On day 14, animals were euthanized and their eyes collected for either histology or combined aqueous humor cytokine analysis and flow cytometry. For histology, whole eyes were fixed in 10% neutral buffered formalin (Sigma-Aldrich Corp., St. Louis, MO, USA) for at least 24 hours. Paraffin block sections (4 µm) were stained with hematoxylin and eosin (H&E) and scored by a single grader (author K.P.), masked to treatment using an established grading system.
46 Three sections per eye were scored and averaged. For animals receiving systemic treatment, final score was assigned per animal and calculated as the average score of both eyes.
Aqueous humor was collected in an ethylenediaminetetraacetic acid (EDTA)-containing capillary tube (Sarstedt, Nümbrecht, Germany) after corneal paracentesis with a 30-gauge needle (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Ten to 15 µL of aqueous was collected from each eye and stored at −80°C, in combination with 1X protease inhibitor (Sigma-Aldrich Corp.), until assayed. Aqueous protein was quantified using Pierce 660 nm Protein Assay Reagent (Thermo Fisher Scientific, Madison, WI, USA) for colorimetric detection on the Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific); 1 µL aqueous was used for total protein concentration determination. The remaining aqueous (9–14 µL) was diluted in an equal volume of radioimmunoprecipitation assay (RIPA) buffer containing phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail, according to the manufacturer's protocol, and divided equally for testing in duplicate. For cytokine analysis in the systemic treatment study, aqueous from the right and left eye of the same animal were pooled into a single sample (16–36 µL per sample). Aqueous from the local treatment study was not pooled. Aqueous cytokine concentrations were determined using the Milliplex MAP rat cytokine/chemokine premixed 27-plex immunology multiplex assay (EMD Millipore Corp., Billerica, MA, USA). The cytokines measured were granulocyte-colony stimulating factor (G-CSF), eotaxin (CCL11), granulocyte monocyte-colony stimulating factor (GM-CSF), IL-1α, macrophage inflammatory protein-1α (MIP-1α/CCL3), IL-2, epidermal growth factor (EGF), IL-13, IL-12p70, IL-5, monocyte chemoattractant protein-1 (MCP-1/CCL2), IFNγ–induced protein 10 (IP-10/CXCL10), fractalkine (CX3CL1), lipopolysaccharide-induced CXC chemokine (LIX/CXCL5), MIP-2 (CXCL2), leptin, IL-4, IL-6, IL-10, IFN-γ, IL-17A, IL-18, growth-related oncogene/keratinocyte chemokine (GRO/KC/CXCL1), VEGF, TNFα, and regulated on activation, normal T cell expressed and secreted (RANTES/CCL5). Due to failure of the GRO/KC controls, results for this cytokine are not reported. Samples were analyzed using the MAGPIX system (Luminex, Austin, TX, USA) with xPonent software version 4.2 (EMD Millipore). Data analysis was performed using Milliplex Analyst Standard version 5.2 software (Vigenetech). Statistics analysis and graphing was performed using GraphPad Prism 9.0 software (GraphPad Software, La Jolla, CA, USA).
For flow cytometry analysis, the retina, retinal pigmented epithelium (RPE), choroid, and vitreous were removed from the eyes following aspiration of the aqueous (for cytokine analysis) and collected in 1X phosphate-buffered saline (PBS) with 2% fetal bovine serum (FBS; Cytiva, Marlborough, MA, USA). A single cell suspension was generated, as described previously,
44 with mechanical disruption using microsurgical scissors, followed by digestion with Collagenase D (Roche, Switzerland) at 37 degrees Celsius (°C), and filtration through 70 mm mesh (Elko Filtering Co., Switzerland). Live cell counting was performed using acridine orange (AO)/propidium iodide (PI) and a Nexcelom cell counter (Auto2000). For cell surface marker identification, cells were stained with live-dead blue viability dye (Invitrogen; 1:1000), following by anti-human IgG (BioLegend [BL] 409318; 1:100), and antibodies to the following rat markers: CD28 (Becton Dickinson [BD] 742583; 1:100), CD3 (BD 563949; 1:100), ICOS (BL 313534; 1:100), CD25 (BL 202103; 1:100), CD11b/c (BL 201820; 1:100), RT1B (BD 205308; 1:200), CD4 (BL 201516; 1:100), FoxP3 (BL 320014; 1:40), CD8 (BD 741771; 1:100), and CD45 (BL 202216; 1:100). For intracellular cytokine staining, cells were incubated with phorbol 12-myristate 13-acetate (PMA) and ionomycin with Golgi block for 4 hours at 37°C prior to staining with live-dead blue viability dye (Invitrogen; 1:1000), and antibodies against rat markers CD8 (BD 741771; 1:100), CD3 (BD 563949; 1:100), CD4 (BL 201516; 1:100), CD45 (BL 202216; 1:100), IL-17A (eBiosciences 11-7177-81; 1:40), and IFNγ (BL 507806; 1:20). For samples in the systemic treatment study, right and left eye samples from each animal were pooled prior to staining and divided into two samples for surface and intracellular staining. For the local treatment study, samples were not pooled. Stained samples were collected on a Beckman Coulter Cytoflex LX or a BD LSRII flow cytometer and data analyzed using FlowJo software (version 10.7.1; Ashland, OR, USA).