Figure 1.
Intraocular pressure (IOP) measurements in normal and glaucomatous eyes.
IOP of normal eyes and eyes from a chronic glaucoma model with and without AG (25 mg/kg; i.p.) and SNC-121 (1 mg/kg) treatment for 7 days (once daily) was measured as described in the Methods. IOP was elevated in one eye of Brown Norway rats by injecting approximately 50 µL of 2.0 M hypertonic saline, while the contralateral eye served as the control. Rats were maintained for up to 6 weeks post-surgery. Data are mean ± SE. *P<0.05; n = 6–8 for each group.
Figure 2.
Pattern ERG recordings in normal and glaucomatous eyes.
Changes observed in Pattern ERGs of untreated, AG or SNC-121-treated glaucomatous rat eyes are shown as a percentage of the contralateral control eye. In these experiments, Brown Norway rats were treated with AG (25 mg/kg, i.p.) or SNC-121 (1 mg/kg, i.p.), individually or together simultaneously, 30 minutes after hypertonic saline injections. Animals were treated with naltrindole 30 minutes after glaucomatous injury followed by SNC-121 treatment. The drug treatment was continued once daily for 7 days in all groups. Data are mean ± SE; *P<0.05; n = 7–8.
Figure 3.
Retina flat mounts of normal and glaucomatous eyes.
Fluorescence micrographs of flatmounted retinas depicting Fluorogold-labeled retina ganglion cells (RGCs) in normal and glaucomatous eyes in the absence or presence of drugs (A). Briefly, 3 µL of a 5% solution of Fluorogold was injected into the superior colliculus of anesthetized animals. Seven days post injection, animals were euthanized and retinas were prepared as flatmounts, vitreous-side facing up. Fluorescent RGCs were visualized under Zeiss microscopy. Bar = 20 µm. Total RGC tallies in glaucomatous eyes in the absence or presence of drugs. RGCs were counted in 8 microscopic fields of identical size (150 µm2 area) for each retina, using Image J software (B). *P<0.05; n = 6 for each group.
Figure 4.
Measurements of nitrate-nitrite in normal and glaucomatous eyes in the presence of SNC-121.
Animals were treated with SNC-121 (1 mg/kg, i.p.) 30 minutes after glaucomatous injury, and continued once daily for 7 days. A separate group of animals was pretreated with naltrindole (Nal; 3 mg/kg, i.p.) followed by SNC-121 (1 mg/kg, i.p.) treatment 30 minutes after glaucomatous injury, and continued for 7 days, once daily. The absorbance was read at 550 nm and absorbance was plotted against the nitrate standards. The amount of nitrate-nitrite is expressed as millimolar nitrates-nitrites per milligram protein. Data are expressed as mean ± SEM, (*P<0.05; n = 4–7).
Figure 5.
Measurements of nitrate-nitrite in normal and glaucomatous eyes in the presence of AG.
Animals were treated with AG (25 mg/kg, i.p.) 30 minutes after glaucomatous injury and continued for 7 days, once daily. The absorbance was measured at 550 nm and absorbance was plotted against the nitrate standards. The amount of nitrate-nitrite is expressed as millimolar nitrates-nitrites per milligram protein. Data are expressed as mean ± SEM, (*P<0.05; n = 4–6).
Figure 6.
Measurement of protein nitrosylation in retina by Western blotting using nitrotyrosine antibodies in response to glaucomatous injury.
Animals were treated with SNC-121 (1 mg/kg, i.p.) 30 minutes after glaucomatous injury and continued for 7 days, once daily. Retina extracts were collected at the 7th day, post injury, and analyzed using anti-nitrotyrosine antibodies and appropriate secondary antibodies (HRP-conjugated; dilution 1∶3000). The signal was captured using enhanced chemiluminescent reagent and the Biorad Versadoc imaging system. Data shown are representative of four independent experiments. Data are expressed as mean ± SEM. *P<0.05; n = 4.
Figure 7.
Changes in iNOS expression in glaucomatous optic nerve at 7 days, post injury.
The optic nerve (non-myelinated; 2 mm post globe) of Brown Norway rats was removed 7-days post glaucomatous injury. Contralateral optic nerve was used as the normal control. Cryosections were immunostained by anti-iNOS antibodies as indicated horizontally. Ocular treatments are indicated vertically. Green color indicates staining for iNOS and blue nuclei for DAPI. There was no positive staining when primary antibodies were omitted (data not shown). Data shown here are representations of at least four independent experiments. A total of 10 animals were used in this experiment. Comparable staining for iNOS was seen in at least 4 animals in each treatment group.
Figure 8.
Changes in iNOS expression in glaucomatous optic nerve at 42 days, post injury.
The optic nerve (non-myelinated; 2 mm post globe) of Brown Norway rats was removed 42-days post glaucomatous injury. Contralateral optic nerve was used as the normal control. Cryosections were immunostained by anti-iNOS antibodies as indicated horizontally. Ocular treatments are indicated vertically. Data shown in this Figure are a representation of at least four independent experiments. A total of 10 animals were used in this experiment. Comparable staining for iNOS was seen in at least 4 animals in each treatment group.
Figure 9.
Changes in iNOS expression in glaucomatous retina at 7 and 42 days, post injury.
(A) Brown Norway rats were treated with SNC-121 (1 mg/kg, i.p.) 30 minutes after glaucomatous injury and continued for 7 days, once daily. Retina extracts were collected at the 7th day, post injury, and analyzed using anti-iNOS antibodies and appropriate secondary antibodies (HRP-conjugated; dilution 1∶3000). The signal was captured using enhanced chemiluminescent reagent and the Biorad Versadoc imaging system. Data are expressed as mean ± SEM. *P<0.05; n = 4. (B) The eyes of Brown Norway rats were removed 42-days post glaucomatous injury. Cryosections of retina were immunostained by anti-iNOS antibodies as indicated horizontally. Ocular treatments are indicated vertically. Data shown in this Figure are a representation of at least four independent experiments.