Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 
  • Users Online: 1678
  • Home
  • Print this page
  • Email this page


 
   Table of Contents      
ORIGINAL ARTICLE
Year : 2000  |  Volume : 48  |  Issue : 4  |  Page : 291-4

Keratocyte loss in Acanihamoeba Keratitis: Phagocytosis, necrosis or apoptosis?


Ophthalmic Pathology Service, L.V. Prasad Eye Institute, L.V. Prasad Marg, Banjara Hills, Hyderabad-500 034, India

Correspondence Address:
G K Vemuganti
Ophthalmic Pathology Service, L.V. Prasad Eye Institute, L.V. Prasad Marg, Banjara Hills, Hyderabad-500 034
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


PMID: 11340887

Rights and PermissionsRights and Permissions
  Abstract 

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 2017 Sep 21];48:291. Available from: http://www.ijo.in/text.asp?2000/48/4/291/14841



Click here to view


Click here to view
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.[9] 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.[14] 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.[15] 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.[16] 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 Top


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[16] 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 37 C. The reaction was terminated with a stop buffer solution and detected using the diaminobenzidine (DAB) reaction.


  Results Top


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 Top


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 Top


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.[20]

The histological changes observed in our cases were similar to the findings from other studies.[6-9] Kinota et al[8] 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.[21] These observed histological changes support the pathogenic mechanism proposed by Garner.[8] 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.[22] 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.[16] 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.[23] 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.[14] It has also been observed in experimental studies on rabbit cornea infected with Herpes Simplex Virus (HSV-1).[24] 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.[15] 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.[25] The mechanism of cell death may influence the tissue repair process and therefore affect scar formation. With the availability of agents that modulate apoptosis,[26] and existing knowledge of epithelial-stromal interactions,[27] therapeutic strategies can be developed to modulate such tissue damage.


  Acknowledgements Top


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.



 
  References Top

1.
Cohen EJ, Parlato CJ, Arentsen JJ, Genvert GI, Eagle RC, Wieland MR, et al. Medical and surgical treatment of Acanthamoeba keratitis. Am J OphtMmol 1987;103:615-25.  Back to cited text no. 1
    
2.
Aversa G, Sten GA, Driebe WT. Diagnosis and successful medical treatment of Acanthamoeba keratitis. Arch Ophthalmol 1995;113:1120-23.  Back to cited text no. 2
    
3.
Flicker LA, Kirkness C, Wright P. Prognosis for keratoplasty in Acanthamoeba keratitis. Ophthalmology 1993;100:105-10.  Back to cited text no. 3
    
4.
Osato MS, Pyron M, Eliondo M, Brown E, Wilhelmus K, Jones D. Adherence and penetration of human epithelium by Acanthamoeba. Invest Ophthalmol Vis Sci 1990;31(suppl):420.  Back to cited text no. 4
    
5.
Niederkorn JY, Ubelaker JE, McCulley JP, Stewart GL, Meyer DR, Mellon JA, et al. Susceptibility of corneas from various animal species to in vitro binding and invasion by Acanthamoeba castellani. Invest Ophthalmol Vis Sci 1992;33:104-12.  Back to cited text no. 5
    
6.
Kremer I, Cohen EJ, Eagle RC, Udell I, Laibson PR. Histopathologic evaluation of stromal inflammation in Acanthamoeba keratitis. CLAO 1994;20:45-48.  Back to cited text no. 6
    
7.
Blackman HJ, Rao NA, Lemp MA, Visvesvara GS. Acanthamoeba keratitis successfully treated with penetrating keratoplasty: Suggested immunogenic mechanism of action. Cornea 1984;3:125-30.  Back to cited text no. 7
    
8.
Kinota S, Wong KW, Biswas J, Rao NA. Changing patterns of infectious keratitis: overview of clinical and histopathological features of keratitis due to Acanthamoeba or atypical mycobacteria, and of infectious crystalline keratopathy. Indian J Ophthalmol 1993;41:3-14.  Back to cited text no. 8
    
9.
Garner A. Pathogenesis of Acanthamoeba keratitis: Hypothesis based on a histological analysis of 30 cases. Br J Ophthalmol 1993;77:366-70.  Back to cited text no. 9
    
10.
Dohlman CH, Gasset AR, Rose J. The effect of the absence of corneal epithelium or endothelium on stromal keratocytes. Invest Ophthalmol Vis Sci 1968;7:520-34.  Back to cited text no. 10
    
11.
Wilson SE, Kim WJ. Keratocyte apoptosis: Implication on corneal wound healing, tissue organization, and disease. Invest Ophthalmol Vis Sci 1998;39:220-26.  Back to cited text no. 11
    
12.
Helena ML, BaerVeldt, Kim WJ, Wilson SE. Keratocytic apoptosis after corneal surgery. Invest Ophthalmol Vis Sci 1998;39:276-83.  Back to cited text no. 12
    
13.
Kim WJ, Shah S, Wilson SE. Difference in keratocyte apoptosis following transepithelial and laser-scrape photorefractive keratectomy in rabbits. J Refract Surg 1998;14:526-33.  Back to cited text no. 13
    
14.
Podskochy A, Gan L, Fagerholm P. Apoptosis in UV-exposed rabbit corneas. Cornea 2000;19:99-103.  Back to cited text no. 14
    
15.
Kerr JFR, Wyllie AH, Currie AR. Apoptosis: Basic biological phenomenon with wide range implications in tissue kinetics. Br J Cancer 1972;26:239-57.  Back to cited text no. 15
    
16.
Gavrieli Y Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labelling of nuclear DNA fragmentation. J Cell Biol 1992;119:493-501.  Back to cited text no. 16
    
17.
Samples JR, Binder PS, Luibel FJ, Font RL, Visvesvara GS, Peter CR. Acanthamoeba keratitis possibly acquired from a hot tub. Arch Ophthalmol 1984;102:707-10.  Back to cited text no. 17
    
18.
Hirst LW. Green WR, Merz W, Kaufmann C, Visveswara GS, Jensen A, et al. Management of Acanthamoeba keratitis. Ophthalmology 1984;91:1105-11.  Back to cited text no. 18
    
19.
Sharma S, Srinivasan M, George C. Acanthamoeba keratitis in non-contact lens wearers. Arch Ophthalmol 1990;108:676-78.  Back to cited text no. 19
    
20.
Bacon AS, Dart JKG, Flicker LA, Matheson MM, Wright P. Acanthamoeba keratitis: the value of early diagnosis. Ophthalmology 1993;100:1238-43.  Back to cited text no. 20
    
21.
Osato M, Robinson N, Wilhelmus K, Jones D. Morphogenesis of Acanthamoeba castellanii: Titration of the Steroid effect. Invest Ophthalmol Vis Sci 1986;27(suppl):S37.  Back to cited text no. 21
    
22.
Larkin DFP, Berry M, Easty DI. In vitro pathogenicity of Acanthamoeba. Eye 1991;5:560-68.  Back to cited text no. 22
    
23.
Fabre T, Belloc F, Dupuy B, Schappacher M, Soun A, Bertrand-Barat J, Baquey C. Polymorphonuclear cell apoptosis in exudates generated by polymers. J Biomed Mater Res 1999;44:429-35.  Back to cited text no. 23
    
24.
Wilson SE, Peroza L, Beuerman R, Hill JM. Herpes Simplex virus type-1 infection of corneal epithelial cells induces apoptosis of the underlying keratocytes. Exp Eye Res 1997;64:775-779.  Back to cited text no. 24
    
25.
Wilson SE., He Y-G, Weng J, Li Q, McDowall AW, Vital M, Chwang EL. Epithelial injury induces keratocyte apoptosis: Hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization and wound healing. Exp Eye Res 1996;62:325-38.  Back to cited text no. 25
    
26.
Kuo IC, Seitz B, LaBree I., MeDonnell PJ. Can Zinc prevent apoptosis of anterior keratocytes after superficial keratectomy? Cornea 1997;16:550-55.  Back to cited text no. 26
    
27.
Wilson SE, Liu JJ, Mohan RR. Stromal-epithelial interactions in the cornea. Prog Retin Eye Res 1999 May: 18(3):293-309.  Back to cited text no. 27
    


    Figures

  [Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4]
 
 
    Tables

  [Table - 1]


This article has been cited by
1 Histomorphological and immunofluorescence evaluation of bimanual and coaxial phacoemulsification incisions in rabbits
Johar, S.R.K., Vasavada, A.R., Praveen, M.R., Pandita, D., Nihalani, B., Patel, U., Vemuganti, G.
Journal of Cataract and Refractive Surgery. 2008; 34(4): 670-676
[Pubmed]
2 Granulomatous inflammation in Acanthamoeba keratitis: An immunohistochemical study of five cases and review of literature
Vemuganti, G.K., Pasricha, G., Sharma, S., Garg, P.
Indian Journal of Medical Microbiology. 2005; 23(4): 231-238
[Pubmed]
3 Keratocyte loss in corneal infection through apoptosis: A histologic study of 59 cases
Vemuganti, G.K., Reddy, K., Iftekhar, G., Garg, P., Sharma, S.
BMC Ophthalmology. 2004; 4
[Pubmed]
4 A clinico-pathological case report of necrotizing ulcerating keratopathy due to topical anaesthetic abuse | [Nekrotisierende ulzerierende keratopathie nach lokalanästhetikamissbrauch. Ein klinisch-pathologischer fallbericht]
Ardjomand, N., Faschinger, C., Haller-Schober, E.-M., Scarpatetti, M., Faulborn, J.
Ophthalmologe. 2002; 99(11): 872-875
[Pubmed]



 

Top
 
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

  Materials and Me...
  In this article
Abstract
Results
TUNEL Assay
Discussion
Acknowledgements
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed3458    
    Printed114    
    Emailed4    
    PDF Downloaded0    
    Comments [Add]    
    Cited by others 4    

Recommend this journal