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Infection and Immunity, June 2004, p. 3245-3251, Vol. 72, No. 6
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.6.3245-3251.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Induction of Morphological and Electrophysiological Changes in Hamster Cornea after In Vitro Interaction with Trophozoites of Acanthamoeba spp.

Maritza Omaña-Molina,¹ Fernando Navarro-García,² Arturo González-Robles,¹ José de Jesús Serrano-Luna,² Rafael Campos-Rodríguez,³ Adolfo Martínez-Palomo,¹ Víctor Tsutsumi,¹ and Mineko Shibayama^1*

Departments of Experimental Pathology,¹ Cell Biology, Center for Research and Advanced Studies,² Department of Biochemistry, School of Medicine, National Polytechnic Institute, Mexico City, Mexico³

Received 3 September 2003/ Returned for modification 8 October 2003/ Accepted 17 February 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Acanthamoeba castellani and Acanthamoeba polyphaga are free-living amebae that cause keratitis and granulomatous encephalitis in humans. We have analyzed the early morphological and electrophysiological changes occurring during the in vitro interaction of cultured amebae with intact or physically damaged corneas obtained from hamsters. Both species of Acanthamoeba produced similar cytopathic changes, as seen by light microscopy and scanning electron microscopy. After adhesion to the epithelial surface, trophozoites formed clumps and migrated toward the cell borders, causing the separation of adjacent cells at 1 h of coculture. At later stages (2 to 4 h), some amebae were found under desquamating epithelial cells whereas others were seen associated with damaged cells or forming amebostome-like structures to ingest detached epithelial cells. Control corneas incubated in culture medium conditioned with amebae showed a cytoplasmic vacuolization and blurring of the epithelial-stromal junction. The early stages of corneal epithelial damage caused by amebae were also analyzed by measuring the transepithelial resistance changes in corneas mounted in Ussing chambers. Both species of Acanthamoeba caused a rapid decrease in electrical resistance. The present observations demonstrate that under in vitro conditions, Acanthamoeba trophozoites rapidly cause significant damage to the corneal epithelium. Furthermore, in our experimental model, previous physical damage to the corneas was not a prerequisite for the development of amebic corneal ulcerations.

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Free-living amebae belonging to the genus Acanthamoeba are protozoan organisms living in a wide variety of habitats and are considered to be the most abundant free-living protozoa in nature (6, 37, 39-43). These amebae have gained in medical importance since they are the causative agents of granulomatous amebic encephalitis (24, 26, 27) and amebic keratitis (22). The corneal disease is a progressive sight-threatening infection that is particularly prevalent among contact lens users (13, 44-46). Clinically, the disease is characterized by severe pain, formation of a paracentral ring infiltrate with granular stromal opacity, and recurrent epithelial breakdown (22, 47). However, the diagnosis of keratitis caused by Acanthamoeba spp. fails frequently due to difficulties in isolating amebae from corneal specimens (2). In addition, amebic keratitis may be misdiagnosed as keratitis caused by herpes simplex virus (14, 25, 36). During the ocular infection, amebae cause thinning and ulceration of the corneal epithelium, eventually leading to necrosis, edema, and a stromal inflammatory reaction (10, 18, 22, 47).

Experimental models of amebic keratitis have been produced in Chinese hamsters and pigs. In these models, ameba-laden contact lenses were placed on abraded corneas for up to 7 days. Although these animal models partially reproduced human amebic keratitis, they showed a self-limited course that did not reach the chronic stages of infection (12, 19, 38). Other models in rabbits and rats (3, 20) have included the injection of amebae directly into the corneal stroma, avoiding the initial and natural route of corneal infection that starts with the adhesion of trophozoites to the corneal epithelial surface.

In vitro studies have shown that adherence of Acanthamoeba trophozoites to the corneal epithelium is an important step in the establishment of infection (38). Amebic adhesion may be mediated by mannose recognition sites localized on the target cells (48), and the adhesion capacity of amebae cultured axenically varies depending on the animal species used (35). It has been suggested that amebae adhere to the corneal surface when a minor trauma to the corneal epithelium is experimentally produced (13, 30). However, none of these studies described stages earlier than 12 h of interaction between amebae and the corneal epithelium.

We report here the early events occurring during the interaction of Acanthamoeba trophozoites with isolated corneas obtained from hamsters. To analyze this interaction, we monitored the sequence of morphological and electrophysiological changes caused by trophozoites of A. castellanii and A. polyphaga. Axenically cultured amebae were found to invade the epithelium of corneas with an undamaged surface. Our observations suggest that the existence of previous trauma to the corneal surface is not a prerequisite for the initiation of amebic keratitis.

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Amebal cultures. Trophozoites of A. castellanii and A. polyphaga species isolated from humans with keratitis were kindly provided by Simon Kilvington (Public Health Laboratory, Bath, England). Initially, strains were grown on nonnutrient agar plates enriched with heat-killed Enterobacter aerogenes (7). A. mauritaniensis, a nonpathogenic strain isolated from water, was used as a control. Amebae were axenized in Chang's medium supplemented with 10% fetal bovine serum (Equitech-bio, Kerville, Tex.) by a modified technique (41). Trophozoites were harvested at the end of the logarithmic growth phase (72 h) by chilling at 4°C and concentrated by centrifugation for 5 min at 300 x g.

Interaction with hamster corneas. Adult male golden hamsters (Mesocricetus auratus) weighing 120 to 130 g were used. Experiments were based on protocol 002/02, approved by the Institutional Animal Care and Use Committee, in accordance with norm-062-Zoo-1999, based on the Guide for the Care and Use of Laboratory Animals. After anesthesia with sodium pentobarbital (Sedatphorte) at 4.72 mg/100 g of body weight, both corneas were removed. Each cornea contained a peripheral rim of scleral tissue, leaving the lens untouched.

For comparative purposes, superficial lesions were produced in another set of hamster corneas by slightly scratching three preestablished sites with a sterile needle prior to introduction of amebae.

Scanning electron microscopy. Corneas were placed in 96-well styrene plates and infected with 10⁶ trophozoites in 0.2 ml of culture medium for 1 or 2 h. Control corneas were treated in a similar manner, except that only culture medium was added. After coincubation, samples were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, dehydrated with increasing concentrations of ethanol, critical-point dried with liquid CO₂ using a Samdri 780 apparatus (Tousimis Research Corp.), and coated with a thin layer (30 nm) of gold in an ion-sputtering device (Jeol-JFC I 100). Specimens were examined with a Zeiss DSM-982 Gemini scanning electron microscope.

Light microscopy. Corneas were cocultured with 10⁶ Acanthamoeba trophozoites in 0.2 ml of culture medium for 1, 2, 4, 8, and 12 h. At the end of each period, samples were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer and post-fixed with 1% osmium tetroxide for 1 h. After being dehydrated with ethanol, samples were embedded in epoxy resin. Semithin sections stained with toluidine blue were examined with a light microscope (Optiphot, Nikon).

Electrophysiological measurements. Corneas removed from anesthetized hamsters were placed in ice-cold Ringer's solution (physiological mammalian cell solution) gassed with a mixture of 95% O₂ and 5% CO₂ as reported previously (31-34). The corneal tissue was separated from the lens and mounted on a disk with a circular opening (3 mm) placed between two adjacent hemi-chambers (Ussing chamber). Each hemi-chamber was filled with 2.5 ml of Ringer's solution kept at 37°C with constant O₂-CO₂ bubbling. The transmembrane potential difference (PD) and the short-circuit current (I_sc) were recorded at various intervals by means of a voltage clamp apparatus. The electrical resistance (R) values were obtained from PD and I_sc data by using Ohm's law equation.

Four experimental groups were established. Group 1 included corneas cocultured with 10⁶ Acanthamoeba trophozoites for 1, 2, 4, 8, or 12 h before being mounted in Ussing chambers. Corneas in group 2 were incubated with ameba-conditioned medium for 1, 2, 4, 8, or 12 h before being subjected to electrophysiological recordings. Corneas in group 3 were mounted in Ussing chambers and cocultured with 10⁶ Acanthamoeba trophozoites in 0.1 ml of medium added to the hemi-chamber facing the concave side of the cornea. Group 4 consisted of corneas cocultured with freeze-thawed amebic extracts (10⁶ trophozoites) for 2 h and then mounted in Ussing chambers. Corneas incubated with culture medium or with previously fixed amebae (2.5% glutaraldehyde) served as controls. Corneas placed in a 25 mM NaOH solution served as positive controls. At the end of each electrophysiological measurement, corneas were removed from the Ussing chambers and fixed as mentioned above. Corneas were cut into 1-mm-thick fragments, postfixed in 1% osmium tetroxide, and embedded in epoxy resin. Semithin sections stained with toluidine blue were observed under a light microscope.

Statistical analysis. The electrophysiological data were presented as normalized values of electrical resistance, R/R₀ ratio (i.e., time value/initial value); five determinations were carried out. Differences in treatments were evaluated by the interaction of slopes compared with those of the controls.

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Scanning electron microscopy. Scanning electron microscopy studies revealed differences between A. castellanii and A. polyphaga trophozoites cultured under axenic conditions. A. castellanii had a flattened appearance with short acanthapodia, whereas A. polyphaga had a wide body with abundant, large acanthapodia (Fig. 1).

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FIG. 1. Scanning electron micrographs of Acanthamoeba trophozoites used in the present study. (A) A. castellanii has a flat morphology and multiple short acanthapodia. Bar, 10 µm. (B) A. polyphaga has a thicker body covered by abundant long acanthapodia. Bar, 5 µm.

Corneas incubated with culture medium alone showed no evidence of epithelial damage, pitting with wounding, or excessive cell exfoliation. Observation of intact hamster corneas incubated with Acanthamoeba trophozoites for 1 h showed that the trophozoites have established close contact with corneal surfaces. Flat and laterally extended amebic acanthapodia were seen adhering to the epithelial surface (Fig. 2A). At the peripheral borders of the epithelial cells, where clumps of amebae were found, cells became separated at an early stage of epithelial tissue invasion (Fig. 2B). Amebae were observed beneath the superficial cell layers (Fig. 2C), often protruding under the corneal surface (Fig. 2D and E). After 2 h of coculture, irregular areas devoid of epithelial cells were seen (Fig. 2F). Amebostome-like structures, similar to those found on Naegleria amebae for ingestion of epithelial cells, were also found on Acanthamoeba trophozoites (Fig. 3). In samples of amebae interacting with intentionally injured hamster corneas, many trophozoites adhered to the epithelial surface. Also, amebae penetrated areas of the epithelium that had not been damaged previously.

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FIG. 2. Scanning electron microscopy of Acanthamoeba trophozoites interacting with hamster cornea. A. polyphaga and A. castellanii caused very similar sequences of damage to the epithelial cells of hamster cornea. (A) At 1 h of coincubation, acanthapodia of an Acanthamoeba trophozoite (t) are in contact with the corneal epithelial cells (ec). Bar, 5 µm. (B) In another area of the same specimen, a trophozoite (t) is localized between epithelial cells (ec) and partially separates and penetrates the corneal epithelium. Bar, 10 µm. (C) At 1 h of incubation in another specimen, several Acanthamoeba trophozoites (t) are observed in the process of separating and damaging the corneal epithelium. Amebae can be observed beneath the epithelial cells (ec). Bar, 10 µm. (D and E) Also at 1 h of interaction, several trophozoites (t) have invaded the epithelial cell (ec) layer, protruding or destroying the corneal surfaces. Bars, 10 µm. (F) At 2 h of interaction, a trophozoite (t) of Acanthamoeba is seen associated with an epithelial cell (ec) that appears separated from the remaining layer. Bar, 10 µm.

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FIG. 3. Scanning electron microscopy of Acanthamoeba spp. cocultured with hamster cornea for 2 h. The corneal surface shows several damaged cells. A trophozoite (t) forming an amebostome-like structure (arrow) is observed during the process of ingesting a loosened epithelial cell (ec). Bar, 10 µm.

Light microscopy. Examination of semithin sections of the squamous epithelium of corneas incubated in culture medium in the absence of amebae showed a normal morphology (Fig. 4A). Corneas cultured with Acanthamoeba spp. displayed a sequence of events that confirmed and extended the scanning electron microscopy observations. At 1 h, individual or clumped trophozoites adhered to the surface of the corneal epithelium (Fig. 4B). Many amebae showed typical contractile vacuoles. At this stage of coculture, most cellular components of the squamous epithelium showed little evidence of damage. After 2 h of coculture, many trophozoites were located between superficial epithelial cell layers (Fig. 4C and D). Damaged epithelial cells had large cytoplasmic vacuoles and were partially detached from adjacent cells. After 4 h of interaction, amebae migrated to the wing cells and damage extended to the middle layers of the corneal epithelium (Fig. 4E). After longer periods of coincubation (8 and 12 h), the basal cells of the epithelium were disorganized but no amebae were seen in the stroma. Although amebae were not observed penetrating the whole thickness of the corneal epithelium, stromal vacuolization with loss of continuity between the basal cells and the stroma was detected in some areas (Fig. 4F). These latter changes at the epithelium-stroma junction were also detected, but with less intensity, in corneas incubated with conditioned culture medium (data not shown).

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FIG. 4. Light microscopy of epoxy-embedded semithin sections of hamster corneas. (A) Control. Corneal tissue was maintained in culture medium without amebae. A normal epithelium formed by superficial cells (sc), wing cells (wc), and basal cells (bc) is associated with a dense stroma (s). Magnification, x400. (B) At 1 h of coculture of Acanthamoeba with corneas, various trophozoites (t) are observed adhering to epithelial cells. Amebae show typical vacuoles (arrows) and nuclei (arrowhead). Deeper layers of the epithelium are normal. Magnification, x1,000. (C) At 2 h of interaction, trophozoites (t) are seen adhering to or raising the superficial epithelial cells. A vacuole (arrow) and nucleus (arrowhead) are evident. Magnification, x1,000. (D) At 4 h of interaction, Acanthamoeba trophozoites (t) are localized inside the first layers of the epithelium. Cells at deeper layers show altered cell junctions. An ameba vacuole is present (arrow). Magnification, x400. (E) At 8 h, a trophozoite (t) is seen at the wing cell layer. The ameba shows its characteristic morphological features: contractile vacuoles (arrow), nucleus (arrowhead), and nucleoli. Magnification, x1,000. (F) At 12 h of interaction, a trophozoite (t) has penetrated and damaged the wing cell layer. In the lower part of the figure, the stroma (s) associated with basal cells shows vacuolization. An ameba nucleus (arrowhead) and vacuole (arrow) are present. Magnification, x1,000. All specimens were stained with toluidine blue.

Electrophysiology. A. mauritaniensis did not cause significant electrophysiological changes in the cornea. Corneas interacted with either A. castellanii or A. polyphaga showed similar electrophysiological modifications; therefore, only data obtained using A. castellanii are shown below. The method used for the Ussing chamber was originally designed for ion-absorbing epithelia, such as bladder, intestine (32-34), or kidney epithelia. For this reason, a standardization for the corneal epithelium electrophysiological measurements was carried out. The mean initial PD (PD₀ = 0.3 mV) was stable for 90 min and then gradually fell at 120 min. The mean initial I_sc (I_sc0 = 3.8 µA/cm²) was stable for about 90 min and then decreased to zero within about 150 min. The decay rate was proportional to that of PD. In contrast, the mean initial R value (R₀ = 78 cm²) was stable for at least 150 min. These data suggest that little damage to the corneal epithelium occurred during the surgical procedures, since their transepithelial resistance remained constant throughout the experiment (2 h).

As a positive control, 25 mM NaOH was used to damage epithelial cells. Corneas mounted in the Ussing chambers showed a pronounced decrease of transepithelial resistance after 30 min of incubation with NaOH (P < 0.001) and was zero after 50 min (Fig. 5A), whereas untreated controls maintained their initial electrical R throughout the experiment (90 min). When trophozoites were added to intact corneas in chambers, a gradual decrease in the electrical R was observed. Corneas incubated with 10⁶ trophozoites for 2 or 8 h and then mounted in Ussing chambers showed a gradual decrease of the corneal electrical R; this was more evident in corneas incubated with amebae for longer periods (Fig. 5A). The values for ameba-treated corneas were statistically significant (P < 0.001) from those for the untreated controls. Corneas incubated with 10⁶ glutaraldehyde-fixed amebae had no effect on the electrical R (data not shown). Interestingly, corneas incubated with conditioned culture medium for 2 or 8 h and then mounted in Ussing chambers showed a decrease in the electrical R, similar to that produced by living trophozoites (Fig. 5B). Moreover, lysed trophozoites added to intact corneas in chambers caused a rapid drop in R, similar to that caused by the addition of NaOH (Fig. 5B).

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FIG. 5. Electrophysiological effects produced by Acanthamoeba trophozoites or their products on hamster corneas. (A) Corneas treated with trophozoites at different times. Untreated corneas, exposed to culture medium lacking amebae, were used as a control. A. castellanii trophozoites (106) were cocultured with hamster corneas for 2 h or 8 h and then mounted in the Ussing chambers. Intact corneas were also mounted in Ussing chambers, and after 10 min, 106 trophozoites (in situ) or 25 mM NaOH were added to the epithelial side. (B) Corneas were incubated with freeze-thawed amebic lysate. Untreated corneas were used as a control. Hamster corneas were treated with conditioned medium from ameba cultures for 2 h or 8 h and then mounted in Ussing chambers. Intact corneas were also mounted in Ussing chambers, and after 10 min, amebic lysate from 106 trophozoites was added to the epithelial side. The results represent three different experiments, and in all cases P was <0.001 compared with the control. The standard deviation is shown as an error bar on each mean.

Top Abstract Introduction Materials and Methods Results Discussion References

 
There have been several attempts to explain the cytopathic effect of Acanthamoeba spp. on the cornea. Studies aimed at producing amebic keratitis in animal models have been performed by intentionally damaging about 25% of the corneal epithelium. In addition, in vitro studies using human corneas and in vivo studies of experimental animal models have analyzed the damage caused by pathogenic free-living amebae to the corneal epithelium from 4 days to 1 month (12, 23, 29, 31, 36). However, these reports have not dealt with the morphological and functional events that take place before 12 h after adhesion of amebae to the corneal surface.

Although historically Acanthamoeba keratitis has been associated with previous corneal trauma, the present study demonstrates that when Acanthamoeba trophozoites are cocultured with isolated hamster corneas, the amebae can invade and cause damage to the intact corneal epithelium, without the requirement for a previous corneal abrasion.

After adhesion to the epithelial surface, trophozoites formed clumps and migrated toward the cell borders, causing the separation of adjacent cells at 1 h of interaction. At later stages (2 to 4 h), some organisms were found under desquamating epithelial cells whereas other amebae caused damage by ingesting detached corneal cells. Control corneas incubated in conditioned culture medium showed a cytoplasmic vacuolization and blurring of the epithelial-stromal junction.

Previous studies reported by Moore et al. (31) in which trophozoites of A. castellanii were incubated for 12 h with isolated human corneas showed adhesion of trophozoites followed by epithelial damage mainly at the periphery of the cornea and close to the sclera. In our study using hamster corneas, damage was randomly distributed on the corneal surface without preferential sites of invasion. Moore et al. (31) observed that during the process of tissue invasion, trophozoites differed morphologically from amebae maintained in liquid culture. Khan (18), using a corneal epithelial cell culture from rabbits, evaluated the cytopathogenicity of several Acanthamoeba species and reported that pathogenic Acanthamoeba spp. had a larger number of acanthapodia than did nonpathogenic species. In our scanning electron microscopy observations, no morphological differences between trophozoites in culture media and those interacting with target cells were observed.

An interesting observation was that Acanthamoeba showed amebostome-like structures in contact with some target cells, suggesting that ingestion of cells plays a role in the pathogenesis of corneal ulcerations caused by free-living amebae. At present, it is not known whether amebae ingest only dead cells or are also capable of ingesting living epithelial cells. This phenomenon may be similar to that reported for N. fowleri organisms, which ingest target cells in a piecemeal manner, using amebostomes, in a process termed trogocytosis (4). In addition, Díaz et al. (8) have reported the presence of amebostome-like structures in Acanthamoeba lenticulata when amebae were cocultured with Vero cells.

The initial adhesion to the target cell could be mediated by specific receptors such as those described by other investigators (15, 16, 38, 48). Cao et al. (5) showed that mannose-mediated adhesion of amebae to the target cells also contributes to induction of the cytopathic effect produced by the parasite. This phenomenon may contribute to the subsequent early process of penetration and desquamation of corneal epithelium layers.

During the process of corneal invasion by Acanthamoeba, in addition to the mechanical action of the trophozoites, their enzymatic activities may play a significant role in the cytolytic mechanisms. Amebic proteolytic enzymes include serine proteases (11, 28, 29), contact-dependent metalloproteases (17), elastases (9), cysteine proteases (11), and cytotoxic proteinases, induced by mannose-mediated adhesion (21). The use of gelatin gels has shown the presence of several proteolytic enzymes secreted by the amebae in medium conditioned by cultured amebae (1).

After adapting the Ussing chamber for our purposes, we proved that amebae and their conditioned culture media induced a decrease in the electrical transepithelial resistance of the cornea, suggesting that the ameba alters the corneal epithelial barriers, facilitating the transport of ions and other molecules through the inner layer of corneal tissue. This process may contribute to the edema observed in corneal epithelium. Both species of Acanthamoeba caused a rapid decrease in electrical resistance, possibly as a result of the opening of tight junctions linking the corneal epithelial cells. Comparison of the observation of the changes involved in the early stages of invasion and damage to hamster corneas by the two species of amebae showed no differences between the cytopathic effects of A. castellanii and of A. polyphaga.

In conclusion, we think that the hamster cornea appears to be an excellent model to study the pathogenic mechanisms of Acanthamoeba spp. on the corneal epithelium. It remains to be elucidated whether soluble factors are participating actively in the pathogenesis of amebic keratitis. Currently, we are performing studies to analyze longer interaction times and the activity of proteases in this experimental model.

 
This work was supported in part by grant 85891 from CONACYT.

We express our gratitude to Francine Marciano-Cabral for her critical and helpful comments. We also thank Leticia Neri Bazán, Martha Dueñas-Mejía, and Angélica Silva-Olivares for their valuable technical assistance and Arístides Gómez for his help with the statistical data.

 
* Corresponding author. Mailing address: Department of Experimental Pathology, Center for Research and Advanced Studies, Av. IPN 2508, Mexico City 07360, Mexico. Phone: 52-55-50 61 38 00, ext. 3348. Fax: 52-55-57 47 71 07. E-mail: mineko{at}mail.cinvestav.mx.

Editor: W. A. Petri, Jr.

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Infection and Immunity, June 2004, p. 3245-3251, Vol. 72, No. 6
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.6.3245-3251.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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