Animals
Mice were obtained from breeding colonies maintained at the Animal Facility of Medical School, University of Pecs (Pecs, Hungary). Wild-type and homozygous PACAP-deficient mice were used. PACAP-deficient mice were generated and maintained on CD1 background as previously described (Hashimoto et al. 2001; Hashimoto et al. 2009); they were backcrossed for ten generations with the CD1 strain. Each nursing mum with their litters was housed in individual cages, fed and watered ad libitum under 12/12 hours light/dark cycles. Animal housing, care and application of experimental procedures were in accordance with the ethical guidelines approved by the University of Pecs (BA02/2000-31/2011) and directives of the National Ethical Council for Animal Research, the European Communities Council (86/609/EEC), and ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Experimental design
To induce retinopathy, wild-type (NOIR-Wt=15) and PACAP-deficient (NOIR-KO=14) mice pups with their nursing mother were kept in an oxygen chamber (Biospherix Ltd., NY, USA) supplied by an oxygen sensor (ProOx 110, Biospherix Ltd., NY, USA) to monitor and maintained continuous 80 % of oxygen concentration from PD7 to PD12. Then they were returned to room air until PD16. Control litters of both genotypes (NCont-Wt=15, NCont-KO =15) were room-air reared during the whole experiment.
Electroretinography
Some of the animals from each group (NCont-Wt= 4, NCont-KO =3, NOIR-Wt= 3, NOIR-KO= 4) underwent electroretinography (ERG) examinations to assess visual function on PD15 after an overnight dark adaptation. The measurements were performed as previously described by Danyadi and co-workers (Danyadi et al. 2014). Briefly, after systemic anesthesia, the pupils were dilated with 0.5 % cyclopentolate (Humapent-Teva, TEVA Ltd., Hungary) and topically anesthetized with Oxybuprocaine 0.4 % (Humacain-Teva, TEVA Ltd., Hungary) eye-drops. ERGs were recorded by surface electrodes from the center of the cornea with a negative electrode placed under the cheek and a ground electrode inserted under the skin of the neck. The responses to light flashes (5.0 cd/m2, 0.25 Hz, 503 nm green LED light) were pre-amplified, amplified and recorded by an A/D converter (Ratsoft-Solar Electronic). Responses were averaged with the software of the A/D converter. The selected parameters were measured by OriginPro 2016 software (OriginLab Corporation, MA, USA) and statistically analyzed by ANOVA with Fisher’s post hoc test after test for homogeneity of variance (STATISTICA, StatSoft Inc., OK, USA).
Immunohistochemistry
Isolectin immunohistochemical staining was performed as previously described by Connor and co-workers (Connor et al. 2009). Briefly, after anesthesia on P16±1, eyes were removed and immediately placed into 4% PFA for fixation at room temperature. After 1 h, eyes were washed three times in PBS and retinas were isolated under dissecting microscope. To stain the retinal vasculature 500 µl fluoresceinated isolectin solution (Isolectin GS-IB4 from Griffonia simpliciforia, Alexa Fluor 568 conjugate; Thermo Fischer Scientific Inc., MA, USA) was added to the isolated retinas. After an overnight rocking in lectin solution at room temperature, retinas were rinsed three times in PBS. Finally, 4 incisions were made to flatten the retinas onto microscope slides and were covered with a coverslip with mounting media (Fluoromount, Sigma-Aldrich Co., MO, USA). Digital photographs were taken with a Nikon Eclipse Ci fluorescence microscope (Nikon, Melville, NY, USA).
Assessment of vessel morphology
A trained observer blinded for the groups evaluated the ratio of central avascular retinal territory, vessel density and neovascular tufts. Measurements were executed by AdobePhotoshop CS6 (Adobe Systems Inc., CA, USA) and ImageJ softwares (National Institutes of Health, Bethesda, MD, USA). Central avascular retina was outlined, measured and its percentage to the whole retina was given. Vessel density was calculated by marking out all the vessels in the intact retina and giving their portion to the whole vascularized territory. To define neovascular tufts formation, flat mounts were divided into 12 approximately equal areas - the so called “clock hours” - and were assigned a number 1-12 depending on the number of clock hours with neovascular tufts.
Results are represented in mean ± SEM. Statistical analysis was performed by independent T-test after Levene’s F-test for equality of variance using STATISTICA software (StatSoft Inc., OK, USA).
Angiogenesis array analysis
After euthanasia on PD 17±1 the eyeballs were removed, retinas were carefully separated and quickly frozen in dry ice. Angiogenesis proteins were investigated from pooled tissue homogenates by semi-quantitative Mouse Angiogenesis Array Kits (R&D Systems, Bio-Techne Ltd., MN, USA). In these arrays, the sample proteins bind to selected captured antibodies spotted on nitrocellulose membranes. The kits contain all buffers, detection antibodies and membranes necessary for the measurement. The arrays were performed as described by the manufacturer’s protocol. In brief, after blocking the membranes for 1 hour and adding the detection antibody cocktail for another 1 hour at room temperature, the membranes were incubated with 1.5 ml tissue homogenates at 2-8 °C overnight on a rocking platform. After washing with buffer 3 times, membranes were incubated with horseradish peroxidase-conjugated Streptavidin at room temperature and exposed to chemiluminescence detection reagent to develop X-ray films. The arrays were repeated two times. For data analysis, films were scanned and mean pixel densities of interested proteins, selected by eye control, were measured by ImageJ software and were normalized to the reference spots. To compare the possible differences between angiogenetic profiles of the different groups, we determined the relative density change of the selected spots. Only those proteins are represented which showed at least 1.3-fold change.
Western Blot Analysis
For Western blot experiments tissue homogenizates of four retinas per group were used. Frozen tissues were homogenized with ultra turrax and Potter homogenizer in 150 μl lysis buffer (50 mM TRIS, 50 mM EDTA, 0.5% protease inhibitor cocktail (Sigma-Aldrich) and 0.5% phosphatase inhibitor cocktail (Sigma-Aldrich), pH = 7.4). The homogenate was sonicated and the protein concentration was determined with a DC™ Protein Assay kit (Bio-Rad) according to the manufacturer’s description. The tissue lysate was diluted in Laemmli buffer, boiled for five minutes, centrifuged (13300 rpm, 10 min) and the clear supernatant was used for further investigations. Tissue extracts were separated with SDS-PAGE with protein loads of 20 μg/lane and transferred onto nitrocellulose membrane. The membranes were blocked with 5% non-fat dried-milk proteins in Tris-buffered saline (TBS) and 0.1% Tween, incubated with anti-Akt (No:9272), anti-Akt1 Ser473 (No:9271) antibody (both from Cell Signaling Technology) at 4°C overnight at a dilution of 1:1000. The secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit IgG. Peroxidase labeling was visualized with the Pierce ECL Western Blotting Substrate (Thermo Scientific) detection system. Quantification of band intensities of the blots was performed by ImageJ software. Pixel volumes of the spot were normalized to the internal controls. Data are represented by pixel density in arbitrary units. Statistical analysis was performed by ANOVA test with Fisher’s post hoc analysis after test for homogeneity of variance using STATISTICA software (StatSoft Inc., OK, USA).