Corneal endothelium cell density changes in patients with primary glaucoma attending Bansara Eye Care Centre

Examination performed

All subjects underwent a complete comprehensive eye examination, which comprised a detailed history taking, visual acuity assessment of both aided and unaided vision for distance and near, and both objective and subjective refraction. IOP was measured using Goldmann applanation tonometry. Slit-lamp bio microscopy and gonioscopy were performed. The required parameters- including minimum/maximum/average cell area, central corneal thickness (CCT), cell density, hexagonal cells, standard deviation, and coefficient of variation- were obtained by the use of a Specular Microscope (Topcon SP 3000-P).

Criteria

Exclusion

Individuals with a history of ocular surgery, trauma, intraocular inflammation, current or past contact lens use, diabetes mellitus, or pre-existing corneal pathologies were excluded to avoid confounding the results.

Specular microscope

The specular microscope provides a non-invasive, high-resolution imaging of the corneal endothelium. Parameters typically assessed include cell area (± SD, μm²), ECD (cells/mm²), polymegathism (CV), and pleomorphism (% hexagonal cells).¹³

The following equation determines the cell density:

$\text{ECD}=\frac{{10}^6}{\text{Average}\text{Cell}\text{Area}\left(\mu m^2\right)}$

The coefficient of variation (CV) is calculated using the equation:

$CV=\left(\frac{\text{SD}\text{of}\text{Cell}\text{Area}}{\text{Mean}\text{Cell}\text{Area}}\right)\times 100$

Age and gender distribution

The age and gender distribution of the five groups have been summarized in Table 2 and Figures 1 and 2. No significant age difference was observed (56.1±9.1) among the five groups (p = 0.245). However, a statistically significant gender difference was noted (p = 0.021), with the female (53.8%) outnumbering the male (46.2%).

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Figure 1: Age Distribution of all the groups. POAG: Primary open angle glaucoma; PACG: Primary angle closure glaucoma; NTG: Normal tension glaucoma; OHTN: Ocular hypertension

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Figure 2: Gender of all the groups. POAG: Primary open angle glaucoma; PACG: Primary angle closure glaucoma; NTG: Normal tension glaucoma; OHTN: Ocular hypertension

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Table 3 and Figure 3 summarize the ophthalmic findings across the five groups, and the comparison between the groups with glaucoma has been presented in Tables 4–7 and Figures 4–7.

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Figure 3: The comparison between normal controls to glaucoma patients in terms of corneal endothelium characteristics and intra-ocular pressure. POAG: Primary open angle glaucoma; PACG: Primary angle closure Glaucoma; NTG: Normal tension glaucoma; OHTN: Ocular hypertension,

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Figure 4: The comparison between Primary Open Angle Glaucoma and the other glaucoma groups in terms of corneal endothelium parameters and intra-ocular pressure. POAG: Primary open angle glaucoma PACG: Primary angle closure glaucoma NTG: Normal tension glaucoma OHTN: Ocular hypertension

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Figure 5: The comparison between Primary Angle Closure Glaucoma and the other glaucoma groups in terms of corneal endothelium parameters and intra-ocular pressure. IOP: Intraocular pressure, CV: Coefficient of variation, CCT: Central corneal thickness.

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Figure 6: The comparison between Primary Normal Tension Glaucoma and the other glaucoma groups in terms of corneal endothelium parameters and intra-ocular pressure. IOP: Intraocular pressure, CV: Coefficient of variation, CCT: Central corneal thickness, CECD: Corneal endothelial cell density.

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Figure 7: The comparison between Ocular Hypertension and the other glaucoma groups in terms of corneal endothelium parameters and intra-ocular pressure.

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IOP

IOP was markedly higher in glaucoma cases relative to the healthy control group (p <0.001). Within the glaucoma subtypes, the POAG, PACG, and OHTN had significantly higher IOP compared to the NTG (p = 0.029). There was no significant difference in IOP between POAG and PACG (p = 0.974). Furthermore, the OHTN patients had significantly higher intra-ocular pressure compared to the POAG (p = 0.013) and PACG (p = 0.012) patients.

CCT

Our analysis showed that CCT did not vary significantly when comparing individuals with glaucoma to the healthy control group (p = 0.372). Similarly, comparisons among the glaucoma groups revealed no significant differences.

Mean cell area

A statistically significant disparity in mean cell area was identified between the healthy controls and the glaucoma group (p <0.001). Among the glaucoma categories, the mean cell area of PACG patients was substantially more extensive than that of the POAG (p = 0.002), NTG (p = 0.015), and OHTN (p = 0.016). There was no statistical significance between POAG, NTG, and OHTN patients.

ECD

ECD was significantly lower in the glaucoma group compared to healthy controls (p <0.001). Within the glaucoma subtypes, PACG patients exhibited the lowest cell density compared to the other glaucoma groups: POAG (p <0.001), NTG (p <0.001), and OHTN (p = 0.005). The differences between POAG and NTG (p = 0.257) and OHTN (p = 0.903) and between NTG and OHTN (p = 0.449) were not statistically significant.

Coefficient of variation

No significant differences were identified in the CV (p = 0.579) or Hexagonality (p = 0.845) across the normal controls and glaucoma patients, nor between the glaucoma groups.

Anti-glaucoma medication

The 79 Glaucoma patients (cases) were further categorized into two subgroups: patients who had not yet received any treatment (21.5%, n = 17) and patients receiving ophthalmic medications (78.4%, n = 62) at the time of the study. Table 8 and Figure 8 summarize the findings of the two subgroups. The analysis revealed no statistically significant differences between the CCT (p = 0.439), mean cell area (p = 0.506), CV (p = 0.057), and hexagonality (p = 0.523). Whereas, comparison of IOP (p <0.001) and corneal endothelium cell density (p = 0.039) demonstrated a major difference between the two subgroups. Endothelium cell density was considerably lower in individuals using medicine (2183.9 ± 435.2 cells/mm²) than in those not taking it (2560.1 ± 243.5 cells/mm²). Both subgroups have lower density than the normal group (2804.4 ± 142.2 cells/mm²). The IOP of the patients receiving medication (16.4 ± 5.6) was significantly lower than that of the patients not receiving medication (19.7 ± 3.9).

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Figure 8: The comparison of glaucoma patients with and without medication. IOP: Intraocular pressure, CV: Coefficient of variation, CCT: Central corneal thickness, CECD: Corneal endothelial cell density.

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Duration of disease

The glaucoma group was classified into three groups based on the duration of the disease, namely: <2 years (n = 35), 2–4 years (n = 33), and >4 years (n = 11). Our analysis showed a high degree of similarity between CCT (p = 0.601), coefficient of variation (p = 0.349), and hexagonality (p = 0.918). However, glaucoma duration significantly impacted corneal morphology. Patients with the disease for >4 years had the lowest ECD (1899.1 ± 419.1; p <0.001) and the largest mean cell area (558.3 ± 161.5; p = 0.003) compared to those with shorter durations. Additionally, IOP was also significantly higher in those with a duration of <2 years (18.1 ± 4.5) compared to those with longer histories (p <0.001) as shown in Table 9 and Figure 9.

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Figure 9: The corneal morphology and intra-ocular pressure based on the duration of the disease. IOP: Intraocular pressure, CV: Coefficient of variation, CCT: Central corneal thickness, CECD: Corneal endothelial cell density.

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Corneal endothelium cell density

According to our data, the corneal endothelium cell density of the glaucoma patients showed a significant decrease compared to that of the normal controls. Waring.² proposed three primary theories for this: first, direct mechanical compression from high IOP; second, shared congenital vulnerabilities in the endothelium and trabecular meshwork; and third, the toxic effects of long-term medication use. Our study found that glaucoma patients had considerably higher IOP than normal controls, which was associated with a significant reduction in CEC. High IOP is not associated with changes in CEC, according to some researchers.^14,15 Our results showed that elevated IOP found in glaucoma significantly correlates with reduced endothelial density, which is similar to the findings of other studies.^5,11,16

According to research by Verma et al.,⁹ individuals with angle-closure glaucoma exhibit substantial changes in CEC compared to healthy participants. Their findings suggest that prolonged exposure to elevated IOP results in progressively greater harm to the corneal endothelium. Specifically, both the length of an acute episode and the level of IOP were linked to a reduction in ECD and a corresponding expansion in the average area of CECs. Meanwhile, those with a history of acute episodes had considerably larger cell areas and different shapes. Cho et al.¹⁷ reported that POAG patients have a significantly lower ECD than NTG patients, which correlates with the difference in IOP. Chawla and Kumar.¹⁸ reported that PACG patients have the least ECD and similar ECD between POAG and OHTN. Within the groups of glaucoma of our study, PACG has the least amount of ECD, followed by POAG, OHTN, and NTG, respectively. NTG has the highest ECD among the subgroups, which is similar to the findings stated above.

CCT

Aghaian et al.¹⁹ reported that the CCT of glaucoma suspects, POAG, PACG, and NTG patients was significantly thinner than that of the normal controls; OHTN patients had a significantly thicker CCT compared to the normal controls. Lee.²⁰ also reported that CCT was thickest in OHTN, thinnest in NTG, and similar in eyes with POAG and normal patients. In our study, CCT did not differ significantly between glaucoma patients and normal controls, which may be attributed to the limited sample size.

In the groups of glaucoma, OHTN had the highest CCT, and NTG had the lowest CCT, but the variance was considered statistically negligible, and a further large-scale study is required. These findings are consistent with a study by R. Thomas¹⁷ in Vellore, which reported a thicker CCT in OHTN patients, leading to IOP overestimation. They advised using a correction factor to determine the actual IOP. Similarly, the numerically lower CCT in our NTG group mirrors findings by Morad,²¹ highlighting the risk of underestimating IOP in these patients.

Mean cell area

The significantly larger mean cell area in our glaucoma cohort is a clear compensatory response to cell loss, as the endothelium cannot replicate. This is consistent with findings by Cho et al.¹⁷

Coefficient of variation and hexagonality

Both the coefficient of variation and the percentage of hexagonal cells remained statistically uniform across all studied cohorts or among the glaucoma subgroups. Studies have shown that CV and hexagonality of the corneal endothelium indicate acute corneal endothelial damage.^9,22 Bourne and McLaren.¹⁶ reported that contact lens wear, trauma, disease, and surgery can be responsible for corneal endothelium changes. According to Arnavielle et al.,²³ procedures, including trabeculectomy, deep sclerectomy, and cataract surgery, may lead to endothelial cell loss and changes in cell morphology. We excluded patients with surgery, trauma, intraocular inflammation, or contact lens wear. As a result, the coefficient of variation and hexagonality did not show significant differences.

Anti-glaucoma medication

It has been shown that the toxicity of anti-glaucoma medication can produce CEC damage.^24,25 Benzalkonium chloride, a preservative frequently found in anti-glaucoma medications, is known to significantly contribute to the deterioration of the ocular surface. The detrimental impacts of this compound are linked to both the concentration and the duration of exposure, potentially leading to endothelial cell damage and alterations in CCT. Research conducted by Yu et al.⁵ identified a marked decline in CEC density among patients undergoing glaucoma therapy in comparison to untreated subjects. Our findings align with this, showing that the group receiving treatment had a significantly lower CEC than the untreated cohort, which may be related to medication toxicity. Although our study did not directly assess specific formulations, previous literature suggests preservatives like benzalkonium chloride may contribute to this deterioration. However, our findings did not achieve a level of statistical significance in the average cell area between these two groups. Further large-scale studies are warranted to validate these findings.

Duration of disease

Our findings demonstrated significant progressive alterations in corneal endothelial morphology with increasing disease duration. Patients with a disease duration of less than 2 years demonstrated the highest ECD and the smallest mean cell area. This was followed by patients with a duration of 2-4 years, while those with a duration of more than 4 years exhibited the lowest ECD and the largest mean cell area. Yee RW.²⁴ has observed that long-term use of glaucoma medications can contribute to CEC damage, which may explain the progressive decline in ECD and the corresponding increase in mean cell area with longer disease duration. Chawla and Kumar.¹⁸ also observed that sustained IOP elevation and long-term IOP fluctuations could result in CEC loss and thinning of the CCT. In our study, ECD significantly decreased as disease duration increased; however, no statistically significant difference in CCT was detected. This lack of significance may be attributable to the relatively small number of patients in the >4-year disease duration group. Further large-scale studies are therefore required to validate these findings.

This study has certain limitations that must be considered. Firstly, the small sample size raises the possibility of sampling error, which could influence the strength of our conclusions. Specifically, the relatively small sample sizes in the NTG and OHTN subgroups may limit the statistical power of these specific comparisons, and the findings should be interpreted with caution. To address this, further prospective research on a larger population is necessary. Secondly, IOP was measured post-treatment, meaning recorded values may not reflect the historical peak IOP that contributed to endothelial damage. Furthermore, the correlation between medication and cell loss is confounded by disease severity, as patients with advanced glaucoma typically require more medications.²⁶