGC damage. METHODS: Bevacizumab, ranibizumab and pegaptanib were intravitreally injected each at two different concentrations. Respective vehicles of the three substances served as controls. In a different group, additionally a rat anti-VEGF antibody was injected after NMDA treatment. Retrogradely labelled RGC were counted on retinal wholemounts 1 week or 2 months after intravitreal introduction of the VEGF inhibitors. Electron microscopy (EM) was performed on normal rat eyes 2 months after introduction of the VEGF inhibitors. RESULTS: RGC counts in healthy rat eyes were essentially unchanged from those of the control animals after the administration of both low and high concentrations of bevacizumab, ranibizumab or pegaptanib. Compared to the other two substances, however, high doses of pegaptanib and its respective vehicle significantly decreased RGC after 1 week and led to a marked increase of mitochondrial swelling in EM. In eyes with NMDA-induced RGC damage, no changes of RGC numbers were detected after rat anti-VEGF antibody or bevacizumab, ranibizumab and pegaptanib at both tested concentrations. CONCLUSIONS: Even at higher doses, bevacizumab and ranibizumab showed no toxic effects on RGC in vivo in either untreated rats or in the NMDA-induced RGC damage model. Also a rat anti-VEGF antibody showed no adverse effects after NMDA. Anti-VEGF therapy therefore appears safe even for eyes with additional excitotoxic RGC damage. Potential harm from the pegaptanib carrier solution at very high local concentrations cannot be excluded.

Neuroprotective effects of tempol on retinal ganglion cells in a partial optic nerve crush rat model with and without iron load.

Iron overload can contribute to oxidative stress in many tissues. We studied the effects of pretreatment with iron dextran on RGC loss in a calibrated partial optic nerve crush (PONC) model in rats, along with the protection offered by tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxyl, a membrane-permeable superoxide dismutase mimetic and free-radical scavenger), in the same experimental paradigm. A total of 40 rats in 6 groups of 5-8 animals each underwent PONC in one eye and sham crush in the other. Animals were pretreated with a single iron dextran load 24 h prior to PONC, and treated with tempol 6 h before and then once daily after PONC. Control animals were treated with PBS. RGC were retrogradely labeled with a fluorescent marker; all data are expressed in percent of the RGC count in the respective sham-treated eye. Immunohistochemistry was performed to visualize 3-nitrotyrosine, a marker of nitroxidative stress. PONC without iron pretreatment resulted in the survival of only 31.4% of labeled RGC after 7 days. Even fewer RGC (12.7%) survived after PONC with iron pretreatment. However, tempol in doses of 20 mg/kg of body weight (BW) significantly attenuated this effect when given as described above; in the group without iron pretreatment the number of surviving RGC doubled from 31.4% to 62.1%. In the group with iron pretreatment the survival rate of RGC increased even more pronouncedly, from 12.7% without tempol to 46.2% with tempol. Tempol in doses of 1 mg/kg BW and 5 mg/kg BW showed no significant rescue of RGC. Immunostaining showed nitrotyrosine-positive RGCs in PONC but not in sham-treated eyes and an increase in positive cells after iron load. Tempol treatment reduced nitrotyrosine staining in both the iron and non-iron groups. Our results demonstrate that PONC results in significantly greater RGC damage when iron pretreatment is performed, and that the compound tempol may provide additional protection for RGC in cases of neuronal damage both with and without prior iron treatment.

In vivo toxicity testing of methyl blue and aniline blue as vital dyes for intraocular surgery.

PURPOSE: To investigate the biocompatibility of methyl blue and aniline blue as vital dyes for vitreoretinal surgery in an in vivo rat model and to evaluate the effect of these dyes on retinal structure and function. METHODS: Adult Brown-Norway rats received intravitreal injections of 0.1%, 0.2%, and 2% methyl blue or aniline blue dissolved in balanced salt solution with balanced salt solution serving as a control. Retinal toxicity was assessed 7 days thereafter by means of retinal ganglion cell counts, light microscopy, and electroretinography. RESULTS: No significant decrease in retinal ganglion cell counts at concentrations up to 0.2% was observed. At 2%, however, a significant retinal ganglion cell loss was detected with both dyes (more pronounced for aniline blue). Light microscopy showed no structural changes in the central retina for concentrations up to 0.2%. Electroretinographies detected no adverse effects of methyl blue or aniline blue on rod- or cone-driven responses at concentrations up to 0.2%. CONCLUSION: Methyl blue and aniline blue are very biocompatible and may, therefore, be usable for intraocular surgery. Further testing with other animal models will be necessary to confirm this. The safety margin of methyl blue is possibly higher than that of aniline blue.

In vivo toxicity study of rhodamine 6G in the rat retina.

PURPOSE: To investigate the intraocular effect of rhodamine 6G (R6G) on retinal structures and function in an in vivo rat model and to develop an in vivo method for accurate evaluation of new dyes for intraocular surgery. METHODS: R6G in physiologic saline solution (PSS) was injected into the vitreous of adult Brown Norway rats at concentrations of 0.0002%, 0.002%, 0.02%, 0.2%, and 0.5%. Control animals received only PSS. Retinal toxicity was assessed by retinal ganglion cell (RGC) counts, light microscopy 7 days later, photopic electroretinography (ERG), and measurement of scotopic sensitivity and recovery of dark adaptation 48 hours and 7 days after intravitreous injection. RESULTS: R6G at concentrations of 0.2% and 0.5% led to a dose-dependent loss of RGC. The most significant loss occurred at 0.5%. Lower concentrations (0.0002%, 0.002%, and 0.02%) produced no statistically significant retinal ganglion cell loss. Analysis of the eyes by light microscopy showed no structural changes in the central retina, although injections of 0.5% R6G were followed by impressive degenerative changes adjacent to the injection sites. ERGs showed no effects of the highest R6G concentration on rods, kinetics of rhodopsin recovery after bleaching, or cone-driven responses. CONCLUSIONS: R6G can be safely injected in doses of up to 0.02% in rats, but has a toxic effect on retinal ganglion cells at higher concentrations. Accumulation of R6G may be a problem at higher concentrations, particularly at the injection site.