Experimental Induction of Acute Acanthamoeba castellanii Keratitis in Cats.

Purpose: To develop a feline model of acute Acanthamoeba keratitis using methods that replicate natural routes of infection transmission. Methods: Corneal Acanthamoeba castellanii inoculation was performed by three methods: topical inoculation with Acanthamoeba solution following corneal abrasion, placement of a contaminated contact lens for 7 days, and placement of a contaminated contact lens for 7 days following corneal abrasion. Sham inoculations with parasite-free medium and sterile contact lenses were also performed. Cats were monitored by ocular examination and in vivo corneal confocal microscopy for 21 days post-inoculation. Corneal samples were collected at intervals for microbiologic assessment, histopathology, and immunohistochemistry. Results: All cats in the corneal abrasion groups developed clinical keratitis. Clinical ocular disease was inconsistently detected in cats from the contaminated contact lens only group. Initial corneal lesions were characterized by multifocal epithelial leukocyte infiltrates. Ocular lesions progressed to corneal epithelial ulceration and diffuse stromal inflammation. After 14 days, corneal ulcerations resolved, and stromal inflammation consolidated into multifocal subepithelial and stromal infiltrates. Corneal amoebae were detected by culture, in vivo confocal microscopy, histopathology, and immunohistochemistry in cats with keratitis. Neutrophilic and lymphocytic keratoconjunctivitis with lymphoplasmacytic anterior uveitis were identified by histopathology. Coinfection with aerobic bacteria was detected in some, but not all, cats with keratitis. Ocular disease was not detected in the sham inoculation groups. Conclusions: Feline Acanthamoeba keratitis is experimentally transmissible by contaminated contact lenses and topical inoculation following corneal epithelial trauma. Translational Relevance: Experimentally induced acute Acanthamoeba keratitis in cats is clinically and histopathologically similar to its human counterpart.

Determining the efficacy of the bovine amniotic membrane homogenate during the healing process in rabbits' ex vivo corneas.

OBJECTIVE: To evaluate the efficacy of bovine amniotic membrane homogenate (BAMH) on wounded ex vivo rabbit corneas. PROCEDURE: Eighteen corneas obtained from normal rabbit eyes were wounded equally using a 6 mm trephine and cultured into an air-liquid interface model. Corneas were treated with phosphate-buffered saline (PBS) (n = 6, control group), 0.2% ethylenediaminetetraacetic acid (EDTA; n = 6), or BAMH (n = 6). All treatments were applied topically 6 times/day. Each cornea was macrophotographed daily with and without fluorescein stain to assess epithelialization and haziness. After 7 days, corneal transparency was evaluated, and the tissues prepared for histologic analysis of viability, and total and epithelial thickness. RESULTS: The mean epithelialization time was 6.2 ± 0.82 days for the control group, 6.2 ± 0.75 days for the EDTA-treated group, and 5.1 ± 0.40 days for the BAMH-treated group, demonstrating a significant difference between the BAMH and the other groups. The corneas that received EDTA had better transparency compared with the other groups. Histologically, all corneas had adequate morphology and architecture after healing. Analysis of corneal and epithelial thickness revealed no significant difference among groups. CONCLUSION: Bovine amniotic membrane homogenate is an effective and promising treatment for stromal and epithelial ulcers.

Determining MMP-2 and MMP-9 reductive activities of bovine and equine amniotic membranes homogenates using fluorescence resonance energy transfer.

INTRODUCTION: Matrix metalloproteinases (MMPs)-2 and -9 are present in corneal ulcers, and an imbalance between MMPs and tissue inhibitors of metalloproteinases (TIMPs) leads to further corneal degradation. Amniotic membrane homogenate (AMH) has proteolytic properties beneficial for corneal healing, but it is unknown whether AMH possesses TIMPs or effectively inhibits MMP-2 and MMP-9 activity. OBJECTIVE: To determine if bovine and equine AMH reduce in vitro MMP-2 and MMP-9 activities associated with the presence of TIMPs. PROCEDURES: Undiluted and diluted twofold series (0-fold to 16-fold dilutions) of equine amniotic membrane homogenates (EAMH, n = 8) and bovine amniotic membrane homogenates (BAMH, n = 8) were subjected to fluorescence resonance energy transfer, and the fluorescence emitted was recorded over time. Average fluorescence was calculated versus recombinant concentration. Enzyme-linked immunosorbent assays for TIMPs 1-4 were applied to quantify TIMPs in the samples. RESULTS: AMH from both species were able to inhibit MMP-2 and MMP-9 activities in vitro, and the inhibition efficacy decreased gradually with dilution. BAMH was significantly more effective than EAMH at inhibiting MMP-2 and MMP-9 in vitro. TIMPs -2 and -3 were present in EAMH and BAMH. TIMP-1 was detected only in BAMH, and TIMP-4 was not detected in any samples. CONCLUSION: Both EAMH and BAMH directly inhibited MMP-2 and MMP-9 in vitro without dilution, and BAMH showed better inhibition of MMP-2 and MMP-9 before and after dilution compared to EAMH.

Proteome Composition of Bovine Amniotic Membrane and Its Potential Role in Corneal Healing.

Purpose: To investigate the protein profile of bovine amniotic membranes (bAM) and to determine putative associations between protein composition in bAM and known corneal healing pathways. Methods: The bAM were acquired from normal full-term births (n = 10), processed, and stored at -80°C for two days. Subsequently, the frozen membranes were thawed at room temperature and prepared for proteomic exploration using high-resolution liquid chromatography-mass spectrometry, followed by bioinformatics analysis. Recently identified corneal healing pathways were contrasted with protein profiles and pathways present in bAM. Results: The analyses identified 2105 proteins, with an interactive network of 1271 nodes (proteins) and 8757 edges (interactions). The proteins with higher betweenness centrality measurements include microfibril-associated protein 4, HSD3B1, CAPNS1, ATP1B3, CAV1, ANXA2, YARS, and GAPDH. The top four pathways in Kyoto Encyclopedia of Genes and Genomes were ribosome, metabolic pathway, spliceosome, and oxidative phosphorylation. The bAM and cornea shared abundant proteins, genome ontology, and signaling pathways. Conclusions: The high-throughput proteomic profile of the bAM demonstrated that numerous proteins present in the cornea are also present in this fetal membrane. Our findings collectively demonstrate the similarity between bAM and the cornea's protein composition, supporting our hypothesis that bAM can be used to treat corneal diseases.