Introduction
The corneal endothelium is a single layer of hexagonal cells covering the inner surface
of the cornea. It acts as a barrier between the corneal stroma and the aqueous humor,
limiting the passage of water and solutes from the anterior chamber to the interior
of the stroma. The endothelial cells have a crucial transport protein, the metabolic-endothelial
pump of electrolytes, which is Na +/K +/ATPase-dependent. This pump counteracts the
flow of water into the cornea, which is vital in maintaining the normal state of relative
dehydration of the corneal stroma essential for corneal transparency.[1]
Endothelial cells do not reproduce. In adults, the density varies between 2,000 to
3,000 cells/mm2 and declines with age. The minimum level of cells necessary for normal function is
estimated to be between 600 and 900 cells/mm2, from this limit stromal edema appears.[2] The endothelium is metabolically very active and is primarily responsible for corneal
transparency. The cornea maintains a constant thickness throughout life and retains
its aqueous content at a stable level of relative dehydration. The anatomical integrity
of the corneal endothelium is one of the most important factors that directly influence
the cornea's hydration rate.[3]
Specular microscopy is a diagnostic technique that allows us to obtain images with
high magnification of endothelial cells. It provides a clear view of living cells,
without altering their function or morphology. Using this test, an endothelial count
by surface area can be performed to determine any alteration in the shape or size
of endothelial cells. These parameters give us a framework for assessing the functional
capacity of the endothelium. Specular microscopy is a diagnostic test of great clinical
utility, especially for cases that require a second intraocular intervention, such
as operated cases or when a primary endothelial alteration is suspected.[4]
Due to the significant variation in endothelial density found in different ethnic
groups and by age, it is essential to know the normal data in each population.[3]
Thus, this study aimed to investigate the corneal endothelial cell density and morphology
in healthy Libyan eyes at Benghazi Teaching Eye Hospital.
Methods
A cross-sectional, observational study was conducted at Benghazi Teaching Eye Hospital,
Benghazi, Libya, from December 2023 to April 2024, which included 198 eyes of 100
healthy white Libyan individuals (two eyes were excluded; one with corneal scar and
the other one with a pterygium), randomly selected among the hospital's staff, relatives
of patients attending the hospital, and the outpatient department, and anyone who
met the inclusion criteria was examined to exclude any ocular pathology to determine
their eligibility for the study.
Inclusion Criteria
Subjects free of ophthalmological diseases aged 17 years or more with no history of
eye surgery and not a known diabetic or hypertensive.
Exclusion Criteria
Subjects with a refractive error greater than ± 3 diopters, Diabetics, a history of
intraocular surgery, ocular trauma, or a history of any intraocular or systemic pathology
(like hyperlipidemia, minor ischemic stroke, and gout), contact lens wearer, and those
who did not collaborate in performing the examination were excluded.
All participants underwent a full ophthalmological examination, including a measurement
of visual acuity, an intraocular pressure checkup, and a dilated fundus examination
to rule out any pathology.
The study was carried out following the Helsinki Declaration, and the subjects signed
an informed consent form after being informed about the study.
Specular microscopy was done by the same examiner using the noncontact Topcon specular
microscope (SP-1P model).[5] The machine conducted an automatic study of the cornea, it recorded parameters such
as central corneal thickness (CCT), mean cell density (MCD), coefficient of variation
(CV%) in the cell area, and hexagonality (Hex [%]).
Statistical Analysis
The Statistical Package for the Social Sciences (SPSS version 25.0; IBM Corporation,
Armonk, New York, United States) was used. Data were presented as mean ± standard
deviation and frequencies.
The Mann–Whitney test was used instead of the Student's t-test for nonparametric data, the test was run to determine if there were differences
in variables between genders. Visual inspection revealed that the distributions of
engagement scores for males and females were similar. Pearson's chi-square analysis
was used to compare percentages. Spearman's correlation analysis was used to compare
two qualitative variables. The value of r is explained as follows: 0.1 to 0.3, weak correlation; 0.3 to 0.5, moderate correlation;
and 0.7 to 1, strong correlation. A p-value of < 0.05 was considered statistically significant. A linear regression was
performed to determine the effect of age on MCD and corneal endothelial parameters.
Results
The mean age of participants in this study was 47.4 ± 13.8 years (range 21–75 years),
with a male predominance of 60 men (60%), and the number of right eyes was 100 (50.5%).
The mean CCT was 516.45 ± 43.04 μm, the MCD was 2664.30 ± 371.26 cells/mm2, the mean CV% was 32.3% ± 3.7, and the mean percentage of the hexagonal cell was
52.8% ± 9.6 ([Table 1]).
Table 1
Demographic characteristics and variables related to the corneal endothelial cell
characteristics in healthy Libyan subjects
|
Variable
|
|
Number (%)
|
|
Gender
|
Male
|
60 (60)
|
|
Female
|
40 (40.0)
|
|
Eye
|
Right eye
|
100 (50.5)
|
|
Left eye
|
98 (49.5)
|
|
Age
|
20–30 y
|
46 (23.2)
|
|
31–40 y
|
56 (28.3)
|
|
41–50 y
|
48 (24.2)
|
|
51–60 y
|
35 (17.6)
|
|
61-70 y
|
13 (6.7)
|
|
Mean ± SD
|
47.8 ± 13.8
|
|
Median (range)
|
51 (21–75)
|
|
CCT (μm)
|
Mean ± SD
|
516.45 ± 43.04
|
|
Median (range)
|
513 (465–642)
|
|
MCD (cells/mm2)
|
Mean ± SD
|
2664.30 ± 371.26
|
|
Median (range)
|
2622 (1093–3877)
|
|
CV (%)
|
Mean ± SD
|
32.3 ± 3.7
|
|
Median (range)
|
31.2 (16–48)
|
|
Hex (%)
|
Mean ± SD
|
52.8% ± 9.6
|
|
Median (range)
|
55 (20–75)
|
Abbreviations: CCT, central corneal thickness; CV, coefficient of variation; Hex,
hexagonality; MCD, mean cell density; SD, standard deviation.
There was no statistical difference (p > 0.05), in the age, CCT, and MCD between males and females. Whereas CV% and Hex
(%) showed significant gender differences (p < 0.01 for both) ([Table 2]).
Table 2
Comparison of age and endothelial cell characteristics between genders in healthy
eyes of Libyan participants
|
Variables
|
Men (mean ± SD)
|
Women (mean ± SD)
|
p-Value[a]
|
|
Age
|
47.6 ± 15.7
|
47.25 ± 15.3
|
< 0.754
|
|
CCT (μm)
|
515.2 ± 36.4
|
511 ± 39.8
|
< 0.198
|
|
MCD (cells/mm2)
|
2665 ± 34.2
|
2635 ± 39.4
|
< 0.125
|
|
CV in cell area (%)
|
32.7 ± 5.7
|
31.1 ± 4.8
|
< 0.012
|
|
Hex (%)
|
53.3 ± 12.1
|
54.1 ± 11.5
|
< 0.015
|
Abbreviations: CCT, central corneal thickness; CV, coefficient of variation; Hex,
hexagonality; MCD, mean cell density; SD, standard deviation.
a Difference between gender using the Mann–Whitney U test.
There was a significant negative weak correlation between CCT (r = –0.10) and age, as well as a significant negative moderate correlation between
MCD (r = –0.36) and Hex (r = –0.31) with age. CV exhibited a significant moderate positive association with
age (r = 0.35) ([Table 3]).
Table 3
Correlation between age and corneal variables in healthy eyes of Libyan participants
|
Variables
|
Spearman's correlation coefficient (r)
|
p-Value
|
|
CCT (μm)
|
− 0.109
|
< 0.009
|
|
MCD (cells/mm2)
|
−0.365
|
< 0.001
|
|
CV in cell area (%)
|
0.351
|
< 0.001
|
|
Hex (%)
|
–0.312
|
< 0.001
|
Abbreviations: CCT, central corneal thickness; CV, coefficient of variation; Hex,
hexagonality; MCD, mean cell density.
The higher endothelial cell loss rate of 8.4% was in the age group 31 to 40 years,
and ranged between 1.1 and 2.7% in other groups ([Table 4]).
Table 4
Corneal endothelial cell loss in different age groups of Libyan subjects with healthy
eyes
|
Age group (y)
|
Mean cell density (MCD)
|
Loss %
|
|
20–30
|
2929 ± 312.6
|
|
|
31–40
|
2683 ± 275.2
|
8.4
|
|
41–50
|
2654 ± 320.14
|
1.1
|
|
51–60
|
2580 ± 311.3
|
2.7
|
|
60–70
|
2549 ± 412.3
|
1.2
|
Discussion
The endothelial cells of the cornea lack regeneration capacity. Thus, a loss in corneal
endothelial cell density is compensated for through cell spreading, resulting in increased
cellular pleomorphism and a drop in the percentage of hexagonal cells.[6] Many studies have demonstrated that the density of corneal endothelial cells varies
by ethnic origin, age,[6]
[7]
[8] and the model of the instrument.[9]
The causes of endothelial cell insufficiency over time are unknown; however, evidence
suggests that apoptosis and/or necrosis caused by light-induced oxidative damage may
play a role. In addition, the number of endothelial cells declines following stressful
events such as trauma, previous corneal transplantation, stress caused by certain
systemic disorders such as diabetes, glaucoma treatment, cataract surgery, and intraocular
lens implantation.[10] Therefore, the measurement of endothelial cell density and shape is highly significant,
because the decrease in endothelial cell density is a primary indicator of pathological
alteration and reduces the ability of corneal healing.[11]
This observational cross-sectional study was conducted in Benghazi Teaching Eye Hospital,
over 3 months on 198 eyes of 100 healthy Libyan participants, to investigate the corneal
endothelial cell density and morphology in healthy Libyan eyes using the noncontact
Topcon specular microscope (SP-1P model).
The mean age of participants in this study was 47.4 ± 13.8 years (range 21–75 years),
with 60 males constituting 60% of the cases.
Central Corneal Thickness
In the present study, the mean CCT was 516.45 ± 43.04 μm, which is comparable to the
results of the Egyptian[6] (514.45 μm) and Caucasian[9] populations (513 μm), but lower than the Turkish[12] (521 μm) and Indian[7] (533.3 μm) studies ([Table 5]). These could be attributed to differences in measuring tools, as the Egyptian study
used the same noncontact specular microscope as ours, while the other two used different
types of specular microscopes. However, a previous study found that people of North
African origin had statistically significantly thinner corneas than those of other
origins.[13]
Table 5
Corneal endothelial cell characteristics reported in previous studies compared to
the present study
|
Variables
|
Age (y)
(mean ± SD)
|
CCT (μm)
(mean ± SD)
|
MCD (cells/mm2)
(mean ± SD)
|
CV (%)
(mean ± SD)
|
Hex (%)
(mean ± SD)
|
|
Egyptian[6]
|
49.48 ± 15.27
|
514.45 ± 43
|
2647.50 ± 382
|
32.31 ± 5.08
|
53.79 ± 11.00
|
|
Indian[7]
|
48 ± 16.5
|
533.3 ± 49.7
|
2525 ± 337
|
35.8 ± 6.9
|
57.3 ± 7.9
|
|
Caucasian[9]
|
42 ± 17.1
|
513 ± 39
|
2732 ± 305
|
34 ± 7
|
46 ± 8
|
|
Turkish[12]
|
44.3 ± 13.5
|
521 ± 33
|
2671 ± 356
|
34.3 ± 5.3
|
54.9 ± 10.0
|
|
Nigerian[16]
|
50.35 ± 20.13
|
NR
|
2610.26 ± 371
|
43.95 ± 9.50
|
46.52 ± 8.83
|
|
Iranian[17]
|
52.7 ± 19.1
|
NR
|
1961 ± 457
|
24.1 ± 7.1
|
NR
|
|
Chinese[8]
|
44 ± 21
|
NR
|
2932 ± 363
|
33 ± 50
|
59 ± 9
|
|
Present study
|
47.4 ± 13.8
|
516.45 ± 43.04
|
2664.30 ± 371.26
|
32.3% ± 3.7
|
52.8% ± 9.6
|
Abbreviations: CCT, central corneal thickness; CV, coefficient of variation; Hex,
hexagonality; MCD, mean cell density; NR, not reported; SD, Standard deviation.
There was no statistical difference in the CCT (p > 0.05) between males and females, similar to what was previously reported by other
studies.[6]
[12]
[13]
[14]
A statistically significant negative weak correlation between CCT (−0.10) and age
was found, which is similar to other studies.[6]
[15]
The Mean Cell Density
The MCD was 2664.30 ± 371.26 cells/mm2 which is comparable to the Egyptian[6] (2647.50 cells/mm2) and Turkish[12] populations (2671 cells/mm2), higher than Nigerian[16] (2610.26 cells/mm2) and Iranian[17] populations (1961 cells/mm2), and lower than Chinese[8] (2932 cells/mm2) and Caucasian population (2732 cells/mm2) ([Table 5]).
Researchers hypothesized an inversely proportionate link between corneal diameter
and endothelial cell density to explain the variations in MCD across populations.[7]
[18] However, we did not measure the corneal diameter in the current study, and no previously
published article on the normal corneal diameter of Libyans was found.
There was no statistical difference in the MCD (p > 0.05) between males and females in our study, which is consistent with previous
studies.[6]
[12]
[15]
[17] However, Padilla et al[19] found that Filipino females had a statistically significantly higher MCD than males
(p < 0.01), while Yunliang et al[8] found that Chinese males at the age of 61 to 70 years had a statistically significantly
higher MCD than females (p < 0.05).
An inverse relationship between MCD and age (r = –0.36) was found, with the highest rate (8.1%) recorded in the third decade of
life which is consistent with previous research.[6]
[8]
[12]
[17] Some researchers explained this by redistributing endothelial cells in the growing
cornea,[7]
[18] while others attributed it to increasing physical activity in this age group.[6]
Researchers reported that the type and model of the specular microscopy instrument
can impact the measurements of corneal endothelial cell density.[9]
[20]
[21]
The cell loss rate in our study (range 1.1–8.4%) was much higher than in prior studies.
In Egypt,[6] the rate of cell loss was 0.1 to 0.7%, in Turkey[12] 1.9 to 5.9%, in Iran[17] 0.6% per year, in China[8] 0.3% per year, and in Japan[22] 0.42%/year, although the exact cause of this higher cell loss in Libyan population
is not known; this could be due to ethnic variations.
Coefficient of Variation
This is the most sensitive biomarker of corneal endothelial dysfunction.[23]
The current study found a mean CV% of 32.3% ± 3.7, similar to previous studies from
Egyptian,[6] Turkish,[12] and Chinese[8] populations, furthermore, it was lower than Nigerian[16] and higher than Iranian populations.[17]
CV% demonstrated significant gender differences where males had higher CV% than females
(p < 0.01) which goes in line with the Egyptian study,[6] nevertheless researchers from Iranian,[17] Turkish,[12] and Filipino[19] populations reported no significant gender differences in CV%.
CV% showed a significant moderate positive (r = 0.35) correlation with age, which was consistent with many previous researches.[6]
[12]
[17]
Hexagonality
The mean percentage of the hexagonal cell was 52.8% ± 9.6. This was similar to the
Egyptian[6] and Turkish[12] populations, but higher than the Nigerian[16] and Caucasian[9] populations.
Hex (%) in our study exhibited significant gender differences (p < 0.01), similar to previous research.[6]
[24] Abdellah et al linked this gender difference to the smoking effect as males smoke
more than females.[6] Other research revealed the effect of smoking on hexagonally.[25] Meanwhile, reports indicate that there are no gender disparities in Hex (%).[9] In the current study, Hex (%) decreased with aging (r = –0.31) similar to earlier studies.[6]
[8]
[9]
[12]
[19]
The variable morphology and density of endothelial cells among different ethnic groups,
as indicated above, highlight the significance of the current work in establishing
normative Libyan data.
Limitations of the Study
The small sample for subgroup analysis limited this study as well as missing information
about the corneal diameter and its effect on corneal endothelial cell density.
Recommendation
We recommend another study in the future with a larger number of participants, the
use of another instrument model, and to measure the corneal diameter to validate our
findings.
Conclusion
The CCT, MCD, and Hex (%) decrease while the CV% increases with age, there was no
statistical difference in the CCT and MCD between males and females, whereas CV% and
Hex (%) demonstrated significant gender differences.
