Reliability and reproducibility of a rodent model of choroidal neovascularization based on the subretinal injection of polyethylene glycol

Animals

The use of animals in this study was approved by the Animal Research and Welfare Ethics Committee of the University of Valladolid (Valladolid, Spain) and was authorized by the Farming and Ranching Agency of Junta de Castilla y León (Regional Government, Spain) in agreement with European (Council Directive 2010/63/UE) and Spanish regulations (RD 53/2013). This study was in accordance with the recommendations of the Association for Research in Vision and Ophthalmology for the Use of Animals in Ophthalmic and Vision Research. Thirty-two (n=32) wild-type male C57BL/6 mice (specific pathogen-free [SPF] colony, 8±1 weeks old, 24±1.5 g; Jackson Laboratory, Bar Harbor, ME) were used. The animals were randomly divided into Control 1 (PBS-injection, n=5), Control 2 (PBS-injection, n=9), and Experimental (PEG-injection, n=18) groups. The Control 1 animals were immediately sacrificed after the injection while the Control 2 and Experimental groups were further split into 5, 14 and 21-day follow-up groups (Table 1). Thus, four control groups and three experimental groups were used. Each animal group was individually housed in Type II conventional cages inside a specific experimental area at the animal facilities of the University of Valladolid. Each experimental area had restricted access to a single researcher and followed a complete sanitary emptying procedure that includes washing and disinfecting the room and materials in advance to house the animals. The animals were kept under 12 h:12 h light-dark conditions at 22 °C and Lignocel 3–4 S (JRS GmbH + Co KG, Rosenberg, Germany) was used as bedding material. The animals had varied enrichment material in their cages and had free access to food (V1534–000 ssniff® R/M-H; ssniff Spezialdiäten GmbH, Soest, Germany) and water during the study. The selection of the species, type, breed and sex of the animals used in this study has been based on a previous publication [25] that described the rodent CNV model by subretinal injection of PEG and in the absence of evidence of gender difference in AMD prevalence [25,27]. This study was designed to use the minimum number of animals per experimental group that allows an adequate biostatistical analysis of the results and their possible extrapolation to humans.

Surgical technique

The following medical and surgical protocols were selected according to the species, sex, age and weight of the animals, as well as the requirements of the study performed. All the procedures described were performed in an operating room inside the animal facilities of the University of Valladolid. The surgical procedure was performed on both eyes in the Control 1 group. For the Control 2 group (PBS injection) and the Experimental group (PEG injection), only the right eye was injected. To carry out the surgical procedure, the animals were anesthetized by an intraperitoneal injection of ketamine (80–100 mg/kg; Imalgene 1000, Merial, Lyon, France) and xylazine (5–10 mg/kg; Rompún 2%, Bayer HealthCare, Kiel, Germany). Pedal and auricular reflexes were used to monitor the level of anaesthesia. Analgesia was applied by subcutaneous injection of butorphanol (0.5–3 mg/kg; Torbugesic® Vet, Fort Dodge Animal Health, Fort Dodge, IA). The periorbital area was cleansed by a solution of povidone iodine (5% Betadine®; Meda Manufacturing, Bordeaux, France). Cyclopentolate 1% (Colircusí Ciclopléjico®; Alcon Cusí S.A., Barcelona, Spain), phenylephrine 2.5% (Colircusí Fenilefrina®; Alcon Cusí S.A.) and 0.4% oxybuprocaine/0.1% tetracaine (Colircusí Anestésico Doble®; Alcon Cusí S.A.) were administered topically as mydriatic agents and as a local anaesthetic, respectively. An electric heating blanket was used to prevent hypothermia during the surgical procedure.

A single subretinal injection was administered per eye as described by Matsumoto et al. [28]. In brief, surgical procedures were performed under a Leica M844 surgical microscope (Leica Microsystems, Wetzlar, Germany). The conjunctiva was dissected using Vannas scissors (John Weiss & Son Ltd., Milton Keynes, UK). To prevent an increase of intraocular pressure, a corneal paracentesis at 1 mm anterior to the limbus was performed with a 30-gauge needle (Becton Dickinson, Franklin Lakes, NJ). A XenoWorks™ analog microinjection system (Sutter Instrument, Novato, CA) equipped with a NanoFil Hamilton 10 µl syringe (World Precision Instruments, Sarasota, FL) and a 33-gauge blunt needle (World Precision Instruments) was used for injections. A drop of hydroxypropyl methylcellulose (Methocel® 2%; OmniVision, Puchheim, Germany) over the cornea was used to facilitate fundus visualization through the microscope. The sclera was punctured at the pars plana on the superior temporal quadrant to introduce the 33-gauge blunt needle. A tunnel through the sclera and the choroid was then created until the subretinal space, between the neural retina and the RPE, was reached. Advancement of the needle was carefully monitored under the surgical microscope. One milligram of PEG-8 (mean molecular weight of 400; Spectrum Chemicals & Laboratory Products, Gardena, CA) diluted in 2 μl of PBS (Gibco; Invitrogen, Paisley, UK) or 2 μl of PBS (control eyes, Gobco, Invitrogen), were gently and slowly microinjected (in 10 s) into the subretinal space. Fresh PEG-8 dilutions were prepared for each experiment. Successful administration into the subretinal space was confirmed by the appearance of a subretinal bleb in the ocular fundus. Postoperative antibiotic treatment was established with topical ciprofloxacin (Oftacilox®, Alcon S.A.) twice a day for three days after the subretinal injection. Physical examination of the animals (such as activity, interaction with cage mates, general appearance) was performed daily throughout the study.

Histological and immunohistochemical evaluation

At the designated times, the mice were sacrificed by CO₂ overdose according to the species, sex, age and weight of the animals. Control 1 mice were killed immediately after the injection of the second eye without allowing recovery from the anaesthesia. Control 2 (PBS-injected) and Experimental mice (PEG-injected) were killed at 5, 14 or 21 days after subretinal injection. A 10–0 nylon suture (Alcon, Fort Worth, TX) was placed at the central superior sclera to facilitate sample orientation during tissue processing. Enucleation was performed to remove the entire ocular globe. The eyeballs were punctured at the limbus with a needle and immediately submerged in fixative (4% paraformaldehyde in 0.1 M-phosphate buffer; pH 7.4) for 24 h at room temperature. After fixation, samples were processed in an automatic tissue processor (Leica ASP300; Leica Microsystems, Wetzlar, Germany). One paraffin block of each eye was made and vertical serial 4-µm thick sections through the optic disc were obtained in a rotatory microtome (Microm HM340E; Microm International GmbH, Walldorf, Germany) until the identification of the injection site. Sections at the site of injection and around that point were used for this study.

Ocular sections for haematoxylin and eosin (HE) or Fontana-Masson (FM) staining were de-waxed in xylene, rehydrated in a series of descending alcohols (100°, 96° and 80°), rinsed in deionized distilled water, and stained with HE (Merck KGaA, Darmstadt, Germany) or FM. Stained sections were dehydrated in a series of ascending alcohols (96° and 100°), cleared in xylene, mounted, and coverslipped. Ocular sections for immunohistochemistry were de-waxed in xylene, rehydrated as described above, and rinsed in PBS (Gibco, Invitrogen). For antigen retrieval, sections were incubated in 0.01% trypsin (Sigma-Aldrich, Steinheim, Germany) in PBS (Gibco, Invitrogen) for 15 min at 37 °C. Then, the sections were immunostained for the phenotype-specific markers cytokeratin 8/18 (CK8/18; 1:25, Abcam plc., Cambridge, UK), isolectin B4 from Griffonia simplicifolia (IB4; 1:50, Molecular Probes, Eugene, OR), VEGF (1:50, Bioss Antibodies, Woburn, MA), and von Willebrand factor (vWF; 1:50, Millipore, Billerica, MA). Antibodies were diluted in PBS (Gibco, Invitrogen) containing 0.5% Triton X-100 (Sigma-Aldrich), and the sections were incubated overnight at 4 °C. The next day, the sections were washed in PBS (Gibco, Invitrogen). Thereafter, the corresponding species-specific secondary antibody conjugated to Alexa Fluor 488 or 568 (green or red, respectively; Thermo Fisher Scientific, Rockford, IL) was applied at a 1:200 dilution for 1 h. Nuclei were stained with 10 μg/ml 4’,6-diamino-2-phenylindole dihydrochloride (blue; DAPI, Molecular Probes). Finally, sections were washed in PBS (Gibco, Invitrogen), mounted in an aqueous-based fluorescent mounting medium (Fluoromount-G; SouthernBiotech, Birmingham, AL) and coverslipped.

Control samples, in which the primary antibody was omitted, were processed in parallel, and no immunoreactivity was found in any case. The specificity of antibodies and IB4 had been previously tested [35,36] in our laboratory. Samples from a human eyelid cavernous haemangioma (courtesy of the IOBA Ocular Pathology Lab, University of Valladolid, Spain) were used as positive controls for the VEGF and vWF antibodies. Samples were analysed with a Leica DM4000B light microscope (Leica Microsystems) equipped for epifluorescence, and images were acquired with a Leica DFC490 (Leica Microsystems). Comparative studies of immunoreactivity expression were performed on images acquired at the same levels of exposure, intensity and gain. Brightness and contrast were minimally adjusted, and the final figures were composed with Pixelmator 3.4 Twist (Apple, Cupertino, CA).

Immunoreactivity semi-quantification was measured in ocular sections as previously described [29]. Two-channel micrographs were analysed using ImageJ software v. 1.49 (ImageJ; National Institutes of Health, Bethesda, MD). The micrographs were then split into different channels and each channel thresholded. To subtract background in the green channel (corresponding to the protein of interest), negative controls were used to set the threshold value, and the same value was used in all micrographs being analysed from the same experiment at the same time. The threshold for the blue channel, corresponding to the area of nuclei, was set according to the area stained. The mean gray value was measured by redirecting the measurement to the corresponding channel. The results, in arbitrary units, were the ratio of the mean gray value for the green channel to that obtained from the blue channel.

Statistical analysis

All data were collected in a database created in Excel (Microsoft Office Excel 2013, Microsoft Corporation, Redmond, WA) and subsequently analysed by the Student t test for independent samples. Each group of animals previously defined was studied as an independent unit of analysis. In all cases, a p value of less than 0.05 was considered statistically significant. Data were expressed as means ± standard deviations. Normality and homogeneity of variance assumptions were checked by the Shapiro-Wilk test and the robust Brown-Forsythe Levene-type test, respectively.

Surgical technique

Ocular evaluation under a surgical microscope ruled out the presence of ocular alteration before performing the surgical procedure. No adverse effects were detected beyond those induced by the subretinal injection procedure; thus, the previously described protocol was not modified. The subretinal injections were adequately performed in all three animal groups and resulted in localized retinal detachment, confirmed under surgical microscopy by the presence of subretinal blebs (Figure 1A, arrows). Eyes from the Control 1 group were evaluated immediately after the procedure (0 days) to confirm localized subretinal injections. Superior retinal detachments were histologically evident (Figure 1B). Photoreceptor outer segment debris was present between the separated neuroretina and RPE (Figure 1B, arrows). No other damage attributable to the injection procedure was observed in the neuroretina or in the RPE/choroid.

Histological evaluation

At 5 days of follow up, 2/3 PBS-injected eyes had retinal degeneration, disruption of the RPE and migration of pigmented cells (Figure 2A). In 1/3 mice, there was RPE vacuolization. In 4/6 PEG-injected eyes, there was a loss of normal retinal architecture and degeneration of the photoreceptor outer and inner segments (Figure 2B). Enlargement and vacuolization of the RPE cells, similar to that seen in the PBS-injected control eye, and the presence of pigmented clumps in the subretinal space were also evident. At 14 days of follow up, all of the PBS-injected eyes had subtle subretinal vacuolization and pigment cell migration with only a slight disruption of the photoreceptor outer segments (Figure 2C). 2/6 PEG-injected eyes had retinal degeneration and RPE/choroid modifications (Figure 2D). Pigment clumps and cell vacuolization in the RPE/choroid were also noted. At 21 days of follow up, none of the PBS-injected eyes presented either neuroretina or RPE/choroid degenerative modifications (Figure 2E). In some areas, 3/6 PEG-injected eyes had outer retinal degeneration and subtle RPE changes, such as RPE clumps and the migration of pigmented cells (Figure 2F).

To determine the composition of the subretinal pigmented clumps, ocular sections were stained with FM, which specifically identifies melanin granules that reduce silver nitrate to metallic silver. This specific staining revealed, in both PBS and PEG-injected eyes, the presence of melanin in the subretinal pigmented clumps at different time points (Figure 3A, arrowheads), as well as in the RPE layer and in the choroid (Figure 3A, arrows).

Immunohistochemical evaluation

To determine the origin of the subretinal pigmented cells, ocular sections were immunostained for CK8/18, the epithelial-type cytokeratins that are the major intermediate filaments in the RPE. Non-injected and PEG-injected eyes had positive immunoreactivity to CK8/18 in the RPE monolayer (Figure 3B and C, arrows). Positive cellular elements at the subretinal space were also observed in PEG-injected eyes (Figure 3C, arrowheads).

To evaluate the choroidal vasculature, ocular sections were immunostained with Griffonia simplicifolia isolectin B4 (IB4), a ligand that specifically binds terminal α-galactosyl residues expressed by endothelial cells and other cell types. In both the PBS- and PEG-injected eyes, IB4 was present in endothelial cells of the choroid (Figure 4A, B, arrows). PBS-injected eyes had a clearly recognizable network of small and medium arteries and arterioles, while in PEG-injected eyes, some vessel walls appeared diffusely thickened. IB4 immunoreactivity was significantly increased in PEG-injected eyes in comparison with that of PBS-injected eyes at all time points (Figure 4C).

To evaluate neovascularization, ocular sections were immunostained for VEGF, a soluble protein that stimulates vascular endothelial cell proliferation and is a potent inducer of angiogenesis. The sections were also stained for vWF, a glycoprotein that plays a major role in blood coagulation and is a widely used endothelial cell marker for studying angiogenesis/neovascularization. Both VEGF and vWF were present in the control sample from a human eyelid cavernous haemangioma, but neither was detectable in the retina/choroid area of the injected-eyes at any follow-up time (Figure 5).