Experimental allergic conjunctivitis mouse model with interleukin-23 eyelid injectionAnimals and ethical approval

Five-week-old female BALB/c mice were purchased from Charles River, Japan and housed under specific pathogen-free conditions. The mice had unrestricted access to standard food and tap water. All animal experiments were approved by the Animal Care and Use Committee of Nihon University School of Medicine (Approval Number: AP21MED021-1 July,13, 2021) and conducted following the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Murine model of experimental allergic conjunctivitis

Mice were randomly divided into four groups (n = 7 per group): control group (untreated mice), allergy group (mice sensitized to induce EAC, followed by eyelid injection of physiological saline), IL-23 group (EAC-induced mice that received eyelid injections of IL-23 solution [1 µg/mL; GTX85738, GeneTex]), and non-sensitized IL-23 group (non-sensitized mice given topical OVA instillation (5 mg/mL) and eyelid IL-23 injections (1 µg/mL).

Figure 1 summarizes two experimental protocols. Protocol 1 (Fig. 1a) assessed the effect of IL-23 on allergic ocular inflammation. Mice in the allergy and IL-23 groups were sensitized with intraperitoneal (i.p.) injection of an emulsion solution of Alum (15 mg/mL; Cosmo Bio Co. Ltd.) and OVA (5 mg/mL; Sigma-Aldrich Japan) twice on days 1 and 7. On day 14, eyelid injections of either physiological (allergy group) or IL-23 solution (1 µg/ml; IL-23 group) were administered subcutaneously into both the upper and lower eyelids. From days 14–16, OVA eye drops were instilled in the mice in the allergy and IL-23 groups in both eyes once daily. The mice were euthanized on day 17, 24 h after the final OVA instillation, by an overdose of pentobarbital. Blood, eyeballs, and eyelids were collected for further analysis.

Fig. 1

Experimental protocol. a Protocol 1 shows the sensitization, eye drop instillation, and eyelid injection procedures for the allergy, interleukin (IL)-23, and control groups. b Protocol 2 shows the sensitization, eye drop instillation, and eyelid injection procedures for the IL-23 and non-sensitized IL-23 groups

Protocol 2 (Fig. 1b) investigated the IL-23 response in the presence or absence of allergic inflammation. The mice in the non-sensitized group received eyelid injections of IL-23 on day 14 and topical OVA instillation OU on days 14–16 without i.p. sensitization. The mice were euthanized with pentobarbital on days 17 and 24 h after the final OVA instillation, and blood, eyeballs, and eyelids were collected for further analysis.

Quantitation of ovalbumin-specific immunoglobulin E

Serum was prepared from whole-blood samples from each mouse. Levels of OVA-specific immunoglobulin E (IgE) antibodies in the serum were quantified using an Anti-Ovalbumin IgE (mouse) EIA Kit (Cayman Chemical), following the manufacturer’s instructions. Samples were diluted (1:50) in phosphate-buffered saline containing 1% bovine serum albumin and 0.05% Tween-20, and quantified within the 1.56-100 ng/ml (assay range)

Histological and immunohistochemical analysis of conjunctival tissue

The entire conjunctival and corneal tissues from all groups were fixed in 4% paraformaldehyde (WAKO), paraffin-embedded, and sectioned at approximately 6 μm using a microtome (Leica CM1520; Leica Biosystems). Sections were stained with direct fast scarlet (DFS) for eosinophils and processed by immunohistochemistry with the enzyme-labeled antibody method using rat anti-mouse Ly-G6/Ly-C6 (Gr-1) antibodies (BD Pharmingen) with N-Histofine® Simple StainTM Mouse MAX-PO (Rat) kit (NICHIREI BIOSCIENCES) to identify neutrophils.

Eosinophil and neutrophil infiltration in conjunctival tissue

Gr-1- and DFS-stained sections in each group were photographed under a light microscope (BZ-X710; KEYENCE). Subconjunctival tissue areas were quantified in each digital image using a semi-automated image analysis protocol in Adobe Photoshop Elements 14 software (Adobe Systems Inc.). Eosinophil and neutrophil densities (cells/mm2) were calculated for each section, and averages were obtained across 15 thin sections per group.

Laser microdissection quantitative RT-PCRTissue preparation and mRNA extraction

Conjunctival tissues were embedded in OCT compound (Sakura Seiki) without prior fixation, sectioned at 20 μm, and mounted on RNAse-free polyethylene naphthalate-coated glass slides (Leica Microsystems). Tissue sections were fixed for 30 s in cold 100% methanol, immersed in distilled water, stained with 0.05% toluidine blue, and air-dried. Laser-assisted microdissection (LMD) was immediately performed using the LMD6000 system (Leica Microsystems), targeting subepithelial inflammatory regions for mRNA extraction.

The LMD-dissected samples were collected in Thermo-Tube caps (Thermo Scientific Japan,) containing 10 μL mineral oil (Life Technologies Japan). Total RNA was extracted using the magLEAD® automated system (Hitachi). cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies Japan), and samples were stored at −80°C for further analysis.

PCR array and quantitative RT-PCR analysis of conjunctival tissues

For PCR array analysis, conjunctival samples from the allergy and IL-23 groups were used to analyze the expression of 84 genes encoding allergic inflammation-associated factors using the RT2 Profiler™ PCR Array Mouse Allergy & Asthma kit (PAMM-067ZA, Qiagen). Additionally, IL-23 (n = 3) and non-sensitized IL-23 (n = 3) group samples were used to analyze the expression of 84 genes encoding Th17-associated factors using the RT2 Profiler™ PCR Array Mouse Th17 Response kit (PAMM-073ZR, Cat. no. 330231, Qiagen). All procedures followed the manufacturer’s instructions. Fold changes were calculated using the ΔΔCt method with RT2 Profiler™ software (Qiagen).

Quantitative RT-PCR was performed on samples from the allergy, IL-23, and non-sensitized IL-23 groups to evaluate expression levels of IL-5, IL-17A, chemokine (C-X-C motif) ligand (CXCL) 1, and CXCL2. Reactions were performed using TaqMan™ Gene Expression Master Mix (Thermo Fisher Scientific Japan) and predesigned primers/probes, including IL-5 (il5, Mm00439646_ml), IL-17A (il17a, Mm00439618_m1), CXCL1 (cxcl1, Mm04207460_m1), and CXCL2 (cxcl2, Mm00436450_ml) (Life Technologies Japan), on a StepOnePlusTM system (Life Technologies Japan). Gene expression was normalized to β-actin (Mm000607939_s1), and relative expression was calculated using the ∆∆Ct method.

Statistical analysis

Eosinophil and neutrophil densities across the control, allergy, and IL-23 groups were compared using the nonparametric Steel–Dwass test. Differences in mRNA expression between the allergy and IL-23 groups, and between the IL-23 and non-sensitized IL-23 groups, were analyzed using a parametric t-test. Statistical significance was set at p < 0.05. All statistical analyses were performed using the MAC Toukei–Kaiseki version 3 software (Esumi).