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

47

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

48

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

49

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.

50

51

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)

52

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

53

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.

54