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   Table of Contents      
ORIGINAL ARTICLE
Year : 2003  |  Volume : 51  |  Issue : 3  |  Page : 225-230

In-Vivo Slit Scanning Confocal Microscopy of Normal Corneas in Indian Eyes


Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All Institute of Medical Sciences, Ansari Nagar, New Delhi, India

Correspondence Address:
M Vanathi
Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All Institute of Medical Sciences, Ansari Nagar, New Delhi
India
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Source of Support: None, Conflict of Interest: None


PMID: 14601847

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  Abstract 

Objective: To study the cellular populations of healthy corneas of Indian eyes using confocal microscopy and to evaluate the correlation with age, gender and laterality. Methods: The central corneas of 100 eyes of 50 healthy subjects were examined using an i n-vivo slit scanning confocal microscope (Confoscan 2). Images were analysed for cell densities of the epithelium, stroma and endothelium. Results: Good quality images enabling analysis of all cell layer populations were obtained in
74 eyes of 43 healthy subjects (22 males and 21 females) with a mean age of 31.89 ± 13.47 (range 19-71 years). The basal epithelial cell density was 3601.38 ± 408.19 cells/mm2 (range 3017.3 -4231.1cells/mm2). The mean keratocyte nuclei density in the anterior stroma was 1005.02 ± 396.86 cells/mm2 (range 571.6 - 1249.6 cells/mm2) and in the posterior stroma was 654.32 ± 147.09 cells/mm2 (range 402.6 - 1049.1 cells/mm2). Posterior keratocyte nuclei density was 30.76% less than the anterior stromal keratocyte nuclei density. The difference in keratocyte nuclei density was statistically significant (P=0.001). The mean endothelial cell density was 2818.1 ± 361.03 cells/mm2 (range 2118.9 - 4434 cells/mm2) and the mean endothelial cell area was found to be 385.44 ± 42.66 mm2 (range 268.9 - 489.2 mm2). Hexagonal cells formed 22.5 - 69.4% of the endothelial cell populations (mean 42.04 ± 11.81%). Mean coefficient of cell size variation was 32.29 ± 3.06 (range 27.2 - 39.2). No statistically significant differences were found in cell densities of any corneal layer either between female and male patients or between right and left eyes. Basal epithelial cell density, anterior stromal keratocyte nuclei and posterior stromal keratocyte nuclei density were unaffected by age (r= 0.12, 0.07, - 0.12 respectively) (P= 0.001). There was a statistically significant negative correlation between mean endothelial cell density and increase in age (r= - 0.42, P=0.001). Coefficient of cell size variation and age were positively correlated (r=0.73, P=0.001).
Conclusion: In-vivo slit scanning confocal microscopy is useful for the study of corneal cell populations. Our study provides normative data of these cell populations.

Keywords: Confocal microscopy, Indian eyes, corneal cell populations


How to cite this article:
Vanathi M, Tandon R, Sharma N, Titiyal JS, Pandey RM, Vajpayee RB. In-Vivo Slit Scanning Confocal Microscopy of Normal Corneas in Indian Eyes. Indian J Ophthalmol 2003;51:225-30

How to cite this URL:
Vanathi M, Tandon R, Sharma N, Titiyal JS, Pandey RM, Vajpayee RB. In-Vivo Slit Scanning Confocal Microscopy of Normal Corneas in Indian Eyes. Indian J Ophthalmol [serial online] 2003 [cited 2019 Nov 12];51:225-30. Available from: http://www.ijo.in/text.asp?2003/51/3/225/14678



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New bio-imaging techniques have enabled non-invasive analysis of corneal structure and function. Minsky[1] described the first confocal microscope in 1957. Since then, several improvements have occurred and the most modern confocal microscopes use a light source focused onto a small volume within the specimen tissue and a confocal detector to collect the resulting signal. This produces an image with enhanced lateral and axial resolution. This new imaging paradigm and its application in-vivo provides important insights into the structure and function of the cornea. Confocal microscopy finds useful application in observation of the anatomy of the anterior parts of the eye, the investigations of these structures after local administration of drugs and in the diagnosis of ocular diseases.[2] The tandem scanning confocal microscope was first used to examine a human eye in-vitro by Lemp et al [3] in 1985 and in-vivo by Cavanagh et al [4] in 1990. Bohnke and Masters[5] have detailed the optical techniques for ocular biomicroscopy and the theoretical foundations of confocal microscopy. Several studies[6],[7],[8],[9],[10],[11],[12],[13],[14],[15],[16],[17] on various aspects of the normal human cornea, using different types of in-vivo confocal microscopes are available.

No study on the corneal cellular populations in Indian eyes has been reported so far. We have documented the cellular counts in normal healthy human corneas of different age groups using in-vivo scanning slit confocal microscopy.


  Materials and Methods Top


The population for this study consisted of 50 normal human subjects (24 males and 26 females). Those with a history of ocular or systemic disease, contact lens use, pregnant or lactating women, previous eye trauma or ocular surgery were excluded. The patients were positioned at the examination table after instillation of topical anaesthetic (0.5% proparacaine) in the study eye. Confocal microscopy was performed on the central cornea of each eye in the study group with a scanning slit confocal microscope (Confoscan 2, Nidek Technologies Srl, Vigouza, Italy). A front lens (40/0.75 immersion lens) with a working distance of 1.98 mm was used in conjunction with a gel (Viscotirs® gel, CIBA Vision Ophthalmics) which provided an almost optically homogenous coupling between the front lens of the objective and the cornea (distant immersion principle).

The x-y position of the image and the section depth were adjusted by moving the microscope manually, on observing the position of the objective lens in relation to the cornea, and the relative images displayed on the video monitor. A digital video recorder allowed automatic recording of the observed images. The images were viewed on a 21-inch digital video monitor. The corneal cell layers were identified and cell counts done by a single observer. The images of the epithelial cells, anterior and posterior stroma and endothelium were identified. To ensure replicability of the counts, random counts were performed 2 weeks later on the saved images in 10 eyes [Table - 1].

The densities of the epithelial cells, anterior and posterior stromal keratocytes and endothelial cells were analysed with the in-built NAVIS Software 3.1.0. Statistical analysis was performed using STATA 7.0 statistical software. Mean values of corneal cell population counts in two groups were compared by the students' t-test. The Pearson correlation coefficient was calculated to measure correlation between two quantitative variables. Statistical significance was set at P< 0.05.


  Results Top


Analysis of images of all the corneal layers was possible in 74 eyes of 43 patients (22 males and 21 females) with mean age of 31.9 ± 13.47 years (range 19-71 years).

The basal epithelial cells were seen as a distinct mosaic, with light cell boundaries. The confocal image of the Bowman's layer appeared as a homogenous acellular layer and nerve fibres of the subepithelial nerve plexus. Keratocyte count in the stroma was done by identifying the bright reflections from the keratocyte nuclei against a dark background. No cellular processes or collagen lamellae were visualised. Keratocyte nuclei count from stromal images adjacent to the Bowman's layer or endothelium were taken as representative of anterior [Figure - 1] and posterior stromal [Figure - 2] keratocyte nuclei counts respectively. The anterior stromal keratocyte nuclei were more abundant and oval compared to the posterior keratocyte nuclei which were less abundant and more oblong in shape.

The basal epithelial cell density was 3601.38 ± 408.19 cells/mm2 (range 3017.3 - 4231.1 cells/mm2). The average keratocyte density in the anterior stroma was 1005.02 ± 396.86 cells/mm2 (range 571.6 to 1249.6 cells/mm2) and posterior stroma was 654.32 ± 147.09 cells/mm2 (range 402.6 - 1049.1 cells/mm2). The posterior stromal keratocyte density was 30.76% less than the anterior stromal keratocyte density. The difference between the anterior and posterior stromal keratocyte densities was statistically significant (P=0.001).

The mean basal epithelial cell density, the mean anterior stromal keratocyte nuclei density, posterior stromal keratocyte nuclei density and endothelial cell density, repeated two weeks later on 10 eyes, is given in [Table - 1].

Endothelial cells were visible as bright cell bodies and dark cell boundaries [Figure - 3]. Endothelial cell density was 2818.1 ± 361.03 cells/mm2 (range 2118.9 - 4434 cells/mm2). The average endothelial cell area was 385.44 ± 42.66 mm2 (range 268.9 - 489.2 mm2). The percentage of hexagonal cells ranged from 42.04 ± 11.81 (range 22.5 - 69.4%). Mean coefficient of cell size variation was 32.29 ± 3.06 (range 27.2 - 39.2).

No statistically significant differences were measured in cell densities of any corneal layer, either between female and male patients or between right and left eyes [Table - 2][Table - 3]. Basal epithelial cell density, anterior stromal keratocyte nuclei and posterior stromal keratocyte nuclei density were unaffected by age (r= 0.12, 0.07, - 0.12 respectively) (P= 0.001). There was a statistically significant decrease in mean endothelial cell density with increase in age (r = - 0.42, P = 0.001). There was a statistically significant increase in coefficient of cell size variation with increase in age (r= 0.73, P=0.001).


  Discussion Top


Confocal microscopy of the human cornea provides images of high magnification, resolution and contrast in cooperative subjects. In-vivo confocal microscopy allows sequential examination of the cornea. Several studies have been performed in normal and diseased corneas.

We obtained images good enough for analysis of all cell layers in 74 of 100 examined eyes. As observed by Hollingsworth et al [14] the reasons for poor image quality in the remaining eyes were eye movement and inability to fixate properly till the end of the examination. Since confocal microscopic examination of the cornea is more intimidating, the subjects in our study required repeated examinations for good quality image acquisition. This perhaps explains the poor imaging of the superficial epithelial cells in most of our confoscans, as this was the last layer to be imaged.

The basal layer appeared as a uniform mosaic of cells with light borders. A fine beaded nerve plexus was seen adjacent to the basal epithelium. Bowman's layer appeared as an amorphous membrane and the sub-epithelial nerve plexus was seen as beaded nerve fibres. In the corneal stroma, cell nuclei were visible against a dark background. Stromal keratocyte density was significantly higher in the anterior stroma compared to the posterior stroma.

Published reports of the mean basal epithelial density show wide variation. [9],[10],[11],[12] Mean basal epithelial density measured by Paul et al [9] was 14,442 cells /mm2. Kauffmann et al[10] examined 20 healthy corneas using an in-vivo slit scanning confocal microscope and observed a mean epithelial cell density of 6248 ± 378 cells /mm2. Mustonen et al[12] reported a mean epithelial cell density of 5699 ± 604 cells /mm2. The mean basal cell density in this study was lower than in earlier studies. Wide variations seen in epithelial cell populations are perhaps due to the difference in the study populations reported. Similar to previous reports[9],[11],[12] mean epithelial cell densities showed no relationship with age and gender.

As observed by Mustonen et al[12] stromal keratocyte nuclei densities in our study showed no correlation with gender and age. Several studies[13],[18],[19],[20],[21]have found a decrease in the cell density over the anteroposterior stromal thickness varying between 30-46.3%. As with previous studies [12],[13],[14] we also observed higher keratocyte nuclei density in the anterior stroma of the normal central cornea with in-vivo confocal microscopy. The posterior stromal keratocyte nuclei density in our study was 30.76% less compared to the anterior stroma. Berlau et al[13] found a lesser keratocyte nuclei density adjacent to the Bowman's layer and the Descemet's membrane in subjects lesser than 50 years of age. Hollingsworth[14] et al found no difference in the counts of the corneal cell populations between the right and left eyes.

Moller-Pederson [21] performed sDNA content analysis and found a direct correlation between keratocyte nuclei density and age with a physiologic decline of 0.3% per year throughout life. Hollingsworth et al [14] observed a decrease of 0.48% per year of anterior stromal keratocyte nuclei density and 0.22% per year of posterior keratocyte density.

Specular microscopy evaluation of the endothelium has been widely studied.[22],[23],[24],[25],[26],[27],[28],[29],[30] Endothelial cell evaluation with confocal microscopy has been reported in two earlier studies.[12],[14] Automated endothelial cell analysis was found reliable and reproducible in both interobserver and intraobserver groups in a study by Imre and Nagymohaly. [31] Our study of the normal central corneal endothelial cell densities and mean cell areas showed results comparable with previously reported studies with specular microscopy. Corneal endothelial cell density and morphology in Indian eyes has been studied by Rao et al[32] by specular microscopy. They have observed that the endothelial cell density in Indian eyes is less than in American and Japanese populations. Our study has also demonstrated a lower endothelial cell density in Indian eyes compared to earlier studies using confocal microscopy[12],[14] [Table - 4]. Hollingsworth et al[14] studied healthy endothelial cells of 120 European subjects and found a mean endothelial cell density of 3061 ± 382 cells/mm.2 Mustonen et al[12] examined the central corneas of 58 healthy American eyes with an in-vivo slit scanning confocal microscope and observed a mean endothelial cell density of 3055 ± 386 cells/mm.[2] A study of the ethnic subgroups in Southern Asia [33] found statistically significant differences of the endothelial coefficient of cell size variation and cell hexagonality between gender and ethnic groups. We observed a statistically significant negative correlation of mean endothelial cells density with increasing age and a statistically significant increase in coefficient of cell size variation with age.

Our study documents cell densities of the various corneal cell layers obtained by slit scanning confocal microscopy in normal Indian eyes. Wide variations in epithelial cell densities disallow a possible comparison with Western literature. Keratocyte nuclei density in Indian eyes is comparable to that reported in the literature. Mean endothelial cell density is lower than the reported studies with confocal microscopy. This corroborates with the observation of Rao et al.[32] Our data can be used as a reference for further studies in confocal microscopy of diseased corneas.

 
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    Figures

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

  [Table - 1], [Table - 2], [Table - 3], [Table - 4]


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