AMD Mouse Models
Both knock out AMD mouse models, Apolipoprotein (Apo)E knock out (-/-) and Nuclear factor erythroid 2-related factor 2 (NRF2) -/- have been described in detail (22,26,27). ApoE -/-, NRF2 -/- and C57BL6/J control mice were purchased from the Jackson Laboratories (Bar Harbour, ME, USA). The homozygous genotype and screening for Crumbs homologue1 (CRB1) retinal degeneration (rd)8 mutation, known to interfere with the AMD phenotype of NRF2-/- mice (28), was confirmed by PCR from tail clips. Mice were kept on a regular 12 hours night and day cycle and fed standard murine diet and water ad libitum. All animal experiments were conducted in accordance with the EU directive 2010/63/EU for animal experiments. They were approved by the animal ethics and welfare committee (approval number: V 242-7224.121-12 (61-5/14)) located at the ministry of energy transition, agriculture, environment and rural areas in Schleswig-Holstein according to German federal and European law. Animal experiments adhere to the NIH Guide for Care and Use of Laboratory Animals.
Animal Maintenance and Anesthesia During Experiments
All examinations and laser treatments were conducted under general anesthesia, like described before (22).
Anesthetized animals were placed on a rigid examination platform and body temperature was maintained within normal limits using a heating mat. Pupils were dilated and eyes were covered with a protective moisturizing gel. After examinations, the anesthesia was antagonized, like described before (22). Anesthesia was uneventful in all mice. Animal wellbeing was evaluated by a standard score sheet and was uneventful in all included mice. After the final examination animals were euthanized by cervical dislocation at the day of enucleation under deep anesthesia.
All examinations were conducted under general anesthesia. All mice were examined by funduscopy (MICRON III, phoenix research labs, Pleasanton, CA, USA), to assess integrity of retina, hallmarks of AMD (drusen-like retinal spots (DRS)), RPE atrophy and CNV.
Optical coherence tomography (OCT) (small animal OCT, thorlabs, Lübeck, Germany) was applied to evaluate retinal structure, confirm retinal integrity after laser treatment and to confirm CNV.
All examinations were repeated at the day of enucleation, thus 1 day or 1 week after laser treatment. Untreated controls also were examined twice, at inclusion and at enucleation day.
For both SRT and TSR a frequency doubled Neodym-Vanadate (Nd:VO4) experimental laser (Carl Zeiss Meditec AG, Jena, Germany) with a wavelength of 532 nm was used. The light was coupled to an optical multimode fiber with a 70 x 70 µm² core profile. The laser light was applied via contact laser-injector (Phoenix) attached to the Micron III camera. The pilot laser was controlled visually via live fundus imaging. Spot size was fixed to 50 µm².
For TSR duration of irradiation was fixed to 10 ms continuous wave mode. For SRT duration of irradiation was fixed to 300 ms, pulse-duration was ~1.4 µs at 100 Hz, creating 30 pulses per spot. Comparable to our previous work (22,23), the intended effect was titrated visually by decreasing energy at the peripheral retina from a clearly visible white burn at higher energy to a barely visible spot at lower energy. The barely but instantly visible spot was classified as threshold of definite retinal burn/RPE destruction with visible neuroretinal involvement. Power was reduced by 70 % to ensure neuroretina-sparing temperature increase for TSR, or an RPE-selective laser damage for SRT respectively. The invisible 50 µm TSR/SRT laser spots were distributed uniformly across the retina at 1 spot interspot spacing to an optic disc centered approx. 50° field of view. No laser spot was applied to vasculature or the optic disc.
RT² profiler PCR array (Qiagen®, Frederick, Maryland, USA; Mouse Inflammatory Response & Autoimmunity; PAMM-077Z) was used to determine regulation of inflammation and cell-death related mediators of TSR or SRT treated eyes in comparison with untreated littermates in AMD mouse models. The procedure was described in detail before (25). Briefly, for RNA isolation, posterior cups were homogenized and total RNA was isolated using TRI Reagent® according to the manufacturer’s instructions.
Isolated RNA was converted to cDNA using the RT2 First Strand Kit (Qiagen). The mixture was aliquoted (25 μl) to each well of the same RT2 Profiler PCR Array plate (96-well plate) containing the pre-dispensed gene-specific primer sets. PCR was performed using a 7500 Fast Real Time cycler (Applied Biosystems).
Qiagens online Web analysis tool (Gene globe) was used to calculate the fold change by determining the ratio of mRNA levels to control values using the Δ threshold cycle (Ct) method (2−ΔΔCt). All data were normalized to the housekeeping genes of PAMM007Z panel (Quiagen). PCR conditions used: hold for 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 60 s at 60°C.
Porcine Organ Cultures Preparation
Fresh porcine eyes were acquired from a local abattoir. The preparation has been described in detail elsewhere (29). Briefly, the eye bulbs were cut at the limbus removing the anterior segment, including lens and vitreous body. Eyes were opened by longitudinal incisions and neuroretina was removed. The complex of RPE, BrM and choroid was removed carefully from sclera. A plastic ring-system was inserted, and the RPE/BrM/choroid complex fixed to it. Rings were placed into 12-well-culture plate and kept warm at 37°C in 1.5 ml organ culture medium (see Richert et al. (29)).
TSR and SRT in Organ Cultures
Organ cultures were placed under a slit-lamp adapted laser system in organ culture medium in 12-well plates. Organ cultures were irradiated by either TSR (100 ms duration, 200 µm spot size, power titrated to no instant cell-death and a cell death rate of ~ 2 % one day after TSR), or by SRT (300 ms duration, 100 Hz, 1.4 µs pulse duration, 200 µm spot size, energy titrated to an initial cell-death rate of 80 %). Calcein-assays were performed afterwards to confirm cell-death rates. This method was also applied to check the quality of organ cultures (30).
Calcein assays were conducted to examine integrity and vitality of RPE organ cultures. Calcein fluoresces if cleaved by active enzymes integrated into vital RPE cell membranes. Dead cells do not fluoresce, since enzymatic cleavage does not function in dead cells (31). Organ culture explants were incubated in 2 ml culture medium with 4 µg/ml Calcein at 37 °C for 45 minutes. Afterwards they were rinsed twice in phosphate buffered saline. Cell vitality was measured by fluorescence microscopy (Axiovert 100, Zeiss, Jena, Germany) at λex/λem = 497/517 nm and documented photographically.
Gene Expression by PCR Arrays
Fold changes in gene expression for pairwise comparison using the ΔΔCT method was calculated through Qiagen® Web analysis tool and p-values were provided, at a confidence interval of 95 % and a type-1 error of 5 %.
For comparison of TSR or SRT treated to untreated eyes, one randomized eye of ApoE-/- or NRF2-/- was treated by TSR or SRT. One day or one week after treatment these eyes were compared to entirely untreated age-matched randomized control eyes of the same genotypes in groups of 6 eyes each.
Determination of Cell Size and Number by Calcein Assays
For determination of cell death and regeneration, 1 hour, 1, 3 and 5 days after TSR or SRT laser treatment, calcein assay photographs were analyzed semiautomatically by AxioVision (Zeiss, Jena, Germany). Cell size and number of vital cells within the defined 200 µm laser spot were measured and noted for statistical analysis. The median percentage of non-fluorescent area within the defined laser spot was calculated from 12 spots each.