Establishment of an acanthamoebic keratitis mouse model

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Doctoral Thesis in Philosophy

Establishment of an acanthamoebic keratitis

mouse model

by

Hee Kyoung Kang

Major in Molecular Medicine

Department of Biomedical Sciences

The Graduate School of Ajou University

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Establishment of an acanthamoebic keratitis

mouse model

by

Hee Kyoung Kang

A Dissertation Submitted to The Graduate School of

Ajou University in Partial Fulfillment of the Requirements for the

Degree of Doctor of Biomedical Sciences

Supervised by

Ho-Joon Shin, Ph.D.

Major in Molecular Medicine

Department of Biomedical Sciences

The Graduate School of Ajou University

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-Abstract-Establishment of an acanthamoebic keratitis mouse model

Acanthamoeba castellanii invades the cornea and results in acanthamoebic keratitis (AK). It is mainly triggered by risk factors such as corneal injury, faulty eye surgery, and poor contact lens use. AK is typically chronic and progressive with inflammatory cells infiltrating the corneal stroma. Many in vitro studies of the pathogenesis of AK and development of therapeutic drugs need to be confirmed by the in vivo experiments. In this study, development of AK animal model was attempted. BALB/c mouse cornea was scratched using a syringe needle and ophthalmic surgical blade under anesthesia. A. castellanii (trophozoites equally mixed with cysts) cultures were serially diluted from 1 × 106 to 0.3 × 105 and placed on a 2-mm contact lens to be inserted into the mouse eye. To allow complete contact of A. castellanii with the damaged cornea, the eyelid was sutured. The keratitis symptoms in mouse eyes were grossly observed from day 1 to 7, post-inoculation. In addition, to confirm the AK development, the genomic DNA was extracted from the mouse ocular tissue with AK. For the amplification of acanthamoebic 18S-rRNA, PCR was performed with P-FLA primers. It was observed that the experimental mouse AK, characterized by typical hazy blurring and melting of the mouse cornea, developed on day 1 post-inoculation and was induced with at least 0.3 × 105 amoeba cells. With 0.5 × 105 amoeba inoculation, the experimental mouse AK was prolonged for 2 months (no more extended observation).

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On the contrary, the sham-operated mouse eye was clear during the observation periods. In addition, PCR products amplified from the extracted mouse eye DNA, using P-FLA primers confirmed the AK development during infection periods. Many commercial lens solutions are less effective against amoebicidal effects of Acanthamoeba. To confirm the amoebicidal effect of the commercial lens solutions, 0.5 × 105_amoebae were pre-treated with solutions A or B for 1, 6, 12 or 24 h, and inoculated into the eye of a previously established AK mouse model. AK occurrence by A. castellanii treated with lens solution A or B for 1 or 6 h was not grossly observed on day 1 and 2, but the infection progressed with a typical circulatory edema and keratitis, on days 3, 5, and 7. Upon pre-treatment for 12 or 24 h, AK development was not observed on days 1, 2, or 3 but progressed to corneal infection on days 5 and 7. Finally, the commercial contact lens solutions did not display a protective effect against AK development, although they affected the occurrence by delaying the AK. In conclusion, it is suggested that the present AK mouse model may serve as an important in vivo model for various future studies.

Keywords: Acanthamoeba castellanii, Acanthamoebic keratitis, Encystment, 18S-rRNA amplification, Commercial lens solution

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LIST OF FIGURES

Figure 1. Life of Acanthamoeba species. ··· 2

Figure 2. Acanthamoeba cysts morphological group II & III ··· 4

Figure 3. Acanthamoeba infection in human ··· 6

Figure 4. Procedure of the experimental acanthamoebic keratitis development ··· 13

Figure 5. Morphological changes of A. castellanii trophozoites into pre-cysts and cysts ··· 19

Figure 6.Induction and observation of acanthamoebic keratitis in experimental mouse models ··· 21

Figure 7. Amplification of A. castellanii 18S-rRNA from AK mouse eye tissue ··· 23

Figure 8. The nucleotide sequence and homology analysis of 18s-rRNA gene obtained from keratitis-induced mouse eye ··· 24

Figure 9. Observation of acanthamoebic keratitis development in mice inoculated with serially diluted A. castellanii (I) ··· 28

Figure 10. Observation of acanthamoebic keratitis development in mice inoculated with serially diluted A. castellanii (II) ··· 30

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Figure 11. Observation of Acanthamoebic keratitis infection with 0.5 × 105

A. castellanii, and 18S-rRNA amplification on mice eye ··· 32

Figure 12. The nucleotide sequence and homology analysis of 18S-rRNA gene

obtained from keratitis-induced mouse eye ··· 34

Figure 13. Morphological change of A. castellanii (trophozoites and cysts) treated

with commercial lens solution A or B ··· 38

Figure 14. Observation of acanthamoebic keratitis upon pre-treatment of solution A · 42

Figure 15. Observation of acanthamoebic keratitis upon pre-treatment of solution B · 44

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I. INTRODUCTION

Acanthamoeba spp. are free-living amoebas, belonging to the genus Acanthamoeba. They are ubiquitously found in a variety of habitats, including soil, dust, fresh water, cooling tower of air conditioning, and domestic tap water. They also exist as a contaminant in tissue culture, contact lenses, and lens cases and containers

(YuGuang He, 1922; Auran et al., 1987; Visvesvara and Stehr-Green, 1990; De Jonckheere, 1991; Mergeryan, 1991; Szenasi et al., 1998; Marciano-Cabral et al., 2000; Schuster and Visvesvara, 2004a). Under environmental conditions, Acanthamoeba spp. feed on various Gram-negative bacteria, green algae, and yeast (Marciano-Cabral et al., 2000).

Acanthamoeba spp. exhibit a typical protozoan life cycle, consisting of an amoeboid trophozoite stage and a resting cyst stage. Adverse environmental and nutritional conditions, such as low temperatures, extreme dryness, and exhaustion of nutrition supply, induce the transformation of trophozoites into cysts. The cyst stage of Acanthamoeba spp. is resistant to physical, chemical, and radiological conditions (Aksozek et al., 2002; Moon et al., 2011). The trophozoite moves using pseudopodia of a size 25–50 μm. The resting cyst is 15–30 μm in size, and consists of thick double walls (Fig. 1) (Marciano-Cabral and Cabral, 2003).

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Acanthamoeba species are divided into three types of morphology groups, according to the size and shape of the cyst (Costas and Griffiths, 1985). Group I consist of species characterized by large amoebas, with cysts of 16–30 μm in size. The largest number of amoeba species belongs to Group II, with polygonal cysts of less than 18 μm in size. A. castellanii and A. polyphaga, causing acanthamoebic keratitis (AK) belong to Group II. Group III also has cysts less than 18 μm in size, but with subtle morphological differences (relatively round form of cyst) (Fig. 2).

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Fig. 2. Acanthamoeba cysts morphological group II & III (Walochnik et al., 2015)

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However, morphology-based species classification is unreliable due to changes in the cyst morphology. There have been efforts to use non-morphological criteria, such as protein and isoenzyme profiling, mitochondrial DNA polymorphism, and 18S-rDNA sequencing as adjuncts to species differentiation based on morphological characteristics (Jonckheere, 1983; Daggett et al., 1985; Johnson et al., 1990; Chung et al., 1998; Visvesvara et al., 2007).

Currently, as a result of phylogenetic analysis based on the 18S-rRNA gene, Acanthamoeba exists in more than 24 species and 17 genotypes (T1–T17) (Stothard et al., 1998; Booton et al., 2005; Corsaro and Venditti, 2010; Nuprasert et al., 2010). Among them, T4 type is mainly associated with the human infections, and includes A. culbertsoni, which causes GAE (granulomatous amoebic encephalitis) (Fig. 3), A. castellanii, A. polyphaga which cause acanthamoebic keratitis (AK) (Fig. 3), and A. healyi and A. rhysodes(Byers et al., 1990; Visvesvara and Stehr-Green, 1990; van Klink et al., 1992; Im and Kim, 1998; Stothard et al., 1998; Corsaro and Venditti, 2010; Visvesvara, 2010; Alves et al., 2018).

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Fig. 3. Acanthamoeba infection in human. (A) GAE (granulomatous amoebic encephalitis) due to Acanthamoeba sp. cyst in brain tissue (arrow). (B) Infection of acanthamoebic keratitis (Siddiqui and Khan, 2012).

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AK can occur in non-contact lens users, but most commonly detected in people using contact lenses. The causes of amoebic keratitis include prolonged contact lens wear, lack of personal hygiene, contact lens contamination, and exposure to contaminated water (Khan, 2006; Siddiqui and Khan, 2012). Furthermore, the corneal stroma can be infected due to corneal injury and eye surgery. When a contact lens is worn for an extended time, oxygen is blocked, weakening the basal layer, and causing excessive irritation of the cornea, thereby creating a favorable environment for keratitis (Auran et al., 1987). Also, inadvertent care of contact lenses can cause amoeba contamination, wherein amoebas can infect eye through the contact lens or the lens solution in a contaminated storage case (Lindquist et al., 1988a; Kilvington and Larkin, 1990; Walochnik et al., 2000; Hiti et al., 2002; Hiti et al., 2005; Fears et al., 2018).

In AK, the amoeba infiltrates corneal stroma and epithelial cells, with progressive infiltration of the inflammatory cells. The symptoms of infection are unilateral red eye, severe unbearable pain, foreign body sensation, blurred vision, ring-shaped stromal infiltrates and photophobia, which are often misdiagnosed initially as herpes simplex virus infection, or bacterial or fungal keratitis (Fig. 3). However, once the disease develops, it is not accessible to treatment, and eventually progresses to chronic. AK is a painful infection that threatens the sight with excruciating pain. Early diagnosis and laboratory tests for AK are important for early treatment and prevention of the irreversible corneal damage. If the infection is not treated urgently, it can lead to corneal ulcers, eventual blindness and removal of the nucleus (Jones et al., 1975; Auran et al., 1987; Lindquist et al., 1988b; Bacon et al., 1993; Seal, 2003; Hiti et al., 2005; Khan,

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2006; Behera and Satpathy, 2016).

Diagnosis of AK employs a variety of methods in the laboratory. The use of in vivo confocal microscopy has emerged as a valuable non-invasive tool for the clinical diagnosis of highly sensitive cases of severe infectious keratitis. Confirmatory evidence of infection is to detect amoebas using laboratory-based assays. The most common method is the cultivation of Acanthamoeba from corneal tissue biopsy or contact lenses and cases. Immunofluorescence assays and multiplex real-time PCR methods can also be used (Winchester et al., 1995; Qvarnstrom et al., 2006; Goldschmidt et al., 2009; Vaddavalli et al., 2011).

An effective therapeutic agent for the treatment of AK has not been developed yet. Therefore, it is often treated with antibiotics and antifungal agents. A variety of drugs have been used to treat AK, including propamidine isethionate, dibromopropamidine isethionate, polyhexamethylene biguanide, neomycin, chlorhexidine, polymyxin B, paromomycin, clotrimazole, miconazole, itraconazole and ketoconazole. Brolene, a commercial eye drop containing dibromopropamidine isethionate and propamidine isethionate, is effective in the treatment of Acanthamoeba infection, but may be accompanied by drug toxicity and resistance. A variety of diamidine compounds, synthetic maganins in combination with silver nitrate, triazole and imidazole compounds, phenothiazine, azithromycin and povidone-iodine have been screened in vitro for their efficacy against Acanthamoeba spp. Another drug, miltefosine (hexadecylphosphocholine), has been also shown to have potential activity against the amoeba. Clinically, the infection is treated with polyhexamethylene biguanide (PHMB)

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or chlorhexidine gluconate, with or without brolene. In some cases, chlorhexidine and penetrating keratoplasty are done together when the treatment fails. Currently, the drugs selected for AK are chlorhexidine gluconate, PHMB, and brolene, which have improved the prognosis of AK patients. However, unwanted effects, such as corneal opacity or scarring may occur after treatment (Seal, 2003; Schuster and Visvesvara, 2004b; Schuster et al., 2006; Niyyati et al., 2009).

The incidence of AK is increasing worldwide, and since the first reported cases of AK in 1973, over 5,000 cases of amoebic keratitis have been reported in the United States in 2006 (Sotelo-Avila et al., 1974; Radford et al., 1995; Heffler et al., 1996; Visvesvara, 2010). In Korea, approximately 42 cases of amoebic keratitis were reported by 2008. In Austria, 89 % of AK patients were reported as contact lens users. Of these, 19 % were undergoing corneal transplantation (Walochnik et al., 2015).

The most common cause of AK infection in contact lens users is closely related to disinfection and storage of contact lenses. Most contact lens users use a convenient, commercial lens solution that primarily cleans, rinses, disinfects, and preserves the lens. However, commercial lens solutions are not completely insecticidal for Acanthamoeba. The Acanthamoeba cyst is known to be resistant to disinfection and washing solutions, as it consists of thick cell walls. Therefore, contact lens users should pay attention not only to tap water but also lens preservation containers and solutions. As prevention is most important, the contact lens should not be worn during swimming, underwater sports activities, or relaxing in the Jacuzzi or hot tub (Zanetti et al., 1995; Hiti et al., 2002; Visvesvara et al., 2007).

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Contact lens use is steadily increasing AK incidence every year, raising the social health issues. Many patients also require corneal transplantation after treatment and may be re-infected with resting cysts (Hurt et al., 2003). Eighty percent of patients with keratitis are at constant risk as contact lens users, and the number of patients worldwide has increased over the past twenty years due to the increased contact lens use (Centers for Disease, 1986; Stopak et al., 1991; Srinivasan et al., 1993; Ren and Wu, 2010; Cope et al., 2016; Padzik et al., 2016). Number of patients with caustic amoebic keratitis is expected to increase in the future with the generalization and convenience of contact lenses, and amoebic keratitis has been reported worldwide, including in Korea.

Currently, many studies are under progress to treat AK. However, the results of pathologic mechanisms and lesion studies after inducing AK in in vivo animal models are insufficient. The development and establishment of in vivo animal models is essential to the development of therapeutic agents for the in vivo testing of AK's molecular biology, pathology and immunology. However, the research by foreign and domestic researchers is not adequate (Van Klink et al., 1997; Polat et al., 2007).

In this study, We established the mouse models of keratitis using trophozoites and cysts of A. castellanii to study the pathogenesis of AK in the future. We determined the minimum number of amoebas needed to induce keratitis in mice. and the efficacy of commercial lens solutions on removal of A. castellanii.

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II. MATERIALS AND METHODS

A. Cultivation of Acanthamoeba castellanii

A. castellanii trophozoites were axenically cultured in 75-cm2 culture flask with PYG medium at 30°C (2% protease peptone, 0.2% yeast extract, 0.1 M glucose, 4 mM MgSO47H2O, 0.4 mM CaCl2, 3.4 mM sodium citrate 2H2O, 50 μM Fe(NH4)2(SO4)6H2O, 2.5 mM KH2PO4, 2.5 mM Na2HPO4, pH 6.5), in accordance with the previous reports (Visvesvara and Balamuth, 1975).

B. Encystation of A. castellanii trophozoites

The formation of A. castellanii cysts was induced by cultivation with encystment solution (95 mM NaCl, 5 mM KCl, 8 mM MgSO4, 0.4 mM CaCl2, 1 mM NaHCO3, 20 mM Tris-HCl, pH 9.0) in accordance with the previous reports (Moon et al., 2008; Sohn et al., 2017). Briefly, A. castellanii trophozoites were harvested, washed twice with PBS (pH 7.4) and centrifugated at 1,500 rpm for 3 min. After the final step, the trophozoites were added into 75-cm2 culture flask (concentration adjusted as 1 × 106 cells/ml), and treated with 10 ml of encystment solution for 72 h. To confirm the formation of cysts, morphological changes were observed with an inverted microscope (Olympus, Shinjuku, Tokyo, Japan).

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C. Experimental acanthamoebic keratitis development in mouse

(1) Inoculation of A. castellanii into mouse eye

In order to establish AK mouse model, A. castellanii trophozoites and cysts were inoculated into mouse eyes. The commercial contact lenses (Proclear 1 day lens, Cooper Vision Inc, USA) were cut into 2 mm pieces using a punch, and placed in a 24-well plate with 10 μl PBS (pH 7.4). The harvested trophozoites and cysts of A. castellanii were mixed in equally at 5 × 105 cells/ml concentration. Total 1 × 106 cells/ml were placed in 1 ml tube, and centrifugated at 1,500 rpm for 3 min. The pellet was applied on a 2 mm contact lens, and incubated at 30°C for 1 h. 8 weeks old female BALB/c mice (Orient, Seongnam, Korea) were anesthetized with ketamine and rompun solution (Kang et al., 2015). The eyeballs of mouse were scratched with a syringe needle and ophthalmic blade. The A. castellanii-cultivated lens was put into the mouse eyelid and sutured with 6/0 nylon (Woorimedical, Namyangju, Korea) for the complete contact of A. castellanii (Fig. 4). The sham operation was carried out by inoculating only a clear lens into mouse eye without A. castellanii. And then, following procedures were same to above practices.

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Fig. 4. Procedure for the experimental acanthamoebic keratitis development.

1. Mouse was anesthetized. 2. Eye balls of mouse were scratched. 3. A. castellanii trophozoites and cysts cultivated on the contact lens were inoculated. 4. Mouse eye-lid was sutured. Another eye was used for sham operation as control. (Figure was provided by Prof. Shin).

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(2) Observation of experimental mouse keratitis symptoms

To observe the keratitis development, the sutures of the mouse eyelid were sequentially removed at intervals from day 1 to 7. The eyeballs were observed with the naked eye for symptoms, such as white ring corneal infection, circular edema with increased central corneal thickness, and possible keratitis circle.

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D. Confirmation of experimental mouse keratitis

(1) Genomic DNA extraction from keratitis-induced mouse eyeball

To extract the ocular DNA of the keratitis-induced mouse, experiments were performed as follows. After the development of gross keratitis was observed, mouse eyeballs were removed under anesthesia for the periods of observation. Briefly, the eyeball tissue was chopped with a surgical scissor in 100 μl of PBS (pH 7.4) taken in 1 ml tube. Tissue lysis buffer and proteinase K were added in eyeball tissue-containing tubes, according to the instructions of DNA extraction kit (Qiagen, France). After lysis at 56°C, subsequent DNA extraction was performed.

(2) PCR with A. castellanii-specific primers

To observe whether acanthamoebic 18S-rDNA is amplified in the keratitis-induced mouse eyeball, PCR was performed with P-FLA primers, as described previously (Kang et al., 2015). The P-FLA primers (forward 5’-CGCGGTAATTCCAGCTCCAATAGC-3’ and reverse 5’-CAGGTTAAGGTCTCGTT CGTTAAC-3’) were prepared to amplify the 18S-rRNA gene specific for A. castellanii (Tsvetkova et al., 2004). Total 2 ng of genomic DNA was used as a template, and 10 μl of NoblezymeTM PCR Plus Premix 2x (Noble Bioscience Inc., Suwon, Korea) was

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added to the PCR tube. The PCR conditions with P-FLA primers were as follows; 95°C

for 5 min, 40 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 3.5 min and a final

extension of 72°C for 10 min. After completion of the PCR reaction, the PCR products

were electrophoresed on 1% agarose gel containing ethidium bromide, 0.005% at 100 V for 20 min, and the image was analyzed by Gel Doc (Bio-Rad, USA).

(3) 18S-rDNA sequencing and homology analysis

DNA sequencing was performed to confirm whether the amplified PCR product matched with Acanthamoeba 18S-rDNA. The results were compared to Genebank database with Acanthamoeba 18-rDNA.

E. Optimal numbers of A. castellanii for the development of mouse keratitis

To determine the optimal numbers for the development of AK in mice, A. castellanii trophozoites and cysts (mixed in equal numbers) were serially diluted to 5 × 105, 2.5 × 105, 1.25 × 105, 0.625 × 105, 0.3125 × 105 or 0.1 × 105 cells/ml. The subsequent procedures were same as mentioned for the experimental mouse keratitis development (No. D).

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F. Effect of commercial lens solutions on AK development in mouse

(1) Pre-treatment of commercial lens solutions on A. castellanii

To confirm the efficacy of the commercial lens solutions (called anonymously A and B), AK was induced with amoebas pre-treated with lens solution A or B. Briefly, total 0.5 × 105_{cells of A. castellanii (equal numbers of trophozoites mixed with cysts)} were placed in a 1 ml tube and centrifugated at 1,500 rpm for 3 min. The pellet was applied on a 2 mm contact lens in 24-well plate and incubated with commercial lens solutions A or B for 1, 6, 12 or 24 h.

(2) Effect of commercial lens solutions on AK mouse model

A. castellanii pre-treated with lens solution A or B were inoculated into mouse eye, as described previously (No. D). Also, the development of amoebic keratitis in mice was observed according to the previously described procedures (No. D).

G. Ethics statement

The present mouse experiments were reviewed and approved by the Animal Research Ethics committee of Ajou university school of medicine (AJ-IBC-17-09-10).

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III. RESULTS

A. Morphological observation of A. castellanii cyst formation

In order to induce the formation of cysts in A. castellanii, trophozoites were cultured for 24, 48, 72 h, and observed with an inverted microscope. It was observed that 24 h after the induction of cyst formation, half of the amoebas were transformed into a pre-cyst with single cell wall. Most of the amoebas were found in a round shaped cyst 48 h after induction. After 72 h induction of cyst formation, various forms of polygonal cysts and pre-cysts were observed as acanthamoeba cysts (Fig. 5).

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Fig. 5. Morphological changes of A. castellanii trophozoites into pre-cysts and cysts.

A. castellanii trophozoites were cultured in encystation medium for 24, 48 or 72 h. Arrows indicate polygonal cysts. Scale bar, 20 μm.

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B. The occurrence of acanthamoebic keratitis in mouse model

To induce acanthamoebic keratitis (AK), 2 mm lens cultivated with 1 × 106 A. castellanii (trophozoites and cysts mixed in equal numbers), was inserted into the mouse eye. The occurrence of keratitis symptoms was observed in mouse eyes between day 1 and 3. On the first day of AK induction, small, white, round edema was observed in the mouse eye, and typical round edema was diagnosed on the day 2 and 3. On the other hand, the sham operation did not show any symptoms of AK (Fig. 6).

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Fig. 6. Induction and observation of acanthamoebic keratitis in experimental mouse models. To induce keratitis, 1 × 106 of A. castellanii (trophozoites and cysts mixed in equal numbers) were cultivated on the lens and inoculated into the mouse eye. AK symptoms were observed from day 1 to 3. All experiments were repeated (Exp. I and Exp. II). (Sham: sham operation, Exp: experiment).

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C. A. castellanii DNA amplification from mouse eye tissue developed acanthamoebic keratitis

In order to identify the cause of AK development in mice, genomic DNA was extracted from eye tissues, and PCR with P-FLA primers was performed for the 18S-rRNA gene. DNA samples from the infected mouse eyeballs gave an amplicon of 1,080 bp size on day 1, 2 and 3, which was same as that of A. castellanii used as a positive control. On the other hand, the same amplicon was not obtained from the normal or sham-operated mouse DNA (Fig. 7).

PCR products amplified with P-FLA primers were subjected to DNA sequence analysis and gene homology search. On comparing the results with the 18S-rRNA of A. castellanii, the amplified products on day 1, 2 and 3 showed 97, 96 and 97 % homology, respectively (Fig. 8).

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Fig. 7. Amplification of A. castellanii 18S-rRNA from AK mouse eye tissue. When

AK symptoms appeared, genomic DNA was extracted from mouse tissues on day 1, day 2 and day 3. PCR with P-FLA primer was performed for the amplification of 18S-rRNA gene. A. cast, genomic DNA of A. castellanii trophozoites (1 × 106_{) as a positive control.} Normal, DNA extracted from normal mouse eye tissue as a negative control. Sham, DNA extracted from sham-operated mice tissues on day 1, 2 and 3 as a negative control. M, size marker.

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Fig. 8. The nucleotide sequence and homology analysis of 18S-rRNA gene obtained from keratitis-induced mouse eye. PCR products (bands figures on left) on day 1 (A),

2 (B), and 3 (C) post-inoculation was sequenced and confirmed by NCBI BLAST search (right box), and the homology was analyzed with BLAST search (below box).

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D. Optimal numbers of A. castellanii for keratitis development in mouse

To determine the optimal numbers for the development of AK in mice, serially diluted A. castellanii (trophozoites and cysts equally mixed) were inoculated into the mouse eye.

It was observed that with 5, 2.5, 1.25 and 0.625 × 105 number of A. castellanii, the keratitis symptoms were observed in all mice eyes between day 1 and 3. On the first day of AK induction, weak infection, characterized by cloudy eyeball, was observed in all mice eyes, along with a progressive corneal infection on day 2 and 3. On the other hand, the sham-operated mice did not show any apparent keratitis symptoms (Fig. 9).

In order to identify the cause of AK development in mice, genomic DNA extracted from mice eye tissues was amplified by PCR with P-FLA primers specific for 18S-rRNA gene. As a result, DNA samples of keratitis-induced mouse eyeballs gave 1,080 bp size of amplicon on day 1, 2 and 3, which was same as that of A. castellanii used as a positive control. On the other hand, same amplicon was not amplified from the normal or sham-operated mouse DNA (Fig. 9).

With 0.3125 or 0.1 × 105_{of A. castellanii inoculation, no apparent keratitis} induction in mice eyes was observed on day 1. Subsequently, white circular ulcer with increased central corneal infection of the mouse eye was observed on day 2 and 3. On the other hand, the sham-operated eye did not show any symptom (Fig. 10).

In the results of identification of 18s-rRNA gene for the cause of acanthamoebic keratitis development in mice, DNA samples of mouse eyeballs inoculated with 0.315

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and 0.1 × 105 ofA. castellanii did not show an amplification of 1,080 bp corresponding to 18S-rRNA gene on day 1 and 3. On the other hand, same amplicon was not amplified from the normal or sham-operated mouse DNA (Fig. 10).

PCR products amplified with P-FLA primers were subjected to sequence analysis and gene homology search. On comparing with the 18S-rRNA of A. castellanii, the amplified bands in all samples showed the 87–99 % homology, respectively (Data not shown).

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Fig. 9. Observation of Acanthamoebic keratitis development in mice inoculated with serially diluted A. castellanii (I). A. castellanii (equally mixed trophozoites and

cysts) were serially diluted to 5 × 105, 2.5 × 105, 1.25 × 105, and 0.625 × 105 and inoculated into mice eyes; and AK symptoms were observed from day 1 to 3 (Sham: sham operation). Regardless of the occurrence of keratitis symptoms, genomic DNA was extracted from all mice tissues on day 1, day 2, and day 3. Each band was PCR-amplified with P-FLA primers for the confirmation of AK development in mice. A. cast, genomic DNA of A. castellanii trophozoites (1 × 106) as a positive control; Normal, DNA extracted from normal mouse eye tissue as a negative control; sham, DNA extracted from sham-operated mouse tissues as a negative control. M, size marker.

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Fig. 10. Observation of acanthamoebic keratitis development in mice inoculated with serially diluted A. castellanii (II). A. castellanii (equally mixed trophozoites and

cysts) diluted to 0.3125 × 105_{, and 0.1 × 10}5_{were inoculated into mice eyes to induce} keratitis; and AK symptoms were observed from day 1 to day 3 (S: sham operation). Regardless of the occurrence of keratitis symptoms, genomic DNA was extracted from all mice tissues on day 1, day 2, and day 3. The genomic DNA of mice eye was subjected to PCR with P-FLA primers for the amplification of 18S-rRNA gene (right for figures). A. cast, genomic DNA of A. castellanii trophozoites (1 × 106), as a positive control; Normal, DNA extracted from normal mouse eye tissue as a negative control; Sham, DNA extracted from sham-operated mouse tissues as a negative control. M, size marker.

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E. Establishment of acanthamoebic keratitis mouse model

As a result of confirming the optimal concentration to cause acanthamoebic keratitis (AK) in mice, It was observed that the results of infection with 0.3125 × 105 and 0.1 × 105_{of A. castellanii were not constant in repeated experiments. The sufficient} number of amoebas that can cause mouse eye keratitis was 0.5 × 105 amoebas. Finally, 0.5 × 105_{amoebas on 2 mm lens were inoculated into mice eyes, and the keratitis} symptoms were observed from day 1 to 7 post-inoculation. white ring corneal infection, circular edema the mouse eye was observed on day 1, which progressed to corneal infection on day 2 to 7. On the other hand, the sham-operated mice did not show the keratitis symptoms (Fig. 11A).

To confirm the cause of AK development in mice, PCR products from DNA samples of mouse eyeballs on day 1 to 7 post-inoculation were obtained, giving an amplicon of 1,080 bp size, corresponding to 18S-rRNA gene, which was same as that of A. castellanii used as a positive control. On the other hand, the same PCR product from normal and sham-operated mouse DNA was not amplified (Fig. 11B).

PCR products amplified with P-FLA primers were subjected to sequence analysis and gene homology search. On comparing with the 18S-rRNA of A. castellanii, the amplified bands on day 1, 2, 3, 5 and 7 showed 96, 97, 97, 91, 94 and 93 % homology, respectively (Fig. 12).

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Fig. 11. Observation of acanthamoebic keratitis infection with 0.5 × 105_A.

castellanii, and 18S-rRNA amplification on mice eye. (A) Total 0.5 × 105 A. castellanii (trophozoites with cysts mixed equally) applied on the contact lens were inoculated into mice eye; AK symptoms were observed from day 1 to 7 (S: sham operation, E: experiment). (B) To confirm the causative agent of AK, genomic DNA extracted from the eye tissues on day 1, 2, 3, 4, 5, 6, and 7 was subjected to PCR with P-FLA primers for the amplification of 18S-rRNA gene. A. cast, genomic DNA of A. castellanii trophozoites (1 × 106) used as a positive control; Normal, DNA extracted from normal mouse eye tissue as a negative control; Sham, DNA extracted from the sham-operated eye tissues as a negative control. Exp I, II, III, experiments were practice three times. M, size marker (below bands figure).

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Fig. 12. The nucleotide sequence and homology analysis of 18S-rRNA gene obtained from keratitis-induced mouse eye. PCR products (bands figures on left) on

day 1 (A), 2 (B), 3 (C), 4 (D), 5 (E), 6 (F), and 7 (G) post-inoculation were sequenced and confirmed by NCBI BLAST search (right box), and the homology was analyzed with BLAST search (below box).

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F. Effect of commercial contact lens solutions on the AK development in mice

(1) Morphology of A. castellanii pre-treatment with solution A or B

To investigate the effect of the commercial lens solutions, 0.5 × 105_A. castellanii were treated with either solution A or solution B, and cultured for 1, 6, 12, or 24 h. On the observation with an inverted microscope, round morphological shapes were observed from 1 to 6 h, some pre-cystic form appeared at 12 h, and cysts were observed at 24 h (Fig. 13A, B).

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Fig. 13. Morphological change of A. castellanii (trophozoites and cysts) treated with commercial lens solution A or B. A. castellanii (trophozoite and cyst, mixed equally)

were cultured in commercial lens solution A (A), or B (B) for 1, 6, 12 and 24 h. Arrows indicate pre-cyst and cyst. Scale bar, 20 μm.

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(2) Change of acanthamoebic keratitis development in mice

To investigate the effect of the commercial contact lens solution, 0.5 × 105 of A. castellanii (trophozoites mixed equally with cysts) pre-treated with solution A or B were inoculated into mouse eye, and keratitis symptoms were observed for 1, 2, 3, 5, or 7 days (Fig. 14).

As a result of the pre-treatment for 1 or 6 h, solution A and B did not show any change in mice eyeball, in comparison with the established AK mouse model (Result E in this paper). Very weak keratitis symptoms were observed on day 1, and a progressive corneal infection on day 2 to 5. On the 7th day, it was confirmed that the typical ulcer circle had worsened (Fig. 14, 15). Subsequently, it was confirmed by PCR with P-FLA primers with an amplicon of the same size (1080 bp) as that of A. castellanii. In addition, all PCR products were sequenced and identified as A. castellanii 18S-rDNA gene (data not shown). On the other hand, control mice (0.5 × 105 amoeba infected) showed the keratitis symptoms.

On pre-treatment of lens with solution A or B for 12 h, the mice did not show the keratitis symptoms on day 1, even though a weak corneal infection was present (Fig. 14, 15). A mild infection was observed on day 2 and 3, and the typical ulcer circle developed on day 5 and 7 (Fig. 14, 15). The results were also confirmed by PCR with P-FLA primers (Fig. 14, 15) and DNA sequencing (data not shown). On the other hand, control mice (0.5 × 105 amoeba infected) showed the keratitis symptoms (Fig. 14, 12h).

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symptoms were not observed on day 1 and 2. A mild infection was observed on day 3 and the circular edema with increased central corneal involvement developed on the day 5 and 7 (Fig. 14, 15), which was same as the previous established AK mouse model (Result E) (Fig. 14, 15). Subsequently, it was also confirmed by PCR with P-FLA primers (Fig. 14, 15) and DNA sequencing (data not shown). Otherwise, keratitis symptoms observed in control mice (0.5 × 105 amoeba infected) (Fig. 14, 15).

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Fig. 14. Observation of acanthamoebic keratitis upon pre-treatment with solution A. Solution A was used to treat 0.5 × 105 A. castellanii (trophozoite and cyst equally mixed) for 1, 6, 12 and 24 h. AK was induced in mouse eyes and observed for 1, 2, 3, 5 and 7 days (Sham: sham operation). Genomic DNA of mouse eye tissues on day 1, 2, 3, 4, 5 and 7 was subjected to PCR with P-FLA primers for the amplification of 18S-rRNA gene. A. cast, genomic DNA of A. castellanii trophozoites (1 × 106) as a positive control; Normal, DNA extracted from normal mouse eye tissue as a negative control; (+) control, DNA extracted from AK-mouse eye tissue (inoculated 0.5 × 105 amoeba) as a positive control. M, size marker (below band figures).

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Fig. 15. Observation of acanthamoebic keratitis upon pre-treatment with solution B. Solution B was used to treat 0.5 × 105 A. castellanii (trophozoite and cyst equally mixed) for 1, 6, 12 and 24 h. AK was induced in mouse eyes and observed for 1, 2, 3, 5 and 7 days (Sham: sham operation). Genomic DNA of mouse eye tissues on day 1, 2, 3, 5 and 7 was subjected to PCR with P-FLA primers for the amplification of 18S-rRNA gene. A. cast, genomic DNA of A. castellanii trophozoites (1 × 106) as a positive control; Normal, DNA extracted from normal mouse eye tissue as a negative control; (+) control, DNA extracted from AK-mouse eye tissue (inoculated 0.5 × 105 amoeba) as a positive control. M, size marker (below band figures).

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IV. DISCUSSION

Ubiquitous free-living A. castellanii and A. polyphaga cause acanthamoebic keratitis (AK) in animals and in humans, and 85–88% of patients with AK use contact lenses. Furthermore, the number of AK patients is increasing because of the increasing use of contact lenses worldwide (Cerva et al., 1973; Lyons and Kapur, 1977; Brown et al., 1982; Auran et al., 1987; Visvesvara and Stehr-Green, 1990; De Jonckheere, 1991; Paszko-Kolva et al., 1991; Abjani et al., 2016).

Contact lens users are mainly exposed to AK because Acanthamoeba species can attach themselves to the soft surface of contact lenses and the cornea, resulting in progression of infection. The amoebae attached to the surface of the cornea destroy epithelial cells, penetrate the corneal stroma, and cause infection deep inside the stroma (Hadas and Mazur, 1993; Illingworth et al., 1995; Cao et al., 1998; Cho et al., 2000; Marciano-Cabral and Cabral, 2003; Por et al., 2009). The symptoms of AK include photophobia, ring-like stromal infiltrates, ulceration, development of retinal lesions, epithelial defects, lid edema, and severe pain (Kilvington and Larkin, 1990; Lorenzo-Morales et al., 2015).

Since AK is mostly chronic, early diagnosis and treatment are important. Sometimes, AK is misdiagnosed as a fungal or herpes simplex infection, which only involves 10–23% of coinfection by Acanthamoeba (Bacon et al., 1993; Tu et al., 2008b; Szentmary et al., 2012; Bouheraoua et al., 2013). Currently, there is no effective

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treatment for AK; imidazole, voriconazole, polymyxin B, and polyhexamethylene biguanide as well as mixed drugs that contain antibiotics, antifungals, antiprotozoals, and antivirals are mainly prescribed (Lim et al., 2008; Dart et al., 2009; Lorenzo-Morales et al., 2013).

Furthermore, preventing AK infection is important, which can be done by keeping the contact lens case clean and not wearing lenses while swimming or showering. In addition, exposure to contaminated water and injury to the cornea should be avoided (Maycock and Jayaswal, 2016).

Although there has been some progress in research regarding the infection mechanism of AK in vitro, in vivo studies using animal models have rarely been conducted. In addition, in these previous studies, AK was induced by direct intrastromal injection of the amoebae into mouse cornea using a microneedle (Matthaei et al., 2012). However, this procedure is complicated because the intrastromal cornea needs to be injected precisely, for which ophthalmologists are required. Moreover, this method of inducing keratitis is artificial. Therefore, in this study, I attempted to construct an animal model of AK which can be used easily and quickly. In this model, AK can be induced in mice using contact lenses in a manner similar to that of natural infection.

Other previous studies used pigs, rabbits, and rats as animal models for AK (Alizadeh et al., 1995; Said et al., 2004; Awwad et al., 2007; Vural et al., 2007). In this study too, experiments were first performed using rats. However, these animals were difficult to handle, only a limited number of the animals were available, and the cornea

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was too thick for AK induction (data not shown). Therefore, the experiment was conducted with mice, which are widely used, cheap, and easy to handle and can be used in large numbers in experiments (Ren and Wu, 2010).

In this study, I constructed an AK mouse model by mixing trophozoite and cyst forms of A. castellanii because in natural AK infections, both trophozoite and cyst forms of A. castellanii are detected in contact lenses. The eyes of the mice were scratched with a syringe needle and ophthalmic blade during AK induction because it is difficult to infect the cornea without any damage. Previous studies have shown that AK incidence is affected by corneal damage before infection (van Klink et al., 1993).

After scratching the mouse eyeball, contact lenses with A. castellanii were placed on the eyes. Next, mouse eyelids were sutured. This is because it is difficult to naturally attach contact lenses to mouse eyeballs as they are rounder than human eyeballs, and it was necessary to ensure complete attachment of the lenses to the cornea. To detect the incidence of AK symptoms, the eyelid sutures were sequentially removed on the days of observation.

To determine the minimum number of Acanthamoeba necessary to induce AK in mice, equal numbers of trophozoites and cysts of A. castellanii were mixed and serially diluted from 1 × 106 to 0.1 × 105 to induce AK. Infection was induced with a minimum of 0.3 × 105 and 0.1 × 105 cells. However, it was not the same as the result of grossly observation and PCR results using the ocular DNAs that caused keratitis in repeated experiments. Therefore, 0.5 × 105 was determined to be the most stable number

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of A. castellanii for AK induction. After AK induction, symptoms were observed for up to two months and were confirmed to persist and worsen (data not shown). AK induction experiments were at least practiced over three times. Therefore, it was determined that 0.5 × 105 A. castellanii can be used to establish an AK mouse model.

The diagnosis of AK is mainly based on in vitro experiments, in which lenses and lens solution in lens case of patients with AK are cultured or corneal biopsies are performed (Stothard et al., 1998; Stothard et al., 1999; Khan, 2001; Schuster and Visvesvara, 2004a; Qvarnstrom et al., 2006). For accurate diagnosis, PCR can also be used to confirm the amplification of Acanthamoeba DNA (Stothard et al., 1998; Stothard et al., 1999; Khan, 2001; Schuster and Visvesvara, 2004a; Qvarnstrom et al., 2006; Tu et al., 2008a; Tu et al., 2008b). In this study, eye tissues and contact lenses of the mice induced with AK were cultured on PYG medium and non-nutrient (NN) plates. However, the presence of A. castellanii was not confirmed because of rapid fungal growth, even after treatment with antifungal reagents. In addition, the number of amoebae remaining in the tissues and contact lenses was too small to culture. Next, eyes of mice that developed keratitis were stained with hematoxylin-eosin (HE) to confirm the presence of amoebae between the cornea and underneath the cornea. However, A. castellanii was not observed on the mouse eye balls (Fig. 17). It is impossible to observe amoebae under these conditions because the cornea of AK mice is too thin, and it would be difficult to obtain a tissue specimen.

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Fig. 16. Histology of AK-induced mouse eye.

Corneal stroma and polymorphic inflammatory infiltrates consisting of many lymphocytes were observed on tissue slides with Hematoxylin-eosin staining (100 µm and 200 µm)

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To address these problems, DNA was extracted from the eye tissue of the AK mice, and PCR was performed with P-FLA primers that amplify the 18S rRNA of Acanthamoeba (Tsvetkova et al., 2004). PCR of DNA from the AK mouse tissue produced an amplicon of the same size (1080 bp) as that of Acanthamoeba 18S-rRNA, and sequence analysis of the obtained PCR products revealed that AK was indeed induced by A. castellanii. The bands below 750 bp were identified as the 18S rRNA of Mus musculus.

Contact lens users mostly use commercial multiple lens solutions. However, commercially available solutions are ineffective in preventing infections because of their poor amoebicidal effects (Radford et al., 2002; Kilvington et al., 2004; Joslin et al., 2006; Lorenzo-Morales et al., 2013). It is known that the effects of antibiotics, antifungal agents, and antiviral agents in commercial lens solutions may affect the trophozoites of the Acanthamoeba but have little effect on the cyst form (Zanetti et al., 1995; Hurt et al., 2001; Hiti et al., 2002). Therefore, to confirm the effectiveness of the AK mouse model established in the present study, the effects of commercial lens solutions A and B, which are widely used worldwide, on AK incidence were tested. After pretreatment with 0.5 × 105 A. castellanii (equal number of trophozoites and cysts) with commercial lens solutions A or B for 1, 6, 12, and 24 h, the shape of all pretreated amoebae changed to that of the pre-cyst and cyst types. The number of amoebae was counted over time to determine if the commercial lens solutions affected amoeba population, but no significant differences were observed between 1 to 24 h (data not

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shown). Then, the AK mouse model was used to test whether commercial lens solutions A or B could inhibit AK over time. AK mice treated with solutions A or B showed delayed development of AK between day 1 and day 3, but AK was induced in mice treated with solutions A or B by day 5 and day 7. These results indicated that the use of commercial lens solutions may be effective in the short term, but they cannot inhibit AK induction over a long period of use.

In this study, an AK mouse model was established. This mouse model will be an important platform for in vivo testing of AK, for in vitro molecular biology, pathology, and immunology studies, and for the development of new pharmacological drug agents for the treatment of AK.

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V. CONCLUSION

Acanthamoeba castellanii causes acanthamoebic keratitis (AK). To decipher the exact pathogenesis of AK and development of therapeutic drugs, in vivo model needs to be established. In this study, BALB/c mouse cornea was scratched using a syringe needle and ophthalmic surgical blade under anesthesia. A. castellanii (trophozoites mixed equally with cysts) were serially diluted from 1 × 106 to 0.3 × 105, placed on a 2-mm contact lens, and put into the mouse eye. After the eyelid was sutured, the occurrence of keratitis symptoms was observed from day 1 to 7 post-inoculation. In addition, to confirm the AK development, the genomic DNA was extracted from the infected mouse ocular tissue. Amplification of acanthamoebic 18S-rRNA gene by PCR using P-FLA primers was performed. In conclusion, the experimental mouse AK, characterized by typical hazy blurring and melting of mouse cornea, was developed from day 1 to 7 post-inoculation with 0.5 × 105 amoeba. PCR products from DNA samples of mouse eyeballs on day 1 to 7 were amplified to be same as 18S-rRNA gene of A. castellanii used a positive control. Subsequently, the amplified bands were identified as 18S-rDNA of A. castellanii, which showed 91–97 % homology, by BLAST search. To confirm the effect of the commercial lens solution, 0.5 × 105 amoebas (trophozoites mixed equally with cysts) were treated with solutions A or B for 1, 6, 12 or 24 h, and inoculated into mouse eye (previously established AK mouse model). Thereafter, AK development was observed for 7 days post-inoculation. AK

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occurrence by A. castellanii treated with lens solution A or B for 1 or 6 h was not grossly observed on day 1 and 2, but the infection progressed gradually with a typical circulatory edema and keratitis on day 3, 5 and 7. Upon pre-treatments for 12 or 24 h, the development of AK was not observed for 1, 2 or 3 days, but progressed to corneal infection at 5 and 7 days. Finally, commercial contact lens solutions did not show the protective effect against AK pathogenesis, although they delayed the occurrence of AK. In conclusion, it is suggested that the present AK mouse model is an important in vivo model for future studies.

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