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. 2017 Jul 31;7(1):6951.
doi: 10.1038/s41598-017-07534-9.

Acute Ocular Hypertension Disrupts Barrier Integrity and Pump Function in Rat Corneal Endothelial Cells

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Free PMC article

Acute Ocular Hypertension Disrupts Barrier Integrity and Pump Function in Rat Corneal Endothelial Cells

Xian Li et al. Sci Rep. .
Free PMC article

Abstract

Acute ocular hypertension (AOH) frequently compromises corneal endothelial cell (CEC) function in clinical practice. This type of stress induces corneal oedema and a decrease in the corneal endothelial cell density (ECD). The anterior chamber of the right eye of Sprague-Dawley rats was irrigated with Balanced Salt Solution (BSS) for two hours, and the left eye served as a control to determine the time-dependent effects of AOH on endothelial cell morphology and function. The average intraocular pressure (IOP) increased to 82.6 ± 2.3 mmHg (normal range: 10.2 ± 0.4 mmHg) during anterior irrigation. Very soon after initiating irrigation, corneal oedema became evident and the cornea exhibited a significant increase in permeability to FITC-dextran. The peripheral ECD was significantly reduced, and the morphology of CECs became irregular and multiform. The structures of the zonula occludens-1 (ZO-1) and F-actin were severely disrupted. In addtion, Na,K-ATPase exhibited a dispersed expression pattern. Two days after irrigation, obvious CEC proliferation was observed, the ECD recovered to a normal level, and F-actin was dispersed throughout the cytoplasm. Seven days later, the CEC structure and function were nearly normalized. Based on the results obtained using this model, an acute IOP crisis exerts transient deleterious effects on CEC structure and function in rats.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Changes in IOP observed in the AOH model and sham-operated rats. IOPs were evaluated in rats once every 20 minutes during irrigation and once every 2 hours after the irrigation using a hand-held, non-contact tonometer. The normal IOP of the rats was 10.2 ± 0.4 mmHg. At the beginning of irrigation, the IOP rapidly increased to 82.6 ± 2.3 mmHg (A). The IOP remained stable at 10.2 ± 1.0 mmHg in rats that underwent the sham procedure (B). After the infusion needle was removed, the IOP decreased to a level below the normal level and recovered to its baseline level after approximately 6 hours (A and B).
Figure 2
Figure 2
Slit lamp microscope examination of corneal images. The corneas of control and sham groups were transparent. Rapidly after irrigation started, the corneas became oedematous and opacified, and the iris borders were poorly defined. Two days later, corneal opacity gradually reversed to become transparent and the iris borders and structures were again well defined.
Figure 3
Figure 3
Peripheral CEC density quantification and histology. AOH decreased the peripheral corneal ECD (P < 0.05, Dunnett’s test) (A). Alizarin red staining revealed the CEC morphology (B). Normal CECs had a regular hexagonal morphology. Rapidly after irrigation, the morphology of CECs became irregular and multiform. Although the peripheral ECD increased to a normal level, the morphology was still not uniform after two days, whereas the morphology and size of the CECs had completely reversed to the normal condition after 7 days.
Figure 4
Figure 4
Changes in CECs induced by AOH. Ki67 immunostaining is used to examine cell proliferation. No Ki67-positive CECs were observed in controls. AOH rapidly decreased the peripheral ECD after irrigation. However, Ki67 staining was still negative. Two days later, Ki67-positive cells were readily apparent, indicating that AOH induced peripheral CECs to proliferate. At Day 7, Ki67-positive cells were not observed.
Figure 5
Figure 5
The integrity of the TJ protein ZO-1 was disrupted by AOH. AOH increased ZO-1 expression, based on a western blot analysis. The blots were run under the same experimental conditions and the images were from the same gel (A). Immunofluorescence staining for ZO-1 showed that ZO-1 formed a regular hexagonal pattern and was expressed in a contiguous pattern around the cell borders in the control group (B). The ZO-1 expression pattern was significantly disrupted by AOH, and ZO-1 became less localized to the cell borders and exhibited a discontinuous distribution. However, 2 days later, the normal ZO-1 distribution around the cell borders was restored.
Figure 6
Figure 6
AOH disrupted the cytoskeletal (F-actin) organization. F-actin microfilaments were contiguous along the apical cell border and formed a circumferential, double-banded structure under control conditions. Rapidly after AOH, the double-banded structure was lost and F-actin exhibited a diffuse distribution. Two days later, the distribution of F-actin was not limited to the cell border but instead was diffusely scattered throughout the cytoplasm. The F-actin distribution only partially recovered and its double-banded structure reappeared in some cells at Day 7.
Figure 7
Figure 7
AOH disrupted the localization of Na,K-ATPase. Na,K-ATPase was localized to the basolateral membrane and was evenly distributed around the cell membrane in control corneas. AOH induced a dispersion of Na,K-ATPase away from its basolateral membrane localization. Two days later, the normal localization of Na,K-ATPase began to appear, but the recovery was still incomplete, even at Day 7 (A). AOH increased Na,K-ATPase expression very slightly, but not significantly, based on the western blot analysis. The blots were run under the same experimental conditions and the images were from the same gel (B).
Figure 8
Figure 8
AOH induced an increase in transendothelial permeability. The influence of AOH on barrier integrity was evaluated by measuring FITC-dextran flux across the endothelium. The accumulation of FITC fluorescence in the entire cornea was detected using a microplate reader. AOH caused the permeability to increase. In contrast, two days later, FITC-dextran accumulation in the endothelial tissue decreased to normal levels. Data are presented as the means ± standard deviations (SD) from three independent experiments. ***P < 0.001 (Dunnett’s test).

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