This case series analyzes the presence of guttae in patients with Fuchs endothelial corneal dystrophy after surgical removal and cultured corneal endothelial cell transplant to determine whether guttae can be removed without Descemet stripping.
Key Points
Question
Is there a decrease in guttae in patients with Fuchs endothelial corneal dystrophy (FECD) following surgical removal and cultured corneal endothelial cell (CEC) transplant therapy?
Findings
This case series revealed that postoperatively, there was no increase in guttae up to 3 years after cultured CEC transplant therapy, even when guttae were removed at the time of cultured CEC transplant therapy.
Meaning
Cultured CEC transplant therapy for Fuchs endothelial corneal dystrophy was associated with a decrease in guttae formation following removal of degenerated CECs and abnormal extracellular matrix during surgery.
Abstract
Importance
Whether guttae in Fuchs endothelial corneal dystrophy (FECD) can be removed by polishing without Descemet stripping and whether postoperative maintenance of reduced guttae can be achieved through cultured corneal endothelial cell (CEC) transplant therapy are critical issues to be addressed.
Objective
To investigate the decrease of guttae through polishing degenerated CECs and abnormal extracellular matrix (ECM) without Descemet stripping and to observe the behavior of guttae following cultured CEC transplant.
Design, Setting, and Participants
This case series prospective observational study was conducted in a hospital outpatient clinic setting. Between December 2013 and January 2019, 22 eyes with corneal endothelial failure caused by FECD received cultured CEC transplant therapy at Kyoto Prefectural University Hospital. Of these, 15 eyes were consistently monitored at the same central corneal area during the preoperative phase, as well as in the early (within 1 year) and late (after 3 years) postoperative phases. The images from these phases were categorized into 3 groups: typical guttae, atypical guttae, and no guttae.
Exposures
Cultured CEC transplant therapy.
Main Outcomes
Proportion of guttae in the observable area was measured, comparing the early and late postoperative phases for each group.
Results
The mean age of the patients at the time of surgery was 69 years (range, 49-79 years). All 15 eyes exhibited the presence of confluent guttae preoperatively (100%). Among these, 3 of 15 eyes belonged to male patients. The early postoperative phase of guttae morphologies was classified into 3 groups: 5 eyes with typical guttae, 7 with atypical guttae, and 3 with no guttae. The decrease in the number of these guttae was achieved by surgical procedures. The median percentage of guttae in the typical guttae, atypical guttae, and no guttae groups was 41.8%, 44.4%, and 16.2%, respectively, in the early phase, and 42.2%, 38.2%, and 18.8%, respectively, in the late phase.
Conclusions and Relevance
The findings demonstrate that in some cases of FECD, guttae can be removed by scraping and polishing abnormal ECM and degenerated CECs, while preserving the Descemet membrane. Furthermore, cultured CEC transplant resulted in no increase in guttae for up to 3 years, providing insights into surgically eliminating guttae.
Introduction
Corneal endothelial cells (CECs) are crucial for maintaining corneal transparency.1 This corneal endothelium continuously secretes the extracellular matrix (ECM) of Descemet membrane throughout an individual’s lifetime.2 This membrane acts as a basal lamina for the endothelial cells. Notably, its thickness is around 4 μm at birth and gradually increases to 10 to 15 μm by age 80 years.3
Fuchs endothelial corneal dystrophy (FECD) is characterized by the disintegration of CECs, marked by the formation of guttae and a decrease in endothelial cell density. In disintegrated CECs, endoplasmic reticulum stress induces unfolded protein responses, activating the intrinsic apoptotic pathway in these cells. Moreover, these disintegrated CECs often undergo an epithelial-mesenchymal transition, contributing to the accumulation of abnormal ECM in Descemet membrane.4,5 This accumulation leads to the membrane’s thickening. Subsequently, CECs tend to experience a functional decline, strained by the localized thickening of Descemet membrane due to ECM deposits.6
FECD is a common cause of corneal transplant in developed countries with predominantly White populations.7 Recently, endothelial keratoplasty, specifically Descemet stripping automated endothelial keratoplasty (DSAEK) and Descemet membrane endothelial keratoplasty (DMEK), has emerged as the preferred surgical treatment.8,9,10 Beyond these transplant procedures, a nontransplant approach known as Descemet stripping only (DSO)11,12,13 has shown effectiveness in treating mild corneal endothelial dysfunctions, especially those with significant corneal guttae at the center of the cornea. Additionally, pharmacological treatments, including Rho-associated protein kinase (ROCK) inhibitor eye drops, have been introduced as therapeutic options, though they have yet to be approved for this indication.14,15
In 2013, a corneal transplant technique involving cultured CEC transplant supplemented with ROCK inhibitor Y-27632 was first administered to patients. Long-term studies indicate that this approach has maintained favorable outcomes for at least 5 years postsurgery.16,17,18 This therapy differs from traditional endothelial transplants like DSAEK and DMEK. Notably, cultured CEC transplant therapy may control corneal edema without necessitating the complete removal of the Descemet membrane, which serves as the basal lamina for CECs. According to findings by Kocaba et al,19 abnormal ECM deposition of FECD creates a hostile environment for CECs. Additionally, guttae exceeding 30 μm in diameter have been found to induce apoptosis in these cells.19
Whether guttae in FECD can be removed by polishing without Descemet stripping and whether postoperative maintenance of reduced guttae can be achieved through cultured CEC transplant therapy are critical issues to be addressed. Here, we investigated the association of cultured CEC transplant therapy with postoperative guttae behavior in patients with FECD. The study aimed to compare images of the same corneal regions, captured preoperatively and at early and late postoperative periods, using slit-scanning widefield contact specular microscopy.
Methods
This case series follows reporting guidelines for uncontrolled case series.20 The surgical protocol and amendments of this clinical research and trial were approved by the institutional review board (IRB) at Kyoto Prefectural University of Medicine (KPUM) and by the Special Committee of the Japanese Ministry of Health, Labor, and Welfare (see the detailed descriptions in eMethods 1 in Supplement 1). The prospective observational study protocol was separately approved by the IRB of KPUM (ERB-C-1376, ERB-C-1838) (UMIN000036422) and was conducted according to the principles of the Helsinki declaration. Before they participated in the study, written informed consent was obtained from all patients without incentives.
Patients
From December 2013 to January 2019, cultured CEC transplant therapy was performed on 65 eyes with corneal endothelial failure at KPUM Hospital. Of these, 22 eyes that had treatment for FECD were selected for further analysis. Panoramic images of CECs were generated from video footage for eyes with imagery available at preoperative and both early phase (within 1 year) and late phase (after 3 years) postsurgery, using the slit-scanning widefield contact specular microscope (CellChek; Konan Medical). However, 2 eyes were excluded because follow-up occurred at a different institute, the National Center for Geriatrics and Gerontology, Obu City. Additionally, 4 eyes were excluded because panoramic images could not be created or the same regions identified due to constant eye movement by aging behavior. In 1 eye, the Descemet membrane was partially ruptured during mechanical scraping, necessitating an additional 5-mm diameter DSO (eFigure 1 in Supplement 1). Consequently, 7 eyes were excluded, and consistent identification of the same CEC region was possible in 15 eyes. The flowchart diagram of the study is shown in eFigure 2 in Supplement 1. The red reflex examination to view the presence of guttae was not performed.
Culture of Human CECs, Surgical Procedure, and Postoperative Care
The methods for culturing human CECs from young donor corneas, their surgical application, and postoperative care were consistent with those previously described.16 A few hours before the surgery, a cell suspension was prepared using cells harvested from the culture flask. This suspension was then transferred into a modified Opti-MEM I reduced serum medium (Thermo Fisher Scientific) supplemented with ROCK inhibitor Y-27632. During surgery, abnormal ECM and/or degenerated CECs on the host Descemet membrane were mechanically removed from the 8-mm diameter central cornea using a silicon cannula needle under irrigation solution to maintain the anterior chamber. The removal was confirmed by VisionBlue staining (eFigure 3 in Supplement 1 and the Video). A 300-μL suspension of cultured CECs (1 × 106 cells for 11 patients, 0.5 × 106 cells for 3 patients, and 0.2 × 106 cells for 1 patient) was injected into the anterior chamber. To promote the adhesion and engraftment of the injected cells, patients were placed in a prone position for 3 hours. Postoperatively, all patients received corticosteroid administration to suppress the innate and acquired immune response previously reported elsewhere.18
Video. Surgical Polishing of the Descemet Membrane Before Transplant of Cultured CECs.
In this video, abnormal extracellular matrix and/or degenerated corneal endothelial cells (CECs) on the host Descemet membrane are mechanically polished and removed from the 8-mm diameter of the central cornea using a silicon cannula needle under an irrigation solution to maintain the anterior chamber. The removal of the abnormal extracellular matrix and/or degenerated CECs was confirmed by VisionBlue staining. After completing this procedure, transplant of cultured CECs was performed.
Classification of Guttae Morphology
The 15 selected eyes were categorized into 3 groups by 3 corneal specialists (Y.T., C.S., S.K.) based on guttae appearance. Typical guttae were characterized by a pronounced elevation, rendering CECs above them invisible. Atypical guttae were identified when cells on the upper portion of the guttae remained visible. No guttae was designated for eyes where the guttae themselves were very few (Figure 1 and eFigures 4 and 5 in Supplement 1).
Figure 1. Representative Cases for Each Group.
This figure shows images captured before and after surgery using slitlamp microscopy and a slit-scanning widefield contact specular microscope in each inset. These images are associated with eyes undergoing cultured corneal endothelial cell transplant therapy. A, An eye from the typical guttae group presurgery, showing massive guttae. B, An eye from the atypical guttae group presurgery, showing massive guttae. C, An eye from the typical guttae group 3 years postsurgery, showing a decreased yet still prominent amount of guttae. D, An eye from the atypical guttae group 4 years postsurgery, showing the disappearance of most guttae on the Descemet membrane but not within the Descemet membrane (scale bar = 100 μm).
CEC Image Analysis From the Same Region at Different Times
Using a slit-scanning widefield contact specular microscope, panoramic images were obtained for preoperative and both the early postoperative phase (within 1 year) and late postoperative phase (after 3 years). Multiple guttae served as reference points to analyze corresponding central regions in both phases (eFigure 6 in Supplement 1), although massive preoperative guttae complicated region identification in subsequent images. Thus, the central corneal preoperative image was considered very close but not identical to these regions.
Image analysis using Fiji (ImageJ)21 with a custom macro extracted guttae and dark banded regions, indicative of extensive ECM. These darker areas were identified using the MorphoLibJ library’s extend minima22 (eMethods 2 in Supplement 1). Ophthalmologists then reviewed the set of dark regions to eliminate unsuitable areas, such as broad shadows that signify wrinkles on the corneal endothelial tissue, isolating abnormal-like regions.
Calculation of Spring Constant K, Guttae Area, CEC Density
Healthy human CECs maintain a high level of collective order. Therefore, using the previously reported method23 to quantify the collective order of the CECs, we analyzed the spring constant K in both the early and late phases. Since the spring constant K represents the sharpness of the spatial confinement of the cells, a higher value of K implies a more ordered CEC structure (eMethods 3 in Supplement 1). Using the original images and extracted abnormal-like regions via mathematical morphology methods, we measured and calculated the ratio of guttae area to the total area, the density of CECs in the observable region, and the spring constant K of CECs in the same area during both early and late phases.
Statistical Analysis
Statistical analyses were performed using Prism version 10.0.2 statistical analysis software (GraphPad Software). The analysis of guttae area, density of CECs, and spring constant K for the early and late phases of each group was performed using the Wilcoxon signed rank test. The CEC densities between groups were compared with Wilcoxon rank sum tests.
Results
Patients
All 15 eyes showed typical guttae across the entire central cornea before the surgery. The postoperative guttae morphologies in 15 eyes were classified into 3 groups: 5 eyes with typical guttae, 7 with atypical guttae, and 3 with no guttae. The mean age of the patients at the time of surgery was 69 years, with a range of 49 to 79 years. Among these, 3 of 15 eyes belonged to male patients. Patient characteristics and relevant clinical data, including visual acuity from both early and late phases, are summarized in the Table.
Table. Patient Characteristics and Clinical Summary Before and After Cultured CEC Transplant Therapya.
| Age, y/sexb | Eye | Dose, ×106 cells | Guttae condition | CEC density, cells/mm2 | CCT, μm | BCVA | Year of phase | No. | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Prephase | Early phase | Late phase | Early | Late | ||||||||||||||||
| Prephase | Early phase | Late phase | Prephase | Early phase | Late phase | Prephase | Early phase | Late phase | Decimal (logMAR) | Approximate Snellen equivalent at 20 ft | Decimal (logMAR) | Approximate Snellen equivalent at 20 ft | Decimal (logMAR) | Approximate Snellen equivalent at 20 ft | ||||||
| 72/F | L | 1.0 | Typical | Typical | Typical | <DL | 2822 | 1255 | 750 | 626 | 532 | 0.3 (0.52) | 20/70 | 0.3 (0.52) | 20/70 | 0.5 (0.3) | 20/40 | 0.5 | 3 | 7 |
| 57/F | L | 1.0 | Typical | Typical | Typical | <DL | 2591 | 2009 | 657 | 490 | 510 | 0.2 (0.7) | 20/100 | 0.5 (0.3)c | 20/40 | 0.7 (0.15)c | 20/30 | 1 | 5 | 8 |
| 71/F | R | 0.5 | Typical | Typical | Typical | <DL | 2199 | 1639 | 655 | 596 | 587 | 0.3 (0.52) | 20/70 | 0.8 (0.1) | 20/25 | 0.9 (0.05) | 20/22 | 1 | 3 | 22 |
| 67/F | R | 1.0 | Typical | Typical | Typical | <DL | 2008 | 1941 | 714 | 620 | 619 | 0.3 (0.52) | 20/70 | 0.3 (0.52)d | 20/70 | 0.4 (0.4)d | 20/50 | 1 | 4 | 25 |
| 76/M | R | 1.0 | Typical | Typical | Typical | <DL | 4386 | 2822 | 690 | 542 | 565 | 0.2 (0.7) | 20/100 | 0.7 (0.15)e | 20/30 | 0.5 (0.3)e | 20/40 | 0.5 | 3 | 54 |
| 58/M | R | 1.0 | Typical | Atypical | Atypical | <DL | 2146 | 1984 | 727 | 540 | 536 | 0.2 (0.7) | 20/100 | 0.7 (0.15) | 20/30 | 1.2 (−0.08) | 20/17 | 0.5 | 3 | 3 |
| 56/F | L | 1.0 | Typical | Atypical | Atypical | <DL | 2331 | 661 | 725 | 561 | 618 | 0.03 (1.52) | 20/630 | 0.9 (0.05) | 20/22 | 0.8 (0.1) | 20/25 | 0.5 | 4 | 11 |
| 68/F | L | 1.0 | Typical | Atypical | Atypical | <DL | 3118 | 1776 | 681 | 556 | 554 | 0.4 (0.4) | 20/50 | 0.8 (0.1) | 20/25 | 1.0 (0) | 20/20 | 1 | 6 | 12 |
| 59/F | R | 0.5 | Typical | Atypical | Atypical | <DL | 2818 | 2512 | 655 | 540 | 549 | 0.3 (0.52) | 20/70 | 1.0 (0) | 20/20 | 1.0 (0) | 20/20 | 0.5 | 5 | 36 |
| 71/M | R | 0.2 | Typical | Atypical | Atypical | <DL | 3441 | 1303 | 703 | 570 | 561 | 0.4 (0.4) | 20/50 | 0.9 (0.05) | 20/22 | 0.8 (0.1) | 20/25 | 0.5 | 5 | 40 |
| 69/F | R | 1.0 | Typical | Atypical | Atypical | <DL | 4496 | 3449 | 719 | 602 | 578 | 0.3 (0.52) | 20/70 | 1.2 (−0.08) | 20/17 | 1.5 (−0.18) | 20/13 | 0.5 | 4 | 55 |
| 77/F | L | 1.0 | Typical | Atypical | Atypical | <DL | 4507 | 4329 | 700 | 620 | 599 | 0.4 (0.4) | 20/50 | 0.9 (0.05) | 20/22 | 0.7 (0.15) | 20/30 | 0.5 | 3 | 58 |
| 79/F | L | 0.5 | Typical | No | No | <DL | 3561 | 2898 | 677 | 665 | 610 | 0.4 (0.4) | 20/50 | 0.4 (0.4)f | 20/50 | 0.5 (0.3)f | 20/40 | 0.5 | 4 | 44 |
| 63/F | L | 1.0 | Typical | No | No | <DL | 4748 | 3818 | 657 | 545 | 541 | 0.15 (0.82) | 20/125 | 0.7 (0.15)f | 20/30 | 1.0 (0) | 20/20 | 0.5 | 4 | 57 |
| 76/F | L | 1.0 | Typical | No | No | <DL | 5364 | 4621 | 798 | 558 | 563 | 0.2 (0.7) | 20/100 | 1.2 (−0.08) | 20/17 | 1.2 (−0.08) | 20/17 | 0.5 | 3 | 60 |
Abbreviations: BCVA, best-corrected visual acuity; CEC, corneal endothelial cells; CCT, central corneal thickness; DL, detection limit of the instrument; F, female; L, left; M, male; R, right.
Sex was obtained via patient self-report.
Early phase was defined as within 1 year postsurgery; late phase, after 3 years postsurgery. Prephase refers to before the surgery.
Glaucoma.
Anterior capsular contraction.
Epiretinal membrane.
Posterior capsular opacification.
Alteration in Guttae Morphology After Cultured CEC Transplant Therapy
For both preoperative and early phases, representative images of typical, atypical, and no guttae are presented in Figure 1 and eFigure 4 in Supplement 1. All groups exhibited the presence of confluent guttae before surgery. The decrease in the number of these guttae was achieved by mechanically removing either the abnormal ECM and/or degenerated CECs from the patient’s central corneal Descemet membrane using a silicone needle. This procedure enhanced the visibility of the landmark CECs in the postoperative images.
Guttae Morphology in the Peripheral Region After Cultured CEC Transplant Therapy
In 1 postoperative peripheral panorama image, there was a demarcation between the area retaining the host’s guttae and the region where guttae on the Descemet membrane have been surgically scraped and replaced with injected CECs. This finding showed the association of surgical removal of abnormal ECM from the Descemet membrane surface with subsequent guttae morphology (Figure 2).
Figure 2. Panoramic Images From Central to Peripheral Regions Captured by Contact Specular Microscopy.
The panoramic images of the posterior surface after corneal endothelial cell transplant therapy were created from the video obtained from the eye with atypical guttae 4 years after surgery. The arrowheads indicate the boundary between the host region and the surgically removed area (scale bar = 100 μm).
Time-Dependent Alterations in Guttae
Preoperatively, all 15 eyes either exhibited a complete presence (100%) of guttae or could not be examined because of corneal edema. However, prior observations indicated a total presence of guttae in that area. In the early phase, the median percentages of guttae in typical guttae, atypical guttae, and no guttae were 41.8%, 44.4%, and 16.2%, respectively. In the late phase, within the same areas as the early phase, these percentages were 42.2%, 38.2%, and 18.8%, respectively (Figure 3 and eFigure 7 in Supplement 1). The mean changes in percentage of guttae from the early phase to the late phase in typical guttae, atypical guttae, and no guttae were −0.57% (95% CI, −9.58% to 8.36%), −2.43% (95% CI, −8.27% to 3.40%), and 0.23% (95% CI, −5.58% to 6.04%), respectively (Figure 4A-C).
Figure 3. Representative Images for Each Group in the Early Phase and Late Phase.
Representative images for each group in the early phase and late phase are displayed. A, Image from the early phase 1 year after surgery in the typical guttae group. B, Image from the early phase 6 months after surgery in the atypical guttae group. C, Image from the early phase 1 year after surgery in the no guttae group. D, Image from the late phase 3 years postsurgery in the typical guttae group. E, Image from the late phase 4 years after surgery in the atypical guttae group. F, Image from the late phase 3 years after surgery in the no guttae group (scale bar = 100 μm).
Figure 4. Percentage of Guttae Area and Corneal Endothelial Cell (CEC) Density for Each Group.
A-C, Preoperative, early-phase postoperative, and late-phase postoperative guttae areas are calculated by determining the guttae area as a percentage of the total image area in the same region for each group. D-F, CEC density is measured in the same region during both the early and late phases using the center method.
CEC Density in the Same Region During Early and Late Postoperative Phases
Preoperatively, the CEC densities in all eyes were below the detection limit of the instrument, rendering them undetectable. In the early phase, the median CEC densities for typical guttae, atypical guttae, and no guttae were 2591 cells/mm2, 3118 cells/mm2, and 4748 cells/mm2, respectively. In the late phase, these densities were 1941 cells/mm2, 1984 cells/mm2, and 3818 cells/mm2, respectively. The no guttae group exhibited the highest CEC density in both early and late phases (Figure 4D-F). The mean change of CEC densities from the early phase to the late phase in typical guttae, atypical guttae, and no guttae were −868.0 cells/mm2 (95% CI, −1698.9 to 37.1 cells/mm2), −977.6 cells/mm2 (95% CI, −1705.7 to −249.4 cells/mm2), and −778.67 cells/mm2 (95% CI, −1119.1 to 438.3 cells/mm2), respectively.
In the late phase, there was a difference in CEC density between typical guttae and no guttae, while no differences were observed either between typical guttae and atypical guttae or between atypical guttae and no guttae. Additionally, in the early phase, the groups did not exhibit any differences in CEC density.
Spring Constant K of CECs in Early and Late Postoperative Phases
In the early phase, the median spring constants K for typical guttae, atypical guttae, and no guttae were 30.7 × 10−3/μm2, 22.7 × 10−3/μm2, and 73.4 × 10−3/μm2, respectively. During the late phase, these values altered to 19.9 × 10−3/μm2, 16.8 × 10−3/μm2, and 28.7 × 10−3/μm2, respectively. Statistical analysis revealed no difference between the early and late phases across all groups. The no guttae group appeared to have a higher mean spring constant K compared with the other 2 groups (eFigure 8 in Supplement 1).
Discussion
The surgical procedure in this study involved removing abnormal ECM and degenerated CECs while preserving Descemet membrane in eyes with extensive guttae with corneal edema, before cultured CEC transplant therapy. This approach resulted in varying postoperative appearance of guttae, as observed through contact specular microscopy, and led to the categorization into 3 distinct groups. Notably, a no guttae group emerged, characterized by the disappearance of guttae. This study also revealed no increase in guttae following cultured CEC transplant therapy from early postoperative phase to at least 3 years postoperatively.
FECD is commonly associated with the accumulation of ECM by disintegrated CECs on the Descemet membrane. This leads to thickening of the membrane and the formation of guttae.4,5 Our study reveals that the postoperative outcomes of FECD, after cultured CEC transplant therapy, can be classified into 3 distinct categories. These variations in clinical observation may correspond to histopathological differences. Waring et al24 had earlier delineated 4 distinct patterns in FECD through both clinical and histopathological observations, one of which included “simple warts (guttae) protruding into the anterior chamber.” Theoretically, if guttae project toward the anterior chamber and abnormal ECM is present on the surface of the Descemet membrane, mechanical removal without stripping the membrane could be effective. It appears that the “guttae protruding into the anterior chamber” type, as characterized by Waring et al,24 aligns with the no guttae category observed postoperatively in our study.
In the typical guttae category group, guttae remained prominent post-surgery, while the atypical guttae category group was characterized by a preoperative thickening of the ECM. These findings align with the description by Waring et al24 of “warts (guttae) embedded within multilaminar tissue.” During our surgical procedures, the Descemet membrane was conserved, and only abnormal ECM and the degenerated CECs were mechanically removed. Consequently, any ECM embedded within the Descemet membrane was left intact, likely accounting for the thickening of ECM with moderate amount of guttae observed in the atypical guttae group. The difference between atypical and typical guttae may stem from varying degrees of ECM thickening in the Descemet membrane before the surgery.
In our study, the contact specular microscope consistently identified the same regions in both the early and late phases across 15 eyes. However, imaging the peripheral corneal regions was sometimes challenging, resulting in fewer cases where the same peripheral area could be consistently identified. Despite these challenges, we did capture clear images of the periphery in some instances. These peripheral observations revealed areas where the host’s guttae persisted, in contrast to areas where guttae had been mechanically removed and where injected CECs had successfully adhered. The images of these peripheral region highlight that abnormal ECM on the surface of Descemet membrane can be effectively removed through surgery. However, the uneven thickness of Descemet membrane due to abnormal ECM accumulation inside cannot be effectively corrected surgically.
We were able to evaluate the temporal changes in the injected, mature-differentiated CECs and guttae by analyzing the same CEC regions in both the early and late phases. Across all 3 groups in our study, there was no change observed in the proportion of guttae formation during postoperative period, including the very early phase (eFigure 9 in Supplement 1). This observation suggests that CECs, once reorganized on the host Descemet membrane through cultured CEC transplant therapy, do not induce adverse effects such as the formation of abnormal ECM including guttae. Indeed, they appear to rejuvenate the microenvironment of the posterior surface of the cornea. In 1 case excluded from the study, DSO was performed followed by the injection of CECs. In this instance, no guttae or ECM were observed in the CEC images, and notably, there was no recurrence of guttae even 4 years after the operation (eFigure 1 in Supplement 1). This contrasts with findings from Kaufman et al,25 which reported the recurrence of guttae following DSO. A combination treatment for early-phase FECD involving the removal of regional disintegrated CECs with guttae through transcorneal freezing, along with the topical application of Y-27632, has been effective in maintaining corneal transparency for an extended period. This effectiveness is likely due to the migration of relatively healthy endothelium in the periphery.26,27 Thus, the increase or decrease of cornea guttae postoperative probably depends on the biological characteristics of the CECs that cover the CEC defect region after surgery.28
Differences in CEC density were noted between the typical guttae and no guttae groups in the late phase. The adhesion capacity of cells to the ECM and changes in their biological characteristics are influenced by the maturity level of the injected cultured human CECs.29,30 Cultured human CECs characterized as negative for CD24, CD26, CD44, CD105, CD133, and positive for CD166 demonstrate a metabolic tendency toward mitochondrial-dependent oxidative phosphorylation.31,32,33,34,35 Injecting these mature CECs into the anterior chamber is believed to facilitate their adhesion to thickened Descemet membrane and guttae. Furthermore, the study by Rizwan et al36 highlights that dense guttae, particularly those exceeding 20 μm in height, can adversely affect cell adhesion and the formation of a cellular monolayer. Hence, the localized thickening of Descemet membrane, as observed in typical guttae, may impede the adhesion of these cultured human CECs to some extent.
Limitations
Our study has several limitations that warrant acknowledgment. First, it was conducted with a relatively small group of patients. Thus, the results are suggestive but not conclusive, and a repetition study may be necessary. Second, the evaluation of guttae depended on images captured using a slit-scanning contact specular microscope. This method may involve manual focus adjustments, potentially affecting the contrast and clarity of guttae images, especially preoperative image of guttae. The results presented here are the best we have at this moment. Furthermore, it should be explored whether this level of guttae postoperative is visually relevant regarding glare and reduced contrast sensitivity.
Conclusions
This study demonstrated that in some cases of FECD, removal of guttae can be achieved by scraping off abnormal ECM and the degenerated CECs while preserving Descemet membrane. Furthermore, the study indicates that injecting mature-differentiated CECs into the anterior chamber can be associated with the inhibition of new guttae formation for at least 3 years postsurgery. These findings suggest that the injection of well-differentiated cultured CECs can bring about beneficial changes in the anterior chamber microenvironment, thereby presenting a promising therapeutic strategy for FECD.
eMethods 1. Detailed descriptions of the protocol approval
eMethods 2. CEC Image analysis for extracting guttae and darker regions
eMethods 3. Calculation of Spring constant K
eReferences
eFigure 1. Patient who underwent additional Descemet Stripping Only (DSO)
eFigure 2. Flow chart diagram of this study
eFigure 3. Surgical procedure of polishing abnormal ECM and degenerated CECs
eFigure 4. Detailed representative cases for each group
eFigure 5. CEC Images obtained from pre and postoperative CEC-Transplantation
eFigure 6. CEC Images obtained from the same region at different time points
eFigure 7. Extracted abnormal regions for each group in the early phase and late phase eFigure 8. Spring constant K for each group
eFigure 9. CEC Images of No guttae in the early post-operative phase Search strategy for Medline (using PubMed)
Data sharing statement
References
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
eMethods 1. Detailed descriptions of the protocol approval
eMethods 2. CEC Image analysis for extracting guttae and darker regions
eMethods 3. Calculation of Spring constant K
eReferences
eFigure 1. Patient who underwent additional Descemet Stripping Only (DSO)
eFigure 2. Flow chart diagram of this study
eFigure 3. Surgical procedure of polishing abnormal ECM and degenerated CECs
eFigure 4. Detailed representative cases for each group
eFigure 5. CEC Images obtained from pre and postoperative CEC-Transplantation
eFigure 6. CEC Images obtained from the same region at different time points
eFigure 7. Extracted abnormal regions for each group in the early phase and late phase eFigure 8. Spring constant K for each group
eFigure 9. CEC Images of No guttae in the early post-operative phase Search strategy for Medline (using PubMed)
Data sharing statement
