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Observational Study
. 2015 Sep 17;10(9):e0138076.
doi: 10.1371/journal.pone.0138076. eCollection 2015.

Recovery of Corneal Endothelial Cells from Periphery after Injury

Sang Ouk Choi  1 , Hyun Sun Jeon  2 , Joon Young Hyon  3 , Yun-Jung Oh  2 , Won Ryang Wee  4 , Tae-Young Chung  5 , Young Joo Shin  1 , Jeong Won Kim  6
Affiliations
Observational Study

Recovery of Corneal Endothelial Cells from Periphery after Injury

Sang Ouk Choi et al. PLoS One. .

Abstract

Background: Wound healing of the endothelium occurs through cell enlargement and migration. However, the peripheral corneal endothelium may act as a cell resource for the recovery of corneal endothelium in endothelial injury.

Aim: To investigate the recovery process of corneal endothelial cells (CECs) from corneal endothelial injury.

Methods: Three patients with unilateral chemical eye injuries, and 15 rabbit eyes with corneal endothelial chemical injuries were studied. Slit lamp examination, specular microscopy, and ultrasound pachymetry were performed immediately after chemical injury and 1, 3, 6, and 9 months later. The anterior chambers of eyes from New Zealand white rabbits were injected with 0.1 mL of 0.05 N NaOH for 10 min (NaOH group). Corneal edema was evaluated at day 1, 7, and 14. Vital staining was performed using alizarin red and trypan blue.

Results: Specular microscopy did not reveal any corneal endothelial cells immediately after injury. Corneal edema subsided from the periphery to the center, CEC density increased, and central corneal thickness decreased over time. In the animal study, corneal edema was greater in the NaOH group compared to the control at both day 1 and day 7. At day 1, no CECs were detected at the center and periphery of the corneas in the NaOH group. Two weeks after injury, small, hexagonal CECs were detected in peripheral cornea, while CECs in mid-periphery were large and non-hexagonal.

Conclusions: CECs migrated from the periphery to the center of the cornea after endothelial injury. The peripheral corneal endothelium may act as a cell resource for the recovery of corneal endothelium.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Photographs of anterior segments (A) and central corneal thickness data (CCT; B).
A. Chemical injury of the cornea causes severe corneal edema and endothelial damage immediately after chemical injury. Slit lamp examination shows severe edematous and opaque cornea, and Descemet’s membrane folds. Endothelial cells are absent under specular microscopy. Corneal edema is reduced from the periphery to the center over time. Case 1 shows severe corneal edema and Descemet’s membrane fold immediately after injury, central edema and peripheral transparency 1 month after injury, and a clear cornea 6 months after injury. Case 2 shows severe corneal edema and Descemet’s membrane folds immediately after injury; the cornea becomes more transparent from periphery 1 month after injury. Case 3 shows severe corneal edema and Descemet’s membrane folds immediately after injury and the cornea becoming clear from the periphery 1 month after injury. This case also shows transparency of the central edema and periphery 6 months after injury. B. CCT is greater immediately after injury but decreases significantly 6 and 9 months later (p = 0.014 and 0.012, respectively, paired t-test) * Statistically significant by paired t-test
Fig 2
Fig 2. Evaluation of corneal endothelial cells using specular microscopy.
A. Corneal endothelial cells are absent immediately after chemical injury; however, they are present 3 months (case 2) or 6 months (case 1 and case 3) later. B. CECD increased significantly 6 months following injury compared to immediately and 1 month after injury (p = 0.008 and p = 0.008, respectively, paired t-test), and 9 months after injury compared to those immediately or 1 months after injury (p = 0.005 and p = 0.005, respectively, paired t-test). C. Average cell area generally decreases over time, although not significantly. D. CV does not change and hexagonal cells are absent 3 to 9 months after injury. * Statistically significant by paired t-test
Fig 3
Fig 3. Changes in corneal edema and opacity after chemical corneal endothelial injury in rabbits.
A. Corneal edema increases 1 day after injury compared to the control, and then decreases over time; however, corneal opacity does not revert back to levels observed in control rabbits at day 14. Corneal edema decreases from the periphery, and peripheral and central corneas are edematous at day 1 and at day 7. At day 14, the central cornea is edematous (white arrow), while the periphery is transparent (black arrow). B. CCT increases significantly at day 1 and at day 7 after injury compared to the control (p = 0.045 and p = 0.015). CCT generally decreases over time and there are no differences in CCT at day 14 compared to the control, although CCT does not revert to levels observed in control rabbits. * Statistically significant by Student’s t-test
Fig 4
Fig 4. Vital staining of rabbit corneal endothelium with alizarin S red and trypan blue after injection of 0.1 mL of 0.05 N NaOH.
A. Corneal endothelial cells (CECs) in both the central and peripheral areas show damage at day 1. At day 7, CECs appeared at the periphery and scanty cells existed at the midperiphery and center 7 days after injury. The peripheral cells began to migrate from the far periphery and to recover from damage. At day 14, the dense small cells were observed at the periphery, the loose large irregular cells appeared at the midperiphery and no cells were still observed at the center of the cornea. The cell integrity of the peripheral cells recovers while cells migrate from the periphery to the center and also recover. B. Cell counts at ×400 magnification. Corneal endothelial cell numbers in the center and in the periphery are significantly lower compared to the control during the entire observation period (p < 0.001 in the center at all time points; p < 0.001 at day 1, p < 0.001 at day 7 and p = 0.012 at day 14 in the periphery). No corneal endothelial cells are present in the periphery or in the center 1 day after injury. Corneal endothelial cells in the periphery increase significantly over time (p < 0.001, one-way ANOVA). Corneal endothelial cells are detected in the periphery 7 days after injury and increase until day 14 (p = 0.022 compared to day 7), although they are lower compared to the number on day 1 (p = 0.014 and p = 0.010). Corneal endothelial cell counts in the center are not observed until day 14. * Statistically significant by Student’s t-test
Fig 5
Fig 5. Microscopic images of hematoxylin and eosin staining in corneal endothelium (A) and trabecular meshwork (B), and immunofluorescent staining for the proliferation marker Ki-67 (C).
A. With hematoxylin and eosin staining, no cells were observed on the center and periphery of Descemet's membrane 1 day after injury. At day 7, corneal endothelial cells (CECs) were detected at the periphery, but not at the center. At day 14, CECs were detected at the mid-periphery and the periphery. B. With hematoxylin and eosin staining, a lot of inflammatory cells were infiltrated around trabecular meshwork 1 day after injury. At day 7, inflammatory cells disappeared and at day 14, trabecular meshworks were recovered much. C. Ki-67 staining was performed to assess the proliferative cells. Corneal sections were immunostained for Ki-67 (Red) and counterstained with 4',6-diamidino-2-phenylindole (DAPI; blue). Phase contrast images reveals the actual sections. No cells were observed on the center and periphery of Descemet's membrane 1 day after injury. At 7 and 14 days after injury, the cells expressing Ki-67 were observed at the periphery of the cornea. Magnification X 400.
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Grants and funding

This study was supported by the National Research Foundation (NRF) grant (2012R1A1A2040118) funded by the Korea government and Hallym University Research Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.