|Year : 2000 | Volume
| Issue : 4 | Page : 291-4
Keratocyte loss in Acanihamoeba Keratitis: Phagocytosis, necrosis or apoptosis?
GK Vemuganti, S Sharma, S Athmanathan, P Garg
Ophthalmic Pathology Service, L.V. Prasad Eye Institute, L.V. Prasad Marg, Banjara Hills, Hyderabad-500 034, India
G K Vemuganti
Ophthalmic Pathology Service, L.V. Prasad Eye Institute, L.V. Prasad Marg, Banjara Hills, Hyderabad-500 034
Source of Support: None, Conflict of Interest: None
Purpose: Pathogenesis of Acanthamoeba keratitis involves breakdown of epithelial barrier, stromal invasion by Acanthamoeba, loss of keratocytes, inflammatory response and finally stromal necrosis. The loss of keratocytes, believed to be due to the phagocytic activity of the parasite, occurs disproportionate to and independent of the parasite load, thereby suggesting additional modes of cell loss. To test our hypothesis that the loss of keratocytes in Acanthamoeba keratitis is due to apoptosis, we did both histology and histochemistry on the corneal tissues.
Methods: Routine Haematoxylin and Eosin, Gomori's Methenamine Silver and Periodic acid Schiff stained sections of five corneal tissues from penetrating keratoplasty and eviscerated eyes were reviewed. TUNEL staining was done for morphological detection of apoptosis in three cases, using formalin-fixed, paraffin-processed tissues.
Results: Histological changes were epithelial ulceration, loss of keratocytes in all layers, inflammation in anterior two-thirds of the stroma with necrosis, and deeper quiet stroma. Acanthamoeba trophozoites were found in the anterior stroma while the cysts were more in the deeper stroma, with minimal or no inflammatory response. TUNEL staining was positive in keratocytic nuclei in all layers.
Conclusions: This study demonstrates that one of the modes of keratocyte loss in Acanthamoeba keratitis is by apoptosis, possibly in addition to the necrotic process and phagocytic activity of the parasite. The death of inflammatory cells also appears to be mediated by apoptosis.
Keywords: Acanthamoeba, isolation & purification, Acanthamoeba Keratitis, parasitology, pathology, surgery, Animals, Apoptosis, genetics, Corneal Stroma, parasitology, pathology, surgery,
|How to cite this article:|
Vemuganti G K, Sharma S, Athmanathan S, Garg P. Keratocyte loss in Acanihamoeba Keratitis: Phagocytosis, necrosis or apoptosis?. Indian J Ophthalmol 2000;48:291
|How to cite this URL:|
Vemuganti G K, Sharma S, Athmanathan S, Garg P. Keratocyte loss in Acanihamoeba Keratitis: Phagocytosis, necrosis or apoptosis?. Indian J Ophthalmol [serial online] 2000 [cited 2018 Jan 20];48:291. Available from: http://www.ijo.in/text.asp?2000/48/4/291/14841
Acanthamoeba keratitis is a well-recognized chronic progressive ulcerative condition of the eye caused by the ubiquitous free-living protozoan, Acanthamoeba. While there have been several studies made towards the diagnosis and treatment of this condition,[1-3] few are directed towards understanding the pathogenic mechanisms involved.[4-9] The sequence of events suggested in pathogenesis of Acanthamoeba keratitis are: parasite invasion of stroma through an epithelial defect; phagocytosis and depletion of keratocytes; secondary inflammatory cell infiltration of devitalised stroma; and stromal necrosis. The loss of keratocytes, without associated inflammation and necrosis in the deeper stroma, led us to consider the apoptotic cell death, similar to non-inflammatory conditions following epithelial debridement, [10,11] refractive surgeries, [12,13] and UV light irradiation. Apoptosis is defined as an involutional and controlled form of cell death used by an organism to rid itself of unwanted cells without the release of degradative enzymes that would damage the surrounding tissues and cells. We hypothesized that the loss of keratocytes in Acanthamoeba keratitis is caused by apoptosis and evaluated our hypothesis, performing a terminal deoxyribonucleotidyl transferase mediated nick end labelling (TUNEL) assay. This method involves morphological detection of apoptosis by labelling and identifying the 3' and 5' terminal of the DNA strands, characteristic of apoptotic cell death.
| Materials and Methods|| |
We examined five corneal tissues (3 corneal buttons from penetrating keratoplasty; 2 eviscerated specimens) of microbiologically and histopathologically proven cases of Acanthamoeba keratitis. Medical records and clinical photographs were reviewed. Three of the 5 patients received treatment with topical polyhexamethylene biguanide (PHMB) and chlorhexidine digluconate (3-13 days), while two did not receive any of these at the time of surgery. The sections from formalin-fixed, paraffin-embedded tissues were examined with Haematoxylin and Eosin (H&E), Periodic acid Schiff (PAS) and Gomori's Methenamine Silver stain (GMS). In addition, TUNEL staining was performed on three specimens using a commercially available kit (Oncogene Research Product, CA, USA.). In brief, the deparaffinized sections were rehydrated to water, permeabilized with Proteinase K (2mg/ml diluted 1:100 in 10mM Tris). Endogenous peroxidase activity was blocked with hydrogen peroxide (30% H2O2 diluted 1: 10 in methanol). The slides were incubated in a humidified chamber with terminal deoxynucleotidyl transferase (TdT) labelling mixture for 90 minutes at 37ø C. The reaction was terminated with a stop buffer solution and detected using the diaminobenzidine (DAB) reaction.
| Results|| |
The five corneal buttons evaluated showed epithelial ulceration, destruction of the Bowman's layer, inflammation in the stroma, necrosis, and trophozoites and cysts of Acanthamoeba within the corneal stroma. The degree and depth of inflammation, load and distribution of the parasite in corneal stroma in each case is given in [Table - 1].
Inflammatory cells in the stroma consisted of neutrophils with occasional eosinophil and nuclear debris. Two cases of eviscerated tissues with peripheral rim of cornea showed presence of lymphocytes and plasma cells at the limbus. The inflammation was restricted to the anterior two-thirds of corneal stroma in three cases, while it extended to the deep stroma in two. Vascular channels were noted in one case. There was loss of keratocytes in all layers of cornea.
The trophozoites (intact and degenerating) and occasional cysts were noted in the superficial and mid stroma, associated with inflammatory cellular response [Figure - 1], while the cysts (intact) were noted in the deeper, quiet stroma [Figure - 2].
| TUNEL Assay|| |
TUNEL staining was performed in three of the five cases. In all the three cases there was dense positivity in the nuclei of keratocytes in superficial stroma, accompanied by granular positivity in the background [Figure - 3]. The nuclei of keratocytes in the deeper location were also stained positive [Figure - 4].
| Discussion|| |
Acanthamoeba is an ubiquitous free-living protozoan with a low virulence. The risk factors for corneal infection with Acanthamoeba include contact lens wear, trauma and contamination with dirty water.[17-19] The infection has a chronic waxing and waning course, which responds well to timely medical therapy at the early stage of disease.
The histological changes observed in our cases were similar to the findings from other studies.[6-9] Kinota et al have described the histological features in 10 cases and noted degenerative changes in the parasite at the site of inflammation, but intact parasites in the areas devoid of inflammation and necrosis. Our study confirmed the lack of vascularization in Acanthamoeba keratitis, except in one case that had received partial treatment.
The parasite is noted in its two forms, active trophozoites and inactive cystic form. The trophozoites were found in the anterior two-thirds of the stroma, associated with inflammatory response and necrosis. The cystic forms were found in the deeper stroma with mild or absent inflammatory response, similar to other studies.[7-9] The cystic forms can easily be recognized on routine H & E sections unlike trophozoites, which resemble the macrophages and pose problems in identification, especially when associated with extensive inflammation. There was loss of nuclei in keratocytes of the deeper stroma without stromal loss and necrosis which did not correlate with the number of parasites in the stroma. The parasites probably invade the deeper stroma and undergo morphogenesis into cystic form, possibly as a defense mechanism. The replication and morphogenesis is known to be influenced by stage of the disease, favorable conditions and also by use of corticosteroids. These observed histological changes support the pathogenic mechanism proposed by Garner. He proposed epithelial breakdown induced by trauma or contact lens wear, followed by stromal invasion by parasite, keratocytic loss by phagocytosis, a secondary inflammation of the devitalised stroma and finally necrosis by collagenases secreted by the leucocytes and Acanthamoeba itself.
The loss of keratocytes can possibly occur by three mechanisms: phagocytosis (by parasite), necrosis and apoptosis. The loss of stromal keratocytes due to phagocytosis by Acanthamoeba has been documented in in vitro studies. The loss of keratocytes, in the deeper stroma, independent of inflammatory response and parasite load, however, made us postulate other mechanisms of cell loss, namely necrosis and apoptosis. Necrosis was mostly seen in superficial stroma along with inflammation, similar to the other studies. In the deeper stroma, however, there was minimal or no change in most instances. It was not similar to the necrotic cell death; rather it was suggestive of other modes of cell loss. We hypothesized apoptosis of stromal keratocytes in Acanthamoeba keratitis. The TUNEL staining was performed for morphological identification of apoptotic cell death. The positive staining of the remaining nuclei of stromal keratocytes in all layers confirmed apoptosis of keratocytes. In the superficial stroma with inflammation and necrosis, additional dense background granular positivity was noted. This is probably due to the apoptotic bodies from keratocytes as well as apoptosis of the inflammatory cells, as demonstrated in exudates. In rapidly advancing diseased state, as seen in two of our cases, the inflammation may extend to the deeper stroma, associated with necrosis. In such cases, the chronology of events and the preceding mode of cell death cannot be contemplated.
Apoptosis in cornea has been documented in non-infective conditions like wound healing following epithelial debridement, [10,11] refractive surgeries, [12,13] and ultraviolet irradiation. It has also been observed in experimental studies on rabbit cornea infected with Herpes Simplex Virus (HSV-1). In infectious process, the cell death is mostly by necrosis, triggered by the inflammatory cells and the agent. Though there is evidence of apoptosis and necrosis of inflammatory cells in exudates, the mechanism of cell death in the host tissue is not clear. Apoptosis has been defined as cell death characterized by the lack of inflammatory response in tissues. However, we believe that the two processes are not mutually exclusive. The cornea is different from other tissues in structure, function and immune response.
Our observations suggest that in addition to necrosis, apoptosis is one of the modes of keratocytic loss in Acanthamoeba keratitis. This finding probably has clinical implications, as the tissue response following apoptosis and necrosis is different. The aim of treating corneal infections is not only to cure the infection but to restore vision by minimizing the tissue damage. The mechanism of cell death may influence the tissue repair process and therefore affect scar formation. With the availability of agents that modulate apoptosis, and existing knowledge of epithelial-stromal interactions, therapeutic strategies can be developed to modulate such tissue damage.
| Acknowledgements|| |
We would like to thank Dr Narsing A. Rao, Director, Ophthalmic Pathology Lab, Doheny Eye Institute and Professor of Ophthalmology and Pathology, University of South California for providing the facilities and guidance for TUNEL staining in his Laboratory.
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[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4]
[Table - 1]
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