**1. Introduction**

There has been a significant rise in the incidence of myopia in the past five decades. Furthermore, myopia is rapidly becoming the primary cause of visual impairment worldwide [1–3]. Thus, myopia prevention will avert visual impairment and retinal complications. The molecular basis of myopia has not been completely elucidated. Nonetheless, several researchers have reported on the role of inflammation [4] and remodeling of the scleral extracellular matrix (ECM) in the onset and progress of myopia [5]. Patients with inflammatory diseases, such as type 1 diabetes, uveitis, and systemic lupus erythematous, show a higher incidence of myopia. Topical application of cyclosporine A, an immunosuppressive agent, slows down the progress of the monocular form of deprivation-induced myopia. In contrast, it is accelerated by the topical application of lipopolysaccharide, an inflammation inducer [6]. The concentrations of interleukine-6 (IL-6) and matrix metalloproteinase-2 (MMP-2) in the aqueous humor are greater in highly myopic eyes than in ametropic or mildly myopic eyes. Moreover, the contents are positively associated with axial length [7]. While the content of collagen I, the most abundant structural collagen in the sclera, decreases via suppression of the *COL1A1* gene, it increases because of collagen degradation enzymes, such as MMP-2 and -9 [8–11]. The latter reportedly leads to collagen fiber thinning, followed by scleral thinning and weakening, thus causing axial elongation.

Lactoferrin (LF) is an iron-binding glycoprotein not only abundant in cow's milk but also abundant in body fluids, such as colostrum, saliva, and tears [12,13]. It possesses various biological functions, such as immunomodulation and the prevention of oxidative damage and photo damage [14–17]. LF reportedly affects collagen synthesis and degradation. Bovine LF enhances the transcription of the *COL1A1* gene and collagen I synthesis in human dermal fibroblasts [18]. In addition, LF hydrolyzate attenuates interleukin-1β-induced expression of MMP-1, -3, and -13 in human articular chondrocytes [19]. Furthermore, it has beneficial effects against eye dysfunctions, including dry eye, choroidal neovascularization, and age-related lachrymal gland dysfunction [15,20–22]. Considering the association between inflammation and collagen turnover, and between myopia onset and progress, LF can counteract these effects. Thus, we hypothesized that daily LF supplementation prevents or suppresses myopia development. We showed that oral administration of LF attenuated minus-lens-induced myopia development in C57BL6J mice. Furthermore, LF suppressed lens-induced myopia (LIM)-induced MMP-2 activation, besides increasing *COL1A1* expression.

#### **2. Materials and Methods**

Experimental animals: All animal experiments in this study were approved by the Animal Experimental Committee of the Keio University (Permit number: 16017-3). Our study adhered to the Institutional Guidelines on Animal Experimentation at the Keio University, the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research, and the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines for the use of animals in research. We purchased male C57BL/6J mice (3-week-old) from CLEA Japan (Yokohama, Japan). We maintained five mice in one cage by free intake with standard chow and water. We kept them in an environment with a 12 h/12 h light/dark cycle (the dark cycle extended from 8:00 p.m. to 8:00 a.m.) at 23 ± 3 ◦C. We maintained the light cycle using a 50-lux background according to a previous report on experimental myopia induction [23,24].

Lactoferrin (LF) administration: We purchased LF from bovine milk from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). LF was dissolved in Ringer's solution at a concentration of 160 mg/mL and stored it at -30 ◦C until use. We orally administered 1600 mg/kg/day LF once a day to 3-week-old mice for 4 weeks. We administered a similar volume of Ringer's solution to the control group.

Lens-induced myopia (LIM): We measured the refraction, choroidal thickness, and axial length of all eyes before (4-week-old) and after inducing myopia (7-week-old) using a refractometer (Steinberis Transfer Center, Tübingen, Germany) and spectral domain optical coherent tomography (SD-OCT; Envisu R4310, Leica, Wetzlar, Germany), respectively. We anesthetized the mice with 0.75 mg/kg medetomidine (Sandoz K.K., Tokyo, Japan), 4 mg/kg midazolam (Domitor; Orion Corporation, Espoo, Finland), and 5 mg/kg butorphanol tartrate (Meiji Seika Pharma Co., Ltd., Tokyo, Japan) dissolved in normal saline. Several eyes could not be measured because of corneal abnormalities, which were observed in a certain percentage of cases and not specifically in this study. At baseline for myopia induction, we fixed −30 diopter (D) lenses and frames without lenses onto the right and left eyes (as a control), respectively. Furthermore, we maintained this setup for 3 weeks, until the mice were 7 weeks old. Figure 1 outlines the protocol for LF administration and LIM.

**Figure 1.** Procedure for oral lactoferrin (LF) administration and minus-lens-induced myopia.

**Tissue preparation:** After myopia induction, we harvested the complete choroid and sclera of each eye for gel zymography and cytokine analysis. We pooled the issues from the LIM and control eyes of three mice in 1 tube and subsequently homogenized them to obtain a protein lysate (three samples each of LIM and control eyes). We homogenized the tissues in an RIPA buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 1% NP-40, 0.1% sodium deoxycholate, 1 mM EDTA, 5 mM benzamidine, 10 mM β-glycerophosphate, 1 mM Na3VO4, 50 mM NaF, and 1 mM PMSF) containing a Halt protease inhibitor cocktail (Thermo Fisher Scientific, MA, USA) using Precellys (M&S Instruments Inc., Osaka, Japan). After centrifugation, we collected the supernatant and stored it at −80 ◦C until use.

**Gelatin zymography:** We performed zymography as previously described with some modifications. We added a 6× non-reducing sample buffer (Nacalai Tesque, Kyoto, Japan) to the supernatant and added a 1× non-reducing sample buffer, thus adjusting the concentration to 0.5 g/L. The samples were loaded onto 10% SDS-PAGE gels containing 1 mg/mL gelatin (Sigma-Aldrich Japan, Tokyo, Japan). After electrophoresis, we rinsed the gels twice for 30 min in a washing buffer (2.5% Triton X-100; 50 mM Tris-HCl pH 7.5; 5 mM CaCl2; 1 μM ZnCl2, all chemicals were purchased from Wako chemical, Tokyo, Japan), incubated them for 24 h in an incubation buffer (1% Triton X-100; 50 mM Tris-HCl pH 7.5; 5 mM CaCl2; 1 μM ZnCl2) at 37 ◦C, used a staining solution (0.5% Coomasie Blue; 40% methanol; 10% acetic acid, all chemicals were purchased from Wako chemical, Tokyo, Japan) for 1 h, and destained the samples to expose gelatinolytic bands. We obtained the gel images and quantified them using GELSCAN-2 (iMeasure, Nagano, Japan) and ImageJ software (version 1.63), respectively.

**Western blotting:** We added a reducing sample buffer (Nacalai Tesque, Kyoto, Japan) to the remaining supernatant and boiled it at 95 ◦C for 5 min. The samples were loaded onto a 4–15% gradient SDS-PAGE gel (Thermofisher Scientific, Waltham, MA, USA), transferred to a PVDF membrane (Merck Millipore, Burlington, MA, USA), and blocked by Blocking One (Nacalai Tesque). Furthermore, we incubated them overnight with anti-MMP2, anti-IL-6, anti-Collagen1A1, and anti-GAPDH (Cell Signaling Technologies Japan, Tokyo, Japan) at 4 ◦C. After washing with TBS-T, we incubated the membranes with a horseradish peroxidase-conjugated secondary antibody. We visualized the bands using EzWestLumi plus (ATTA, Tokyo, Japan) and LAS-4000 (GE Healthcare, Chicago, IL, USA). After visualization, densitometric analysis was performed using ImageJ software (version 1.63).

**Statistics:** All data are expressed as means ± standard deviations (SDs). We analyzed the differences between groups by one-way analysis of variance (ANOVA) or Student's *t* test. A *p*-value < 0.05 was considered statistically significant.
