Unless specified otherwise, all reagents were purchased from Sigma (St. Louis, MO) or Fisher Scientific (Waltham, MA). All of the experiments were done in normal room light, with the exception of the NO imaging, which, for technical reasons, was done using dark-adapted retinas. All statistical evaluations were performed using OriginPro 8 software (OriginLab, North Hampton, MA).
Adult (between 3 and 5 months of age), male C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA) and were kept on a standard 12 h:12 h light-dark cycle with free access to food and water. All animals were treated using protocols approved by the Boston University Charles River Campus Institutional Animal Care and Use Committee. Mice were age matched and made diabetic by an intraperitoneal (IP) injection of 75 mg/kg streptozotocin (STZ; Alexis Biochemicals, San Diego, CA) in pH 4.5 citrate buffer; each injection was given after an 8-h fast, one injection per day for three consecutive days. Control animals received three IP injections of citrate buffer alone after an 8 h fast on the same days as the experimental animals. The animals were allowed to recover for two days before their blood glucose levels were tested with a FreeStyle FlashTM blood glucose meter (Therasense, Almeda, CA). Mice were considered diabetic if their fasting blood glucose level was at or over 250 mg/dl. Animals that did not become diabetic were not used for this study. Treatment with insulin, aminoguanidine (AG), or Nω-nitro-L-arginine methyl ester (L-NAME) was given to subsets of animals starting one week after induction of diabetes. Thus, these treatments were given for a duration of 4 weeks up to the time of sacrifice as follows. Once a day, 2 U/kg insulin from bovine pancreas (in sterile saline; Sigma I5500) was administered via IP injection. Also once a day, NOS inhibitor L-NAME (37.5 mg/kg in sterile 0.1 M pH 7.4 phosphate buffer, PB) was administered by IP. Every day, 1 g/l AG, an inhibitor of both NOS and advanced glycation end product (AGE) formation, was administered to animals in their drinking water, which was changed daily. Animal weight and fasting blood glucose levels were recorded at the beginning and end of each experimental cycle. Final blood glucose levels and bodyweights were compared between each group using ANOVA. Post hoc analysis was performed using Tukey’s test. A p<0.05 was considered significant. Animals were euthanized in each experiment by exposing them to IsoFlow™ Isoflurane gas (Abbott Laboratories) until they were deeply anesthetized, and then they were immediately decapitated.
Protein extraction and western blotting
Following sacrifice, the eyes were rapidly removed and placed into balanced salt solution (BSS; 137 mM NaCl, 5 mM KCl, 2 mM CaCl2
, 15 mM D-glucose, 1 mM MgSO4
, 1 mM Na2
, and 10mM HEPES, pH 7.4) and the retinas were then surgically isolated from the eyes. Specimens in solution were immediately placed into 1.5 ml centrifuge tubes, set on dry ice. Six retinas (three animals) were pooled together to ensure an adequate amount of protein for detection by western blotting. The retinas were then suspended in 200 μl of homogenization buffer containing 20 mM Tris-HCl, 0.05% SDS, and 1X Complete Mini Protease Inhibitor Cocktail with EDTA (Roche Applied Science, Indianapolis, IN) before being ultrasonically homogenized. The protein concentration in each sample was determined using the Bradford method. In each lane, 100 μg protein was loaded onto 8% SDS PAGE gels and transferred onto 0.4 μm Immobilon™ PVDF membranes (Millipore, Billerica, MA). A rapid detection method was used, whereby the blocking step was omitted [28
]. The primary antiserum was a rabbit anti-nNOS (sc648; Santa Cruz Biotechnology, Santa Cruz, CA) that was diluted 1:5,000 in 2% BSA in Tris buffered saline w/ 0.25% Tween-20 (TBST). The secondary antibody was a horseradish peroxidase-conjugated goat polyclonal antiserum raised against rabbit IgG (Molecular Probes, Invitrogen, Carlsbad, CA) at a dilution of 1:100,000. The blots were then treated with Immobilon™ HRP substrate (Millipore) and exposed to blue X-ray film (F-BX57; Phenix, Asheville, NC). To confirm equal lane loading, we stripped the blots and reprobed them with mouse anti-β-tubulin. The β-tubulin monoclonal antibody used was developed by Dr. Willi Hafler and was obtained from the Developmental Studies Hybridoma Bank. This antibody was developed under the auspices of the NICHD and is maintained by The University of Iowa (Department of Biology, Iowa City, IA). Quantitative analysis was performed using Image J
image analysis software (Wayne Rasband, National Institute of Mental Health, Bethesda, MD). The difference in protein levels was evaluated using the paired Student’s t
-test. A p<0.05 was considered significant.
After euthanasia, the mouse eyes were rapidly enucleated and placed in ice-cold BSS. The anterior chamber, lens, and vitreous were then removed and the resultant eyecups were immediately placed into 4% paraformaldehyde in PB for 90 min at room temperature. Next, the eyecups were cryoprotected in 30% sucrose in PB, embedded and frozen in Optimal Cutting Temperature embedding media (OCT; Tissue-Tek, Miles, Inc., Elkhard, IN), and cut into 14 µm thick cross-sections using a cryostat and mounted on Superfrost /Plus® slides (Fisher Scientific). The eyecups from the control and diabetic mice were embedded into the same block and cryosectioned onto the same slide to reduce variability when comparing changes in immunoreactivity. The asymmetry provided by this embedding method allowed us to clearly distinguish each of the eyecups in the resultant cryosections. Thus we could embedded one eye from each of 4 separate mice with different treatments in the same block.
Immunocytochemistry was performed on the retinal sections using standard methods as previously described [25
] on retinal cross-sections mounted on slides. Immunochemical procedures were replicated, using at least three different animals (n=3), where each "n" was the average of three trials for a given animal compared to the other retinas on the same slide as described in the previous paragraph. The primary antiserum used was rabbit anti-nNOS (sc648; Santa Cruz Biotechnology) at a dilution of 1:1,000 overnight in a humid chamber at 4 °C. The slides were then washed 3×10 min in 0.1 M PB at room temperature and then incubated for 2 h in an Alexa488-conjugated goat anti-rabbit IgG secondary antiserum (Molecular Probes, Carlsbad, CA) at a dilution of 1:500. Incubation with the primary antiserum omitted served as a control for nonspecific secondary antiserum staining. Slides were washed in 0.1 M PB as before, coverslipped with glycerol, and the fluorescent signal was visualized using an Olympus Fluoview™ 300 confocal microscope (Olympus, Melville, NY). The excitation was done using the 488 nm laser line from an argon laser and the emission was visualized using a 510–550 nm bandpass filter. Ten to 14 optical sections were taken at 1 μm intervals, with the same number of optical sections always being captured and compared for both the control and experimental retinas. The laser intensity and all confocal settings were kept consistent within replicates.
The “z project” function of Image J image analysis software was used to obtain a single image from a collapsed confocal optical stack. The images were then converted to inverted grayscale so that the immunoreactivity would appear black. Mean intensity values were then recorded for each treatment and compared to the control retina on the same slide. These values were averaged together for each animal and compared to the average control mean using the paired Student’s t-test. A p<0.05 was considered significant. The relative intensity of immunoreactivity within each retinal layer was quantified using Image J to produce a horizontal line-profile average of the intensity of the immunoreactivity in the different retinal layers, minimizing the effect of local regional differences.
RNA isolation and reverse transcription
The retinas were isolated in BSS as described in the immunocytochemistry methods section, although all reagents and materials were kept RNase-free. Total RNA was then isolated from the retinas using a standard Trizol™ reagent (Invitrogen, Carlsbad, CA) extraction. To obtain sufficient amounts of RNA to perform these reactions, we pooled three animals (six retinas) together for each extraction. The RNA was then treated with rDNase™ (Ambion, Applied Biosystems, Austin, TX), following the manufacturer’s instructions, to remove any DNA contaminants. The RNA was quantified using a Nanodrop™ spectrophotometer (ThermoFisher Scientific, Waltham, MA). RNA was then converted into cDNA using the Verso™ cDNA kit (ThermoFisher Scientific) and subsequently treated with 2 U of RNase H™ (ThermoFisher Scientific) at 37 °C for 20 min.
Quantitative real-time PCR analysis
Quantitative real-time PCR analysis (qPCR) was performed using cDNA converted from 1 µg of retinal RNA as described in the previous section, using an ABI PrismTM
7900HT Sequence Detection System (Applied Biosystems, Carlsbad, CA). We used a pre-designed TaqMan™ gene expression assay (Applied Biosystems) for nNOS (assay ID: Mm00435173_m1). Our normalizing control was an optimized 18s rRNA primer set that was kindly provided to us by Dr. Ulla Hansen (Boston University, Boston, MA), which was designed to work with SYBR Green™ (Applied Biosystems). All data was obtained in quadruplicate and analyzed using the Microsoft ExcelTM
qGene template [30
NO imaging was performed as reported previously [24
]. Since DAF-FM is a light sensitive dye, all aspects of the NO imaging experiments, beginning with the retinal eyecups, were done in the dark using infrared imaging, except for the actual final light stimulations. To prepare retinal slices, we enucleated the eyes and removed the anterior segment of the eye and lens under dim light. We placed the eyecups in chilled aerated artificial cerebrospinal fluid (ACSF, 125 mM NaCl, 26 mM NaHCO3
, 3 mM KCl, 1.6 mM CaCl2
, 1.5 mM MgSO4
, 1.25 mM NaH2
, and 10 mM glucose) in the dark on ice for 1 h to allow the retinas to recover and fully dark adapt. The eyecups were then sectioned into 250-µm-thick slices. Loading of the NO-sensitive dye, DAF-FM (Molecular Probes), was done as previously described [31
]. Briefly, slices were allowed to recover from slicing for at least 30 min in ACSF at room temperature and then incubated for 60 min in ACSF containing 10 µM of DAF-FM at 37 °C. Slices were then washed 3x 15 min with fresh, aerated ACSF at 37 °C.
Some slices were stimulated for 20 min with a light emitting diode (660 nm, 3Hz, 20 µW, 25% duty cycle), while a matched set of slices were kept in the dark for comparison. Light-stimulated and control sections were then fixed in the dark for 2 h with 4% paraformaldehyde in PB and then they were washed 4×15 min in 0.1 M PB at room temperature. Fluorescent images of the retinal slices were acquired using a FluoviewTM
300 confocal microscope (Olympus Corporation) using a 40× water immersion objective and the FluoviewTM
2.1 software. Image preparation and analysis was conducted using Image J
software as described above.
To ensure unbiased thresholding of the images when comparing the different retinal regions or treatments, we assigned a threshold value, using a custom written Image J plugin, which determined the inflection point for the region of interest (ROI) of each collapsed confocal optical image stack. Briefly, on a plot of arbitrary fluorescent units obtained from the confocal microscope versus the number of pixels in the image, the inflection point was computed as a point on the curve at which the tangent crossed the curve. Fluorescent pixels above this threshold value were quantified for each ROI using the “ROI manager.”
The quantitative analysis of these collapsed image stacks was done using the freehand selection tool in Image J to select specific ROIs. At least 25 ROIs from each animal in each category and at least five animals in each treatment group were analyzed in this manner. The average number of fluorescent pixels of all images from one animal was considered as n=1. The final graphs depict the mean and standard deviation (SD) of at least n=5 animals to average out any minor local intensity differences. The difference between the control and experimental groups was analyzed using ANOVA. Post hoc analysis was performed using Tukey’s test. A p<0.05 was considered significant.