Identification and Analysis of Primary Cilia in the Corneal Endothelial Cells of Patients with Bullous Keratopathy

Abstract Purpose To identify primary cilia in human corneal endothelial cells (CECs) obtained from patients with bullous keratopathy (BK). Methods This study involved CEC specimens obtained from 10 eyes of 10 consecutive patients (three males and seven females; mean age: 74.5 years, range: 68–90 years) with BK who underwent Descemet’s stripping automated endothelial keratoplasty at Baptist Eye Institute, Kyoto, Japan between August 2019 and September 2020. Three corneal buttons obtained from 3 patients who underwent penetrating keratoplasty for keratoconus were used as 'non-BK' controls. All specimens were evaluated with immunofluorescence staining using an antibody against acetylated α-tubulin. Results Ciliary expression was observed in six of the 10 CEC specimens; i.e. in two specimens obtained from BK patients after glaucoma surgery (trabeculectomy), in two specimens obtained from patients with Fuchs endothelial corneal dystrophy, and in two specimens obtained from a patient with BK after laser iridotomy for primary angle closure. There was acetylated α-tubulin staining but no hair-like structures in two specimens, and ciliary expression was unknown in two specimens due to the absence of cells. The length of the primary cilia varied between all specimens. In contrast, no primary cilia were observed in the corneal buttons obtained from the three keratoconus patients. Conclusion The findings in this study clearly demonstrate the expression of primary cilia in the CECs of patients afflicted with BK.


Introduction
][5][6][7][8][9][10][11][12][13][14] Primary cilia form during the G0/G1 phase of the cell cycle, and disappear during mitosis.The basal body, which exists at the base of primary cilia, forms a centriole when mitosis begins.Hence, primary cilia and cell division are mutually exclusive and inextricably related. 4,5Primary cilia are also involved in the regulation of cell migration in interneurons at the cerebral cortex. 6In addition, primary cilia in renal tubular epithelial cells and vascular endothelial cells reportedly sense shear stress and control intrarenal or intravascular flow. 11,12orneal endothelium is comprised of a monolayer of hexagonal-shaped corneal endothelial cells (CECs), and plays a pivotal role in the maintenance of corneal transparency.Thus, any dysfunction or damage of CECs can result in bullous keratopathy (BK), a pathological condition that disturbs corneal transparency and results in a marked deterioration of visual acuity.Primary cilia have reportedly been identified in the CECs of mice, monkeys, and humans (Figure 1). 15,16In a previous study by Svedbergh et al. the authors' findings reported that few CECs located at the center of the human cornea have primary cilia, while CECs located at the periphery of the cornea do express primary cilia. 16Human CECs are mitotically inactive and are arrested at the G1 phase of the cell cycle in vivo, and the healing of corneal endothelial wounds primarily occurs via the migration and enlargement of existing CECs rather than by the proliferation of CECs to replace the dead cells. 17ince primary cilia are associated with cell migration and cell volume, 6,14 the expression of primary cilia should change in cases of cell dysfunction diseases such as BK.
The aim of this present study was to identify and characterize the primary cilia in Descemet's membrane/CEC specimens collected from patients afflicted with BK, with those specimens being analyzed with immunofluorescence staining using an antibody against acetylated a-tubulin.Acetylated a-tubulin is a major component of the primary cilia, 18 and it is commonly used to visualize them in immunofluorescence staining. 3,8,10,11,14,15,19We also investigated the relationship between patient age, cause of BK, central corneal thickness (CCT), eye drop usage, neovascularization, and the expression of primary cilia.

Materials and methods
This study was approved by the Kyoto Ethics Review Board (ERB), Kyoto, Japan (ERB Approval No. 1604) the ERB of Kyoto Prefectural University of Medicine, Kyoto, Japan (ERB Approval No. ERB-C-1006-1), and in accordance with the tenets set forth in the Declaration of Helsinki, written informed consent was obtained from all patients prior to their involvement in the study.

Descemet's membrane/CEC specimens
The Descemet's membrane/CEC specimens used in this study were obtained from 10 eyes of 10 consecutive patients (three males and seven females; mean age: 74.5 years, range: 68-90 years) who were diagnosed with BK and underwent Descemet's stripping automated endothelial keratoplasty (DSAEK) at Baptist Eye Institute, Kyoto, Japan between August 2019 and September 2020.Corneal buttons obtained from three male patients (age 32, 34, and 73 years) who had undergone penetrating keratoplasty (PKP) for severe keratoconus during the same time period were used as 'non-BK' controls.1][22] The Descemet's membrane was removed from the eye after being carefully folded, with the endothelial surface inward, immediately fixed in 4% paraformaldehyde for 15 min, and then transferred to a 2-ml microtube containing phosphate-buffered saline (PBS).

Results
In the 10 patients, the cause of BK was as follows: glaucoma-related BK after trabeculectomy (glaucoma with bleb) (n ¼ three patients), Fuchs endothelial corneal dystrophy (FECD) (n ¼ two patients), BK after laser iridotomy for primary angle closure (LI-BK) (n ¼ two patients), pseudoexfoliation keratopathy (n ¼ two patients), pseudophakic BK (n ¼ 1 patient).In all cases, the CECs could not be observed prior to surgery via specular microscopy due to corneal edema.The eye drop medications used prior to surgery are shown in the Table .The mean CCT was 643 ± 102 lm.Invasion of neovascularization into the periphery of the cornea was observed in Cases 4 and 8.
Immunofluorescence images showed that more than 50 CECs per field of view were observed in two specimens (Cases 1, 2, 3, and 5).However, CECs could not be identified in two specimens (Cases 9 and 10).Structures stained with acetylated a-tubulin were found in two specimens obtained from eyes with FECD (Cases 1 and 2), two specimens obtained from eyes with LI-BK (Case 3 and 4), and two specimens obtained from eyes with glaucoma with bleb (Cases 5 and 6) (Figure 2).In the specimens obtained from Cases 1 and 2, primary cilia were observed in many of the CECs.In the specimens obtained from Cases 2, 3, 4, and 5, the primary cilia observed in the CECs were relatively long.In the specimens obtained from Cases 1 and 6, there were a few CECs in which the staining showed the primary cilia to be very short.Nevertheless, staining showing a distinct cilia length was observable via the analysis of three-dimensional tilted images.Conversely, in the specimens obtained from   Cases 7 and 8, there were a few areas stained with a-tubulin, but they were not hair-like structures that could be considered primary cilia (Supplementary Figure 1).There was no relationship between the cause of BK, the preoperative eye drops used, CCT, neovascularization, and the expression of primary cilia (Table ).
Corneal buttons obtained from three patients with keratoconus and no corneal edema (i.e.Case 11, a 32-year-old male, Case 12, a 34-year-old male, and Case 13, a 73-yearold male) were used as controls.In all cases, no eye drop medications had been used prior to surgery.The area of the corneal buttons with Descemet's membrane and the preserved corneal endothelium structure were used.
Immunofluorescence analysis of the corneal button specimens obtained from the keratoconus cases (Cases 11, 12, and 13) showed more than 200 CECs per field of view when observed.However, no primary cilia expression was found in those three cases (Figure 3).

Discussion
To the best of our knowledge, this is the first study aimed at identifying and characterizing the primary cilia in the CECs of human eyes afflicted with BK, and our findings demonstrated that in six of the eight Descemet's membrane specimens with CECs obtained from patients with BK, primary cilia were observable in the CECs and the length of the primary cilia varied per specimen.On the other hand, we could not find any correlation between the cause of BK, the preoperative eye drops used, CCT, neovascularization, and the expression of primary cilia due to the small sample size.In contrast, no primary cilia were observed in the corneal button specimens obtained from the 3 keratoconus patients.
It should be noted that the specimens used in this study were obtained from only the central region of the cornea, the area where donor corneal grafts are transplanted during DSAEK surgery.Of interest, a previous study reported that in normal healthy human CECs, primary cilia are rarely found in the central region of the cornea, yet are found in the peripheral region. 16Thus, the expression of primary cilia observed in the BK cases may have been induced in the CECs in the central region of the cornea due to corneal endothelial dysfunction, and our findings seem to support that probability.
In our results, we observed the presence (þ/-) of primary cilia in 1 case of glaucoma with bleb and 1 case of PBK, thus suggesting a low expression of acetylated a-tubulin in those 2 cases.The length of primary cilia can change during the cell cycle, and primary cilia disappear during mitosis. 4,5Knockdown of a component protein of primary cilia also shortens primary cilia. 15Currently, we are unable to determine the reason for the presence (þ/-) of primary cilia in the 2 cases described above, so further analysis involving cell cycle staining and the expression of other proteins associated with primary cilia will hopefully help answer that question.CECs and renal tubular epithelial cells are morphologically and physiologically similar, as both are exposed to water flow at their apical side.In addition, they transport water and ions between basolateral and apical membranes. 13,23Recent studies suggest that renal tubular epithelial cells regulate ion and water transport via primary cilia. 13,24Thus, it is not surprising that the primary cilia of CECs function as sensors to receive extracellular changes.
Diseases characterized by dysfunction of primary cilia have recently been categorized as ciliopathies, with autosomal dominant polycystic kidney disease (ADPKD) being one of the ciliopathies most commonly observed. 25Interestingly, in a study by Kurtul et al. the authors reported that the CEC density (CECD) values were lower in ADPKD patients compared to those in healthy control subjects. 26Although that report only focused on the CECD decrease in ADPKD patients, and although the exact mechanism of the CECD change remains unknown, dysfunction of primary cilia may affect CECD.Further studies are needed to more fully elucidate the specific roles of primary cilia in CECs.

Limitations
This study did have limitations.First, invasion during specimen extraction, or the DSAEK procedure itself, may have affected the expression of primary cilia and/or the CECD.Second, there was no normal control group in this study.Since the expression of primary cilia is dynamically changed by temperature, 19 donor corneas harvested and stored at 4 � C for transportation post-donor death are not suitable as controls.In fact, there were no cilia in donor corneas stored at 4 � C. Therefore, corneal button specimens of keratoconus collected under the same conditions as that used for the Descemet's membrane and CEC specimens were used as the 'non-BK' controls.Third, the number of samples was limited, thus making it difficult to discuss the relationship between the cilia and the type of disease, so the inclusion of a greater number of cases in further studies will hopefully help resolve that issue.

Conclusion
The findings in this study clearly demonstrate the expression of primary cilia in the CECs of patients afflicted with BK, and help to further broaden the knowledge of the role of primary cilia in CECs.

Figure 1 .
Figure 1.Schematic model of primary cilia in corneal endothelial cells (CECs).Primary cilia protrude from the CEC membrane toward the anterior chamber, and are exposed to the aqueous humor.

Figure 2 .
Figure 2. Immunofluorescence staining images of CECs in which primary cilia were observed.The causes of bullous keratopathy (BK) were Fuchs endothelial corneal dystrophy (FECD) in Cases 1 and 2, BK post laser iridotomy for primary angle closure (LI-BK) in Cases 3 and 4, glaucoma-related BK post trabeculectomy in Cases 5, 6, and 7, and pseudophakic BK (PBK) in Case 8.Primary cilia were detected with acetylated a-tubulin (green) and nuclei were stained using DAPI (blue).Scale bars: 50 lm (left) and 10 lm (right).

Table .
Patient profiles and immunohistological analysis.