Acanthamoeba spp., clinically isolated and characterized as genotype T4, was provided by the Beijing Eye Institute. The parasites were grown anemically in 25 cm² canted-neck tissue culture flasks containing 5 mL of PYG medium (Peptone-Yeast-Glucose) at 30℃ until 95% was in trophozoite form after one week of culture. Approximately 1×10⁶ cells of Acanthamoeba were harvested from each flask and washed three times with phosphate-buffered saline (PBS) by centrifuging at 500 g for 7 minutes. Each precipitate was suspended in 1 mL of PBS to be used as the stimulating solution.

Sixty soft contact lenses (1 Day ACUVUE, Johnson & Johnson, Vision Care, Shanghai, China) were bought from optical shops. Thirty bigger lenses of suitable size for rats (inner diameter of 4 mm) and thirty smaller ones for mice (inner dismeter of 2 mm) were made from these contact lenses by a sterile corneal trephine (66vision Tech Co., Ltd, Suzhou, China) under an operating microscope (66vision Tech Co., Ltd, Suzhou, China). All procedures were undertaken in a bacteria - free environment. Then, all the contact lenses were washed 3 times and incubated overnight with multi-functional solution. Fifteen bigger lenses and fifteen smaller ones were washed with PBS and incubated with Acanthamoeba solution for 1 hour for the parasites to attach to the lenses as contaminated lenses, whereas the other lenses were washed with PBS to be used as controls.

Forty-five Wistar rats, weighing 280 g to 300 g, and 45 Kunming mice, weighing 28-32 g, regardless of their gender were obtained from the Animal Supply Centre of Shandong University. Animal care and treatment in this investigation conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Slit lamp examination prior to the experiments was used to exclude any injury to the cornea. The rats and mice were randomly divided into groups A, B, and C with 15 rats and 15 mice in each group. Five rats or mice in each group were used for histopathological investigations and the other ten were used for clinical evaluations. Anti-microbial chloromycetin eye drops (Lukang Co., Shandong, China) were applied frequently one day before the inoculation to prevent bacterial co-infection.

The animal models of AK were established in the following 3 ways. Each procedure was performed under an operation microscope.

Microscopic examination of 10% potassium hydroxide wet mounts of corneal scraping was performed to observe Acanthamoeba trophozoites. The tissues from corneal scraping were examined under a light microscope and incubated with PYG medium containing non-nutritive agar to confirm the models and to exclude co-infection with bacteria or fungi.

On days 1, 3, 7, 13 and 21 post infection, the animals were monitored by slit lamp examination. A grade of 0 to 4 was assigned to each, based on the following three criteria: area of opacity, density of opacity and surface regularity (Table 1). A normal untreated cornea was given a score of 0 in each category and thus had a total score of 0. The scores from all three categories were tallied at 1, 3, 6, and 10 days post infection for each eye to yield a possible total score, ranging from 0 to 12. A total score of 5 or less was categorized as mild infection, a total score of 6 to 9 was considered moderate, and a total score of more than 9 was severe. At the end of the experiments, the other corneas were collected and stored at -80℃ for future analysis.

On days 0, 1, 3, 7, 13, and 21 post infection, infected corneas of 5 animals in each group were harvested and paraffin sections were made for Hematoxylin-Eosin (HE) staining to observe pathological process of AK in these animal models under a microscope. At the end of the experiments, the other corneas were collected for future analysis.

In the present study, laboratory tests such as examination of 10% potassium hydroxide wet mount of corneal scraping tissues, HE staining of the corneal sections and incubation of corneal scrapings were used to confirm AK and to exclude co-infections. The tests were negative for bacteria and fungi, and there were no colonies of bacteria or fungi formed in the culture medium. Typical cysts were observed on 10% potassium hydroxide wet mounts of corneal scraping tissues and HE staining of corneal sections under a duplex inverted microscope (Figs. 1 and 2). Active trophozoites grew in PYG medium with corneal tissues after 10 days of incubation.

The clinical features of AK were recorded, scored and graded with the aid of a dissecting microscope and slit lamp examination at 0, 1, 3, 7, 13, and 21 days post infection (Table 2). As rats and mice models developed similar clinical courses, pictures of rat corneas only in group B are presented in Fig. 3. Four rats and 6 mice in group A, 7 rats and 8 mice in group B, and 10 rats and 10 mice in group C developed Acanthamoeba keratitis. In group A, no severe inflammation was observed in any of the rats or mice. The corneas of 6 rats and 4 mice recovered within just 24 hours after the treatment. In group B, slight corneal edema and then complete recovery were observed in 3 rats and 2 mice. The infected corneas developed the typical ring ulcer of Acanthamoeba keratitis. In group C, most animals developed severe inflammations with ring ulcers and obvious stroma abscesses. Two mice developed even severe endophthalmitis and one of them died 13 days after the treatment. From the 13th day to the 21st day after the treatment, angiogenesis appeared in all three groups and the infections in groups A and B were gradually alleviated with decreasing ulcers and repair of scarring, but the inflammation in group C was aggravated with increasing abscesses and the infection spreading to involve nearly the whole cornea. After one month, most of the inflammation was alleviated or healed.

The corneas of the animal models were harvested and treated for pathological analysis. Fig. 4 shows the pathological process of AK in rats of group B. The picture of "0d" is the corneal section of normal rats in which the epithelium and stroma are well-defined, the stroma fibers are arranged regularly, and there are no inflammatory cells infiltrated. In the corneal sections of the AK animal models, the normal structure was destroyed. On the 1st day post infection, a few inflammatory cells were infiltrated just beneath the corneal epithelia. On day 3 post infection, more inflammatory cells were infiltrated in the cornea. On the day 7, most of the epithelia and stroma were infiltrated by a large number of inflammatory cells and the structure of the epithelia was unorganized. Tissue necrosis could be observed and the whole structure of the cornea was destroyed. On the 13th day, the amount of inflammatory cells decreased and neovascularization could be observed. Twenty-one days post infection, the corneal stroma was full of irregular collagen fibers, demonstrating scar healing in the cornea.

To evaluate the effect of the three different treatment methods, the infection rate of each group was analyzed and the results are listed in Table 2.

We used the SPSS12.0 program to do the statistical analysis. The results suggested significant differences among the three groups (p < 0.01).