RESULTS - Ethics statement - Establishment of an acanthamoebic keratitis mouse model

G. Ethics statement

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).

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.

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).

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).

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).

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.

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).

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 × 10⁵number 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

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).

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.

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 × 10⁵ 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.

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).

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).

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).

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).

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.

(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).

As a result of the pre-treatment of lenses with solution A or B for 24 h, keratitis

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).

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).

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;

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

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

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 × 10⁵to induce AK. Infection was induced with a minimum of 0.3 × 10⁵ and 0.1 × 10⁵cells. 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

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.

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)

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

문서에서 Establishment of an acanthamoebic keratitis mouse model (페이지 28-0)