Confocal microscopy can be used to evaluate keratitis and corneal nerves [9,10,11,12,13,14], but there is no study about its use in eyes with AACC. This study aimed to investigate corneal epithelial cells, subepithelial nerve fiber plexus, stromal cells, and endothelial cells of the AACC and fellow eyes using confocal microscopy. The results suggest that AACC can reveal the decreased density of corneal subepithelial nerve fiber plexus, activation of stromal cells, increased endothelial cell polymorphism, and decreased cell density. Confocal microscopy might be superior to specular microscopy to analyze the endothelial cells in AACC with corneal edema.
The confocal microscope can directly obtain images of tissues and cells in each layer of the cornea through continuous confocal scanning, detecting various elements of normal and affected corneas in multiple layers and in vivo, without fixation and staining of tissue sections [9,10,11,12,13,14]. Thus, it is a major method for exploring the pathological mechanism of ophthalmic diseases at the cellular level. In this study, confocal microscopy was used to observe the histological changes for each layer of the cornea in AACC and fellow eyes, which provided a basis and guidance for further exploring the effect of acute ocular hypertension on corneal injury.
Previous studies on corneal injury caused by ocular hypertension have used specular microscopy, which can only observe the number and morphological changes of corneal endothelial cells [7, 8, 18]. Still, the effects of AACC on the changes in corneal subepithelial nerve fibers, stromal layers, and other layers of the tissues remain unclear. The present study showed that acute ocular hypertension in AACC patients not only damaged the corneal endothelial cells but also caused various degrees of pathological changes in other layers of tissues, including corneal epithelial cell swelling, widening of intercellular space, and large vacuoles in epithelial cells in severe cases. The CNFD, CNBD, and CNFL were reduced. The stromal layer was in an activated state, showing swollen stromal cells, enhanced reflection, and cross-linkage into a network.
Previous studies showed that patients with dry eye  and diabetes [14, 19] have different degrees of reduced density of the corneal subepithelial nerve fibers, which can even disappear. Roszkowska et al.  found that corneal subbasal nerve plexus density reduces with age and myopic refractive error in healthy adults. The reduction in the density of the subepithelial nerve fiber plexus in patients with acute angle closure might be related to corneal dystrophy due to decreased blood flow of the corneal limbal vascular network and corneal epithelium injury caused by acute ocular hypertension [20,21,22]. The activation of corneal stromal cells suggested an inflammatory response in the corneal tissue. In AACC, the blood-aqueous humor barrier is destroyed, and the anterior segment shows an acute inflammatory response . The expression levels of interleukin (IL)-6, colony-stimulating factor, and vascular endothelial growth factor (VEGF) in the aqueous humor are then increased . Some studies proposed that the increased expression levels of inflammatory factors may result in an AACC event, participating in recurrence [25,26,27].
Verma et al.  found that the density of corneal endothelial cells in PACG patients was not statistically different from that of normal controls. Other studies supported the negative correlation between ocular hypertension and corneal endothelial cells [27,28,29]. In AACC, long-term ocular hypertension can decrease the density of corneal endothelial cells . The corneal edema is closely related to endothelial cell loss, while in acute angle closure, the corneal edema occurs for the endothelial failure of pomp due to imbalanced imbibition pressure . Sihota et al.  found that the average endothelial cell count was 2016 ± 306 cells/mm2 in patients with the acute attack lasting < 72 h and 759 ± 94 cells/mm2 in those with acute attack lasting > 72 h. This study suggested that compared with the fellow eyes, the polymorphism of corneal endothelial cells in AACC eyes was significantly higher, and the number of cells was significantly lower. In the AACC eyes, endothelial cells could not be analyzed in 17 eyes using specular microscopy due to corneal edema, while confocal microscopy allowed the observation of the morphology and number of endothelial cells in 13 of these 17 eyes. These results indicate that confocal microscopy has the advantages of completing the examination even in the presence of corneal edema, which is a frequent feature of AACC. Using confocal microscopy, the cells were uneven in size, had deposition of punctate high-reflective substances on the cell surface, and the cells were enlarged, deformed, and fused. Furthermore, 10 of the 13 eyes had an endothelial cell count < 1500 cells/mm2, with an average of 620 ± 325 cells/mm2.
After intravenous infusion of mannitol and oral methazolamide combined with carteolol hydrochloride, brinzolamide, brimonidine tartrate, and pilocarpine nitrate eye drops for IOP-lowering treatment, the average IOP of the 17 severe eyes was 37.2 mmHg, and the average duration of the ocular hypertension event was about 13 days. The results indicated that acute ocular hypertension could damage the corneal endothelial cells, and long-term acute ocular hypertension was critical for the decrease in the number of corneal endothelial cells and abnormal morphology. These results are supported by a rat model of acute ocular hypertension. Indeed, Li et al.  observed abnormal morphology and decreased the number of corneal endothelial cells, and these corneal injuries could be reversed after IOP was reduced. Although Verma et al.  demonstrated that the differences in the density of corneal endothelial cells at various phases of PACG were not statistically significant; thus, we speculated that an early rapid reduction of IOP could relieve the corneal injury. That will have to be confirmed in future studies.
Both specular microscopy and confocal microscopy can be used for evaluating the corneal endothelial state. Specular microscopy is easy to operate and has been widely used clinically to evaluate the functional status of the corneal endothelial cells, but it cannot be used when corneal edema is present. On the other hand, confocal microscopy can dynamically observe the corneal tissues in real-time, depicting the correlations among various layers of corneal tissues; it is minimally affected by corneal edema, and hence, has gained increasing clinical attention. Therefore, when routine specular microscopy is used for examining the decreased density of corneal endothelial cells, corneal lesions or endothelial cells cannot be analyzed due to corneal edema, and confocal microscopy should be performed for clinical diagnosis and treatment.
There are some limitations to this study. Firstly, the present study was a cross-sectional study, and cause-to-effect relationships cannot be determined. Secondly, no follow-up was performed for patients with corneal edema, and the corneal endothelial cells were not observed in time. Finally, the interval from acute angle-closure attack to initial visit differed in these patients, which have an influence on corneal morphology changes.