Corneal Buttons Research Articles

Autologous Contralateral and Ipsilateral Rotational Penetrating Keratoplasty - A Case Series and Mini-Review.

Corneal stromal opacities can severely impact visual acuity if they are located in the visual axis. Homologous penetrating keratoplasty (HPK) is usually the preferred surgical option in such clinical circumstances. However, autologous penetrating keratoplasty (APK) could be an immunologically safer alternative. The purpose of this study was to report the surgical technique, indications, and (dis)advantages of ipsilateral rotational and contralateral APK, as well as pre- and postoperative clinical findings of four patients who underwent contralateral APK. In ipsilateral rotational APK, eccentric trephination places the central corneal opacity at the excision edge, whereafter the corneal button is rotated to clear the visual axis. Contralateral APK is suitable for more specific clinical situations with corneal opacity in a functionally much better eye. Clear cornea of the (almost) blind eye suffering from a noncorneal pathology is transplanted to the potentially better seeing eye suffering from a corneal pathology, followed by HPK in the (almost) blind donor eye. After 18 months, potentially better-seeing eyes improved from logMAR 1.3 to 0.6 in visual acuity, with most HPK-treated weaker eyes matching preoperative levels. Considering ipsilateral rotational and contralateral APK before moving on straight towards HPK is crucial in select cases. Autologous contralateral keratoplasty should be preferred for patients with (1) corneal scars in a potentially better seeing eye and (2) a clear cornea in an (almost) blind eye, especially in cases of high risk for graft rejection.

Microsporidial stromal keratitis: treatment outcomes, clinical manifestations, confocal microscopy and histopathology findings: a retrospective observational study

ObjectiveMicrosporidial stromal keratitis (MSK) is an uncommon disease. Only several case series have been reported. We aimed to describe the clinical manifestations, histopathology and treatment outcomes of MSK.Methods and analysisRetrospective...

Novel treatment of infectious keratitis in canine corneas using ultraviolet C (UV-C) light.

To investigate the therapeutic effect of 275 nm wavelength ultraviolet C (UV-C) light for treatment of bacterial keratitis in canine corneas using an affordable, broadly available modified handheld device. UV-C therapy (UVCT) was evaluated in two experiments: invitro using triplicates of three bacterial genera (Staphylococcus, Streptococcus, Pseudomonas spp., and a mix of all species) where the UVCT was performed at a distance of 10, 15, and 20 mm with 1 or 2 doses (4 h apart) for 5, 15, or 30 s; exvivo model where healthy canine corneal buttons were inoculated superficially and deep (330 μm) with the same bacterial isolates and treated at a 10 mm distance for 15 s with one dose of 22.5 mJ/cm2. Fluorescent marker (STYO9-PI) was used to label (green = live bacteria, red = dead bacteria), and confocal microscopy was used to image the bacteria. In vitro results showed all plates treated with UVCT had 100% bactericidal effect for all isolates with single dose of 15 s at 10 mm distance or two doses, 4 h apart at 15 mm and was ineffective with single dose at 15-20 mm. The exvivo results confirmed a significant decrease in bacterial load for all isolates on samples inoculated superficially but were inconclusive for intrastromal ones. UVCT confirmed the therapeutic potential for all tested isolates, for both invitro and exvivo experiments using a single exposure of 15 s. While safety studies are underway, clinical trials are warranted.

Fuchs endothelial corneal dystrophy: aspects of pathogenesis

Fuchs endothelial corneal dystrophy (FECD) – multigenic bilateral disorder, characterized by dysfunction of corneal endothelium cells (CECs) that eventually results in loss of transparency. Purpose: To evaluate the pathogenesis of FECD with histological and immunohistochemical methods, immunofluorescence, scanning electron microscopy (SEM). Methods: Endothelium–Descemet membrane (EDM) complexes, corneal buttons were obtained from 76 patients (85 eyes) with FECD during keratoplasty and divided into 1A, 2A, 3A groups, 15 EDMs (donor tisuue) – 1B, 2B, 3B groups (control). Morphological (hematoxylin/eosin staining) and immunohistochemical (primary antibodies to cytokeratin AE1/AE3 and vimentin) studies (1A, 1B groups), phase-contrast microscopy and immunofluorescence analysis of tight junction protein ZO-1 (2A, 2B groups), SEM (3A, 3B groups) were performed. Results: FECD is characterized by decline of CECs, cell and nuclear enlargement. Nuclear-cytoplasmic ratio of CECs is relative to control. CECs expression ZO-1 is decreased in the area of guttae ingrowth. Coexpression of cytokeratin AE1/AE3 and vimentin is found. Morphological and immunohistochemical studies show DM’ thickening, stromal and epithelial edema and local fibrosis, vimentin (stromal cells) and cytokeratin AE1/AE3 (epithelium) expression in FECD. Conclusion: Pathogenesis of FECD include CECs’ epithelial-mesenchymal transition, followed by guttae formation – the excrescences, which destroy cytosketon and lead to progressive loss of CECs. Changes in permeability of endothelium cause edema and fibrosis of stroma and epithelial layer.

Temporary Pupil Scaffolding Technique for Managing Positive Vitreous Pressure and Impending Intraocular Lens and Vitreous Extrusion During Penetrating Keratoplasty.

Positive vitreous pressure (PVP)secondary to intraoperative acute hypotony during penetrating keratoplasty can result in extrusion of the intraocular lens and vitreous. Currently described techniques are difficult or impossible to apply intraoperatively when positive vitreous pressure is noticed in an"open sky"situation after excision of the host corneal button. We describe a technique where pupil scaffolding is used to prevent intraocular lens or crystalline lens extrusion by simply closing the pupil with a temporary suture that holds the retropupillary contents back. Once the eye is thus stabilized, the donor cornea is sutured rapidly after which the pupillary knot is cut and removed using microscissors and microforceps. Five patients undergoing penetrating keratoplasty under peribulbar anesthesia underwent this technique after experiencing PVP after host corneal button excision. PVP was successfully controlled in all 5 patients, and the optical grafts remained clear in the postoperative period. Temporary pupillary scaffolding can help control PVP and prevent ocular content extrusion during PKP surgeries.

Investigation of mechanical strength and structure of corneal graft-host junction

Dehiscence is a common complication of corneal transplant surgery involving separating the graft from the host eye. The present article aims to investigate fundamental insights into the mechanical and structural aspects of the graft-host junction (GHJ) of a graft that survived in a patient for 13 years after penetrating keratoplasty (PK). Additionally, it adopts the sutur retention strength (SRS) test procedure defined in ISO:7198-2016 and aims to provide a comprehensive test protocol to study the biomechanics of the GHJ in extracted PK buttons. A 9 mm corneal button with GHJ was extracted from a 46-year-old patient who underwent PK 13 years back. The strength of the GHJ was quantified using the SRS test. Corresponding control results were obtained from the SRS tests of a corneoscleral button with no history of any refractive procedure. Birefringence, histological, and scanning electron microscopy (SEM) imaging were used to visualize the microstructural details of the GHJ. The strength of the GHJ was observed to be ten times lower than the native cornea. Histopathological features, such as fragmented Bowman's layer, and fibrosis with a clear demarcation line between host and graft tissue, were observed at the GHJ, suggesting a weak bond across the GHJ. The low strength of the GHJ in PK indicates the high susceptibility of the GHJ towards wound dehiscence.

Evidence for persistent UV-induced DNA damage and altered DNA damage response in xeroderma pigmentosa patient corneas

Xeroderma pigmentosum (XP) is a rare genetic disorder characterized by injury to the ocular surface due to exposure to ultraviolet (UV) radiation. UV-induced damage in the cells leads to the formation of cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts that are repaired by the NER (Nucleotide Excision Repair) pathway. Mutations in the genes coding for NER proteins, as reported in XP patients, would lead to sub-optimal damage repair resulting in clinical signs varying from photo-keratitis to cancerous lesions on the ocular surface. Here, we aimed to provide evidence for the accumulation of DNA damage and activation of DNA repair pathway proteins in the corneal cells of patients with XP. Corneal buttons of patients who underwent penetrating keratoplasty were stained to quantify DNA damage and the presence of activated DNA damage response proteins (DDR) using specific antibodies. Positive staining for pH2A.X and thymidine dimers confirmed the presence of DNA damage in the corneal cells. Positive cells were found in both control corneas and XP samples however, unlike normal tissues, positive cells were found in all cell layers of XP samples indicating that these cells were sensitive to very low levels of UV. pH2A.X-positive cells were significantly more in XP corneas (p < 0.05) indicating the presence of double strand breaks in these tissues. A positive expression of phosphorylated-forms of DDR proteins was noted in XP corneas (unlike controls) such as ataxia telangiectasia mutated/Rad-3 related proteins (ATM/ATR), breast cancer-1 and checkpoint kinases-1 and -2. Nuclear localization of XPA was noted in XP samples which co-localized (calculated using Pearson's correlation) with pATM (0.9 ± 0.007) and pATR (0.6 ± 0.053). The increased presence of these in the nucleus confirms that unresolved DNA damage was accumulating in these cells thereby leading to prolonged activation of the damage response proteins. An increase in pp53 and TUNEL positive cells in the XP corneas indicated cell death likely driven by the p53 pathway. For comparison, cultured normal corneal epithelial cells were exposed to UV-radiation and stained for DDR proteins at 3, 6 and 24 h after irradiation to quantify the time taken by cells with intact DDR pathway to repair damage. These cells, when exposed to UV showed nuclear translocation of DDR proteins at 3 and 6 h which reduced significantly by 24 h confirming that the damaged DNA was being actively repaired leading to cell survival. The persistent presence of the DDR proteins in XP corneas indicates that damage is being actively recognized and DNA replication is stalled, thereby causing accumulation of damaged DNA leading to cell death, which would explain the cancer incidence and cell loss reported in these patients.

An in vitro investigation into the impact of corneal rinsing on riboflavin/UVA corneal cross-linking

BackgroundCorneal cross-linking (CXL) using riboflavin and ultraviolet-A light (UVA) is a treatment used to prevent progression of keratoconus. This ex vivo study assesses the impact on CXL effectiveness, as measured by tissue enzymatic resistance and confocal microscopy, of including a pre-UVA corneal surface rinse with balanced salt solution (BSS) as part of the epithelium-off treatment protocol.MethodsSixty-eight porcine eyes, after epithelial debridement, were assigned to six groups in three experimental runs. Group 1 remained untreated. Groups 2–6 received a 16-min application of 0.1% riboflavin/Hydroxypropyl methylcellulose (HPMC) drops, after which Group 3 was exposed to 9 mW/cm2 UVA for 10 min, and Groups 4–6 underwent corneal surface rinsing with 0.25 mL, 1 mL or 10 mL BSS followed by 9 mW/cm2 UVA exposure for 10 min. Central corneal thickness (CCT) was recorded at each stage. Central 8.0 mm corneal buttons from all eyes were subjected to 0.3% collagenase digestion at 37 °C and the time required for complete digestion determined. A further 15 eyes underwent fluorescence confocal microscopy to assess the impact of rinsing on stromal riboflavin concentration.ResultsApplication of riboflavin/HPMC solution led to an increase in CCT of 73 ± 14 µm (P < 0.01) after 16 min. All CXL-treated corneas displayed a 2–4 fold greater resistance to collagenase digestion than non-irradiated corneas. There was no difference in resistance between corneas that received no BSS rinse and those that received a 0.25 mL or 1 mL pre-UVA rinse, but each showed a greater level of resistance than those that received a 10 mL pre-UVA rinse (P < 0.05). Confocal microscopy demonstrated reduced stromal riboflavin fluorescence after rinsing.ConclusionsAll protocols, with and without rinsing, were effective at enhancing the resistance to collagenase digestion, although resistance was significantly decreased, and stromal riboflavin fluorescence reduced with a 10 mL rinse. This suggests that a 10 mL surface rinse can reduce the efficacy of CXL through the dilution of the stromal riboflavin concentration.

Use of In Vivo Corneal Confocal Microscopy to Guide Excimer Laser With Adjunctive Mitomycin C for Treatment of Recalcitrant Acanthamoeba Keratitis.

The aim of this study was to describe 3 cases of recalcitrant Acanthamoeba keratitis (AK) that were successfully treated using in vivo corneal confocal microscopy (IVCM) to guide excimer laser ablation depth with adjunctive mitomycin C 0.02%. Three patients diagnosed with AK did not respond to several weeks of intensive topical therapy with antiamoebic agents. The patient underwent phototherapeutic keratectomy with topical mitomycin C 0.02% application. The maximum stromal depth of cysts measured by IVCM was 80 μm, 100 μm, and 240 μm, and the stromal ablation depths were 80 μm, 100 μm, and 100 μm, respectively. In all 3 eyes, AK resolved after a single excimer laser application, and topical treatment was gradually discontinued within 6 weeks afterward. In 1 eye, penetrating corneal transplantation was performed 6 weeks after phototherapeutic keratectomy because of ongoing severe corneal pain. IVCM and histology of the corneal transplant did not reveal any Acanthamoeba cysts within the excised corneal button. No recurrence was observed during the follow-up period of 19 to 34 months. IVCM-guided phototherapeutic keratectomy with mitomycin C 0.02% seems to be a safe and successful approach for the treatment of AK, especially in cases of resistance to topical treatment. Corneal IVCM should be performed before laser application to measure cyst depth, determine ablation depth, and assess postoperative treatment success.

Comparative posterior corneal profile of keratoconus hydrops versus Haab's striae in congenital glaucoma.

Keratoconus eyes develop corneal decompensation more often compared to eyes with primary congenital glaucoma (PCG) following Descemet's membrane (DM) tear. This study was conducted to compare the posterior corneal morphology in areas with DM breaks with regards to DM and pre-Descemet's layer (PDL) between the two. In this cross-sectional comparative study, anterior segment optical coherence tomography (AS-OCT) scans of the posterior cornea of advanced keratoconus eyes with hydrops ( n = 12), PCG eyes with Haab's striae ( n = 15), and healthy control eyes ( n = 14) were compared for DM-PDL morphology. These were further corroborated by the histopathology of corneal buttons from keratoconus ( n = 14) and PCG ( n = 13) cases obtained following penetrating keratoplasty and compared with controls (enucleated retinoblastoma globes, n = 6) on light microscopy and collagen IV immunostaining. AS-OCT showed a thicker median DM/PDL complex in PCG (80 µm) versus keratoconus eyes (36 µm, P = 0.01; Kruskal-Wallis test). The median height and length of detached DM-PDL were significantly more in keratoconus versus PCG (145 μm, 1766.1 ± 1320.6 μm vs. 26.5 μm, 453.3 ± 303.2 μm, respectively, P = 0.012; Kruskal-Wallis test). Type-1 DM/PDL detachment (seen as a characteristic taut chord) in keratoconus (90%) was the most common morphological pattern versus intracameral twin protuberance (92%) following DM breaks in PCG. Histopathology confirmed thicker DM in PCG (median: 63.4 μm) versus keratoconus eyes (median: 33.2 µm) or controls (27.1 μm) ( P = 0.001; Kruskal-Wallis test). Greater height/length of DM/PDL detachment compounded by poor healing response (lower DM/PDL thickness) probably causes more frequent corneal decompensation in keratoconus eyes when compared to PCG eyes following DM tears.

Combining Riboflavin/UV-A Light and Rose Bengal/Green Light Corneal Cross-Linking Increases the Resistance of Corneal Enzymatic Digestion.

The purpose of this study was to determine if concurrent riboflavin/UV-A light (RF/UV-A) and rose Bengal/green light (RB/green) epi-off PACK-CXL enhances corneal resistance to enzymatic digestion compared to separate chromophore/light treatments. Ex vivo porcine corneas were allocated as follows. Group A corneas were soaked with riboflavin (RF) and were either not irradiated (A1, controls) or were irradiated with 10 (A2) or 15 J/cm² (A3) UV-A light at 365nm, respectively. Group B corneas were soaked with RB and either not irradiated (B1, controls) or were illuminated with 10 (B2) or 15 J/cm² (B3) green light at 525nm, respectively. Corneas in group C were soaked with both RF and RB and were either not irradiated (C1, controls) or were subjected to the same session consecutive 10 J/cm2 (C2) or 15 J/cm2 (C3) UV-A and green light exposure. Following treatment, all corneas were exposed to 0.3% collagenase A to assess digestion time until corneal button dissolution. A1 to A3 digestion times were 21.38, 30.5, and 32.25hours, respectively, with A2 and A3 showing increased resistance to A1. B1-3 had digestion times of 31.2, 33.81, and 34.38hours, with B3 resisting more than B1. C1 to C3 times were 33.47, 39.81, and 51.94hours; C3 exhibited superior resistance to C1 and C2 (both P < 0.05). Same-session combined RF/UV-A and RB/green PACK-cross-linking significantly increases corneal enzymatic digestion resistance over standalone treatments. Combining RF-based and RB-based PACK-CXL considerably increases corneal collagenase digestion resistance, potentially minimizing ulcer size in clinical contexts.

Case Series: Mixed Infectious Keratitis by Pythium insidiosum and Fungal Species.

This case series is the first to illustrate mixed infection from Pythium sp. and fungal species in corneal ulcer. This case series aimed to alert all toward the possibility of both Pythium sp. and fungal species infection in case of nonresponding corneal ulcer treated with either antifungals or antipythium drugs alone. Increased suspicion of mixed infection in case of nonresponding fungal/ Pythium keratitis may facilitate early and prompt management. Six patients presented with signs of either fungal or Pythium keratitis. They underwent ophthalmological examinations, smear examinations, cultures, and polymerase chain reaction (PCR). Therapeutic penetrating keratoplasty was performed in cases where symptoms worsened after treatment with either antifungal or antipythium drugs. The half corneal button (HCB) was shared for histopathological and microbiological examinations. In the first case, smear examination from corneal scraping (CS) revealed Pythium -like filaments, which were confirmed with PCR; however, Aspergillus nidulans grew in culture. In the second case, iodine-potassium iodide (IKI) staining was positive for Pythium ; however, PCR was positive for both Pythium and fungus, which was further confirmed by DNA sequencing. In the third case, IKI staining and HCB were positive for Pythium ; however, PCR was positive for fungus, which was identified as Candida saitoana with DNA sequencing. In the fourth case, Pythium grew in the CS culture; however, Candida sp. grew in the HCB culture. In the fifth case, Cladosporium sp. grew in culture from CS; however, Pythium insidiosum grew from the anterior chamber exudate after therapeutic penetrating keratoplasty. In the sixth case, smear examination revealed septate fungal filaments, and Cladosporium sp. grew in culture; however, HCB on histopathological examination showed features of Pythium keratitis. In unresponsive cases of Pythium or fungal keratitis, diagnostic modalities such as IKI and PCR should be implemented as a routine practice, in addition to smears and cultures.

Differential expression of antimicrobial peptides in human fungal keratitis.

To analyze a series of antimicrobial peptides (AMPs) in corneal tissue from individuals with fungal keratitis (FK) during the active phase of the fungus infection and after healing. Patients undergone lamellar keratoplasty for the treatment of severe FK or corneal scar had their corneal buttons sampled. Quantitative real-time polymerase chain reaction (PCR) was used to ascertain the gene expression of human beta-defensin (HBD)-1, -2, -3, -9, S100A7, 8, 9, and LL-37. All AMPs' messenger ribonucleic acid (mRNA) expression was considerably elevated in all samples (n=12). In contrast to controls, where HBD-2, -3, and S100A7 mRNAs were expressed at very low levels, it was discovered that HBD-1, -9, S100A8, S100A9, and LL-37 were constitutively expressed in all healed samples (n=4). HBD-1, -2 -3, S100A7, and LL-37 mRNAs were significantly increased in all active FK samples (n=8). The levels of HBD-9, S100A8, and S100A9 mRNAs were moderately upregulated in all active FK samples. Subgroup comparison showed that HBD-2 was significantly increased in Fusarium keratitis samples (n=5), and LL-37 mRNAs were significantly enhanced in Aspergillus keratitis samples (n=3). Whereas there was not significantly increased of HBD-1, -3, -9, S100A7, 8, 9 mRNA in Aspergillus keratitis samples compared with Fusarium keratitis samples. AMPs expression is increased in active FK, but not all AMPs are equally expressed. HBD-2 and LL-37 expression levels are the highest, showing some specificity of AMP expression related to FK. Human AMPs, particularly HBD-2 may play a significant role in Fusarium keratitis and LL-37 might be the key player in Aspergillus keratitis.

OUTCOME OF THERAPEUTIC PENETRATING KERATOPLASTY IN A TERTIARY EYE HOSPITAL IN NEPAL

Background: Therapeutic penetrating keratoplasty is the most common variety of keratoplasty performed in the developing countries. The objective of this study was to assess the outcome of therapeutic penetrating keratoplasty in infective keratitis. Methods: This was a hospital based retrospective study where medical records of patients who underwent therapeutic penetrating keratoplasty between July 2021 and December 2022 were analyzed. Data collected included demographic parameters, indications for surgery, microbiology of the ulcers, donor details. Success was evaluated in terms of eradication of the disease, anatomical stability, graft clarity and visual acuity. SPSS software version 20.0 was used for statistical analysis. The mean, standard deviation, frequency and percentage were calculated Results: Sixty-seven patients of therapeutic penetrating keratoplasty who completed 2 months of follow-up after surgery were included in the study. Corneal scrapings and corneal buttons were positive for organisms in 19 cases (90.47%) and 20 cases (29.85%) respectively. In all,out of 67 eyes, 30 (44.77%) were positive for organisms in smear or culture or both, of which 27 cases showed pure fungus and 3 cases showed pure bacteria. Aspergillus (8, 29.62%) was the commonest fungus isolated. Eradication of infection after the primary procedure was seen in 48 cases (71.64%). Anatomical success was seen in 60 eyes (89.50%), clear grafts were seen in 23 eyes (34.32%). Visual acuity of ≥6/60 was seen in 14 eyes (20.89%). Conclusions: Therapeutic penetrating keratoplasty provided good success in terms of maintaining anatomical integrity and helps in eradication of infection while giving satisfactory functional outcome.

Prevalence of Herpes Simplex and Varicella-Zoster Virus DNA in Corneal Grafts Is Higher than Expected.

(1) To determine the prevalence of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), varicella-zoster virus (VZV), and cytomegalovirus (CMV) DNA in donor corneas; (2) To evaluate the clinical outcome of the grafts with viral DNA and to compare donors with and without viral DNA. We analyzed data from all donors and recipients who underwent penetrating keratoplasty (PK) or Descemet membrane endothelial keratoplasty (DMEK) between September 2022 and March 2023. Donor corneoscleral rims and excised recipients' corneal buttons were tested for the presence of HSV-1, HSV-2, VZV, and CMV DNA by polymerase chain reaction (PCR). The results were known 2-3 days after the surgery. We closely followed up on patients whose grafts tested positive for viral DNA. We compared the medical histories of donors with and without viral DNA. We included 85 corneas from 67 donors. Seven (8.2%) donor corneas tested positive for HSV-1 (n = 3) or VZV (n = 4) DNA. We did not detect any HSV-2 or CMV DNA. In the postoperative follow-up of patients with positive PCR, a graft failure was observed in one and infections in two eyes. Re-operation was necessary in three of these cases (42.9%). Patients without herpes DNA in the donor cornea needed reoperation in 7.7% of the cases. Cultural duration, the cause of the donor's death, and the death-to-explantation interval did not differ significantly between donors with and without viral DNA. Additionally, 3 of the 7 (42.9%) donors with positive PCR were in a septic status at the time of death, compared to 21 of the 78 (26.9%) donors with negative PCR (p = 0.52). The prevalence of herpes DNA in the donor corneas was 8.2% and thus higher than previously reported. We did not notice any evidence for a donor-to-host transmission, but a higher rate of postoperative complications in recipients of the grafts with viral DNA. The donors with and without herpetic DNA did not differ significantly regarding systemic diagnoses or cultural conditions, but sepsis was more frequent in the group with viral DNA.

High-Fluence Accelerated PACK-CXL for Bacterial Keratitis Using Riboflavin/UV-A or Rose Bengal/Green in the Ex Vivo Porcine Cornea.

To investigate and compare the efficacy of high-fluence accelerated photoactivated chromophore for keratitis-corneal cross-linking (PACK-CXL) using either riboflavin/ultraviolet (UV)-A light or rose bengal/green light to treat Staphylococcus aureus or Pseudomonas aeruginosa infections in an ex vivo porcine cornea model. One hundred and seventeen ex vivo porcine corneas were injected with clinical isolates of S. aureus or P. aeruginosa, divided into eight groups, and cultured for24 hours. Then, either riboflavin with UV-A light irradiation (30 mW/cm2; 8 minutes, 20seconds; 15 J/cm2) or rose bengal with green light irradiation (15 mW/cm2, 16minutes, 40 seconds; 15 J/cm2) was applied; unirradiated infected groups served as controls. All corneas were incubated for another 24 hours. Next, corneal buttons were obtained and vortexed to release the bacterial cells. The irradiated and unirradiated solutions were then plated and incubated on agar plates. The amount of colony-forming units was quantified and the bacterial killing ratios (BKRs) resulting from different PACK-CXL protocols relative to non-treated controls were calculated. Riboflavin/UV-A light PACK-CXL resulted in median BKRs of 52.8% and 45.8% in S. aureus and P. aeruginosa, respectively, whereas rose bengal/green light PACK-CXL resulted in significantly greater BKRs of 76.7% and 81.0%, respectively (both P < 0.01). Both accelerated PACK-CXL protocols significantly decreased S. aureus and P. aeruginosa bacterial loads. Comparing the riboflavin/UV-A light and rose bengal/green light PACK-CXL approaches in the same experimental setup may help develop strain-specific and depth-dependent PACK-CXL approaches that could be used alongside the current standard of care. Our study used an animal model to gain insight into the efficacy of high-fluence accelerated PACK-CXL using either riboflavin/UV-A light or rose bengal/green light to treat Staphylococcus aureus or Pseudomonas aeruginosa infections.

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

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.

The relationship between keratan sulfate glycosaminoglycan density and mechanical stiffening of CXL treatment

The corneal stroma is primarily composed of collagen fibrils, proteoglycans, and glycosaminoglycans (GAGs). It is known that corneal crosslinking (CXL) treatment improves mechanical properties of the cornea. However, the influence of stromal composition on the strengthening effect of CXL procedure has not been thoroughly investigated. The primary objective of the present research was to characterize the effect of keratan sulfate (KS) GAGs on the efficacy of CXL therapy. To this end, the CXL method was used to crosslink porcine corneal samples from which KS GAGs were enzymatically removed by keratanase II enzyme. Alcian blue staining was done to confirm the successful digestion of GAGs and uniaxial tensile experiments were performed for characterizing corneal mechanical properties. The influence of GAG removal and CXL treatment on resistance of corneal samples against enzymatic pepsin degradation was also quantified. It was found that removal of KS GAGs significantly softened corneal tensile properties (P < 0.05). Moreover, the CXL therapy significantly increased the tensile stiffness of GAG-depleted strips (P < 0.05). GAG-depleted corneal buttons were dissolved in the pepsin digestion solution significantly faster than control samples (P < 0.05). The CXL treatment significantly increased the time needed for complete pepsin digestion of GAG-depleted disks (P < 0.05). Based on these observations, we concluded that KS GAGs play a significant role in defining tensile properties and structural integrity of porcine cornea. Furthermore, the stiffening influence of the CXL treatment does not significantly depend on the density of corneal KS GAGs. The findings of the present study provided new information on the relation between corneal composition and CXL procedure mechanical effects.

Use of RGP lens as Temporary Keratoprosthesis in Open-Sky Technique for Heterotopic Autokeratoplasty

PURPOSE To present a case of a combined procedure of heterotopic autokeratoplasty and ECCE with posterior chamber intraocular lens (IOL) implantation, and to demonstrate the novel use of an RGP lens as a safe, cost-effective alternative to temporary keratoprosthesis. REPORT OF CASE A 65/F presented with blindness, OS from glaucoma but with a clear cornea, and corneal decompensation with cataract in OD with good visual potential. Heterotopic autokeratoplasty was performed due to the scarcity of corneal allografts and the high-risk nature of the case, with ECCE and IOL implantation in OD due to lens opacity. Temporary keratoprosthesis (TKP) (such as Eckardt and Landers models) was substituted with RGP due to lower cost and accessibility. Pre-placement of two polyglactin perpendicular mattress sutures in the episclera at the horizontal and vertical positions was done and left untied. Next, left corneal trephination was performed using an 8-mm vacuum trephine, followed by the instillation of oculoviscosurgical device (OVD) and placement of a 10-mm diameter RGP lens over the left open globe, secured in place by tying the pre-placed overlying cross sutures. Then, right corneal trephination using a similar-sized trephine proceeded. Cataract was extracted in open-sky technique with IOL in the bag implantation. The clear left corneal button was then transplanted to OD and vice-versa. Postoperatively, VA improved from hand motions to 10/200 (Day 1). The graft remained clear 4 weeks post-op with further improvement in VA. IOP was maintained below 15 mmHg. CONCLUSION Heterotopic autokeratoplasty is a valid option for unilateral cornea blind patients who have clear corneas in the fellow eye with no visual potential. This is useful in countries where corneal transplantation is costly, especially during time of pandemic when corneal allografts are scarce. In our experience, it can be safely performed using an RGP lens as a safe and cost-effective alternative to temporary keratoprosthesis.

Oxidative Stress and Cellular Protein Accumulation Are Present in Keratoconus, Macular Corneal Dystrophy, and Fuchs Endothelial Corneal Dystrophy

The aim of the study was to investigate oxidative stress as well as cellular protein accumulation in corneal diseases including keratoconus (KC), macular corneal dystrophy (MCD), and Fuchs endothelial corneal dystrophy (FECD) at their primary affecting sites. Corneal buttons from KC, MCD, and FECD patients, as well as healthy controls, were analyzed immunohistochemically to evaluate the presence of oxidative stress and the function of the proteostasis network. 4-Fydroxynonenal (4-HNE) was used as a marker of oxidative stress, whereas the levels of catalase and heat-shock protein 70 (HSP70) were analyzed to evaluate the response of the antioxidant defense system and molecular chaperones, respectively. Sequestosome 1 (SQSTM1) levels were determined to assess protein aggregation and the functionality of autophagic degradation. Basal epithelial cells of the KC samples showed increased levels of oxidative stress marker 4-HNE and antioxidant enzyme catalase together with elevated levels of HSP70 and accumulation of SQSTM1. Corneal stromal cells and endothelial cells from MCD and FECD samples, respectively, showed similarly increased levels of these markers. All corneal diseases showed the presence of oxidative stress and activation of the molecular chaperone response to sustain protein homeostasis. However, the accumulation of protein aggregates suggests insufficient function of the protective mechanisms to limit the oxidative damage and removal of protein aggregates via autophagy. These results suggest that oxidative stress has a role in KC, MCD, and FECD at the cellular level as a secondary outcome. Thus, antioxidant- and autophagy-targeted therapies could be included as supporting care when treating KC or corneal dystrophies.