Relationships between the clinical characteristics and copy numbers of DNA of cytomegalovirus determined by real-time PCR.

PURPOSE: To determine whether there is a correlation between the clinicals characteristics including various types of keratic precipitates and the copy numbers of the DNA of cytomegalovirus (CMV) in eyes with CMV corneal endotheliitis. METHODS: We reviewed the medical charts of four cases of corneal endotheliitis that were CMV-positive. We have classified types of clinical phenomenon into four types: coin-shaped KPs, sectoral corneal edema with or without Khodadoust line-like KPs, mutton-fat KPs, and fine KPs and have graded their severity. We also determined the copy numbers of the DNA of CMV in the aqueous humor by real-time polymerase chain reaction before and during the treatment. We evaluated the correlation between the patterns of clinical characteristics and copy number of the DNA of CMV. RESULTS: There were clinical improvements in all eyes following topical ganciclovir in conjunction with low dose of topical steroid treatment, with or without oral valganciclovir. The clinical characteristics and the copy numbers of the DNA of CMV varied during the treatment period. The presence of coin-shaped KPs was correlated with high copy numbers (105-103 copies/ml) of the DNA of CMV. The copy numbers of the DNA of CMV with sectoral corneal edema with or without Khodadoust line-like KPs ranged from 104 to 102 copies/ml, and it was occasionally accompanied by high intraocular pressure. Mutton-fat KPs were observed inferiorly, sometimes together with coin-shaped KPs and sectoral corneal edema, or solely. The copy numbers in eyes with mutton-fat KPs varied and occasionally less than the cutoff level. Fine-pigmented KPs were observed after the resolution of the endotheliitis, and no DNA of CMV was detected in the aqueous humor. CONCLUSIONS: Careful observations of the clinical characteristics such as the KPs and corneal edema might be helpful in estimating the amount of the DNA of CMV in eyes with corneal endotheliitis.

Effects of lactoferrin on the viability and the encystment of Acanthamoeba trophozoites.

Lactoferrin (LF) is an iron-binding basic glycoprotein that has an antimicrobial effect against certain microbes. The purpose of this study is to evaluate the amoebicidal effect of bovine milk LF (bLF) against Acanthamoeba clinical-isolate trophozoites, which cause severe keratitis. Most of the risk factor for Acanthamoeba keratitis is from wearing soft contact lenses (SCLs). Acanthamoeba trophozoites were incubated in bovine LF (bLF) solution, and the ratios of viability and encystment were determined with microscopic analysis of cyst formation. The amoebicidal effect of bLF was assessed by Trypan blue assay. The ratios of viable cells in the presence of iron-free bLF (apo-bLF), native-bLF, and iron-saturated bLF (Fe-bLF) at the concentration of 10 µmol/L for 60 min were 7.7% ± 4.6%, 80.7% ± 10.1%, and 97.3% ± 1.5%, respectively. Apo-bLF showed potent amoebicidal effect against Acanthamoeba trophozoites, but Fe-bLF did not have this effect. After treating with apo-bLF, most dead cells were nonglobular forms of trophozoites but not cystic forms. Encystment of Acanthamoeba was assessed by the sarkosyl-calcofluor white assay. The encystment ratios treated with 0.5% propylene glycol (positive control) and 10 µmol/L apo-bLF for 24 h were 96.12% ± 10.6% and 0.47% ± 0.5%, respectively. These results suggest that the amoebicidal effect of apo-bLF without encystment might lead to the prevention of contamination of Acanthamoeba in SCL stock cases.

The effects of oral and topical corticosteroid in rabbit corneas.

BACKGROUND: To determine the most effective route of administration of corticosteroids in the treatment of ocular surface disease, by characterizing the difference between oral prednisolone and topical dexamethasone administration using an animal model. METHODS: Pharmacokinetic analyses determined the corticosteroid concentrations in the normal ocular tissues of rabbits after oral or topical administration of corticosteroids using LC-MS/MS. In wound healing analyses, the area of the epithelial defect created by keratectomy using a 6-mm trephine was calculated with an image analyzer using an orally or topically steroid-administrated animal model. The average size of basal epithelial cells, the frequency of mitotic basal epithelial cells, the number of squamous cells, and the number of hypertrophic stromal fibroblasts were determined in the enucleated corneal tissues after wound closure. RESULTS: By slit lamp examination, no remarkable differences were observed between orally and topically administered groups. Pharmacokinetic analyses showed that the distribution of dexamethasone after topical administration was superior to that after oral administration in the cornea. In contrast, both concentrations of corticosteroid applied topically and orally were similar with regards to AUCs (area under the concentration-time curve) in the conjunctiva. Although the healing rate was slower in the topical group, all corneas were almost healed within 96 h in the wound healing analysis. According to the histological analyses of epithelial cells, the average basal cell size was larger, the frequency of mitotic basal cells was greater, and the number of squamous epithelial cell layers was lower in the topically administered group although all of these differences were with no statistical significance. However, the number of hypertrophic stromal fibroblasts in the topically administered group was significantly lower than that in the orally administered group. CONCLUSIONS: There are different distributions and effects between orally and topically administered corticosteroids on the ocular surface. The data may provide the useful information in selecting the appropriate route of corticosteroid application for the treatment of ocular surface disease.

[Multicenter Prospective Observational Study of Fungal Keratitis--Identification and Susceptibility Test of Fungi].

PURPOSE: To investigate the causative fungi of fungal keratitis in Japan and their drug susceptibility. METHODS: Identification and antifungal susceptibility test for 8 drugs (micafungin, amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, miconazole and pimaricin) were performed using isolated fungi from patients with fungal keratitis treated at 27 facilities in Japan between November 1, 2011 and October 31, 2013. RESULTS: Fungal strains were detected in 72 (50.7%) out of 142 samples. The major isolates were Fusarium spp. (18), Candida parapsilosis (12), C. albicans (11) and Alternaria spp. (6), in all, fungi of 31 species were identified by gene analysis. In the yeast-like fungi, susceptibility rates were evident for more than 80% in voriconazole, pimaricin, flucytosine, micafungin, amphotericin B and fluconazole. In filamentous fungi, the susceptibility rate was less than 50% except for PMR (90%). Fusarium spp., which were susceptible to amphotericin B and pimaricin, showed lower susceptibility rates compared with other genera. CONCLUSIONS: Although various genera and species of fungi cause fungal keratitis, the obtained drug susceptibility data in this study demonstrates the different susceptibility patterns among the major isolates (Fusarium spp., C. parapsilosis, C. albicans and other groups). This is important evidence useful for fungal keratitis treatment.

[Multicenter Prospective Observational Study of Fungal Keratitis--Current Status of Patients' Background, Clinical Findings, Treatment and Prognosis].

PURPOSE: To investigate the current status of fungal keratitis in Japan. METHODS: The patients with fungal keratitis were examined at 27 facilities in Japan from November 1st 2011 to October 31st 2013, concerning isolates, patient background, clinical findings, treatment and prognosis. RESULTS: Out of 139 cases, 133 were diagnosed as fungal keratitis, of which fungi were isolated from 72 samples of 71 cases (yeast-like fungi 32 strains and filamentous fungi 40 strains). The corrected visual acuity at the first visit of 88 cases (66.2%) was less than 20/200 and 42 cases (31.6%) were involved with deep stromal lesions, indicating high proportion of severe cases in this study. Three months later, 56 cases (42.1%) were still under treatment, and corrected visual acuity of 57 cases (42.9%) was less than 20/200. In cases with yeast-like fungi, there were significantly more cases with past history of corneal diseases, ocular surgery including keratoplasty, and eye drops' use such as steroids than those with filamentous fungi. On the other hand, there were significantly more cases of filamentous fungi, with trauma on the onset and with intervention of previously attending doctors than those with yeast-like fungi. Logistic regression analyses revealed that contact lens wearing was a significant factor of good prognosis, and yeast-like fungi as one of poor outcome compared with no fungal isolation. CONCLUSION: Although the choice of antifungal drugs has been increasing, fungal keratitis is still severe, refractory and vision-threatening disease.

Changes in the Visual Field Test after Descemet Stripping Automated Endothelial Keratoplasty in Advanced Glaucoma.

Background: To evaluate changes in the visual field (VF) after Descemet stripping automated endothelial keratoplasty (DSAEK) in eyes with advanced glaucoma and previous trabeculectomy. Methods: Changes in VF, best-corrected visual acuity (BCVA), intraocular pressure (IOP), and number of glaucoma medications were analyzed before and after DSAEK in 19 eyes. The VFs were evaluated using the 10-2 program of the Humphrey Field Analyzer (HFA) and/or Goldmann perimetry (GP). Results: In nine eyes, the MD improved from -22.24 ± 6.5 dB to -18.36 ± 5.1 dB in HFA. In five out of nine eyes, postoperative MD improved >1 dB compared to preoperative MD. In GP testing, 10 out of 15 eyes showed an improvement, that is, greater than 20° in VF enlargement by the isopter of I-4e and/or new detection of a smaller or darker isopter. Overall, improvement in VF with the HFA and/or GP test was observed in 12/19 (63.2%) eyes after DSAEK. Postoperative BCVA improved by more than two lines in logMAR VA in 18 of 19 (94.7%) eyes. There were no significant differences between the preoperative and postoperative IOP and the number of glaucoma medications. Conclusions: DSAEK may produce subjective improvement in the visual field as well as improved visual acuity, even in advanced glaucomatous eyes.

Classification of secondary corneal amyloidosis and involvement of lactoferrin.

PURPOSE: To classify secondary corneal amyloidosis (SCA) by its clinical appearance, to analyze the demographics of the patients, and to determine the involvement of lactoferrin. DESIGN: Retrospective, observational, noncomparative, multicenter study. PARTICIPANTS: Twenty-nine eyes of 29 patients diagnosed with SCA by corneal specialists at 9 ophthalmologic institutions in Japan were studied. METHODS: The clinical appearance of SCA was determined by slit-lamp biomicroscopy and was classified into 3 types. The demographics of the patients, for example, age, gender, and the duration of the basic disease (trichiasis, keratoconus, and unknown), were determined for each clinical type. Surgically excised tissues were stained with Congo red and antilactoferrin antibody. The postoperative prognosis also was determined. MAIN OUTCOME MEASURES: Clinical appearance of the 3 types of SCA, along with the gender, age, and duration of the basic diseases were determined. RESULTS: Classification of SCA into 3 types based on clinical appearance found 21 cases with gelatinous drop-like dystrophy (GDLD)-like appearance (GDLD type), 3 cases with lattice corneal dystrophy (LCD)-like appearance (LCD type), and 5 cases with the combined type. Patients with the GDLD type were younger (average age: 40.9 years for the GDLD type, 74.3 years for the LCD type, and 46.8 years for the combined type), predominantly women (85.7% for the GDLD type, 33.3% for the LCD type, and 60% for the combined type), and had the basic disease over a longer time (average duration: 22.1 years for the GDLD type, 14.0 for the LCD type, and 11.4 for the combined type). The distribution of the basic diseases (trichiasis vs. keratoconus vs. unknown) was not significantly different for each type. Surgical treatments, for example, phototherapeutic keratectomy, lamellar keratoplasty, and simple keratectomy, resulted in a good resolution in all surgically treated cases. One subject dropped out of the study. Spontaneous resolution was seen in one subject after epilation of the cilia. Amorphous materials in the excised tissues showed positive staining results by Congo red and by antilactoferrin antibody. CONCLUSIONS: Secondary corneal amyloidosis can be classified into 3 clinical types based on its clinical appearance. Larger numbers of females and lactoferrin expression were seen in all 3 types. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Aberrant Corneal Homeostasis in Neurosurgery-Induced Neurotrophic Keratopathy.

The characteristic features of neurotrophic keratopathy have been well documented by in vivo and in vitro studies using animal models. However, case reports of neurotrophic keratopathy induced by neurosurgery are limited. We describe the clinical characteristics, anterior segment optical coherence tomography (AS-OCT) and in vivo confocal microscopy (IVCM) findings of neurotrophic keratopathy induced by surgery for intracranial lesions. This is a case series including 6 eyes of 3 patients (mean age, 69.67 ± 12.50 years) with unilateral neurotrophic keratopathy. The clinical findings of three patients were described and IVCM findings of three patients were analyzed. The duration of neuropathy ranged from 2 to 30 years (median, 22 years). Thickening of the epithelial layer and higher reflection density of the anterior stroma were observed during the healing process using AS-OCT. The mean nerve fiber density of the subepithelial plexus, as determined by IVCM, was 1943 ± 1000 µm/mm2 for neurotrophic eyes and 2242 ± 600.3 µm/mm2 for contralateral eyes (p = 0.0347). The mean respective dendritic cell densities were 30.8 ± 21.8 and 6.25 ± 5.59 cells/mm2 (p < 0.0001), while the mean basal cell sizes were 259 ± 86.5 and 185 ± 45.9 µm2 (p < 0.0001), respectively. These findings suggest that neurosurgery-induced neurotrophic keratopathy may be associated with alterations in the healing process and immune cell distribution in the cornea.

Corneal ulcers with non-infectious appearance caused by nasolacrimal duct obstruction or canaliculitis.

Purpose: To describe the clinical features of corneal ulcers with non-infectious appearance due to nasolacrimal disease in a retrospective case series. Observations: Eight eyes of 8 patients (aged 74.4 ± 11.1 years) with corneal disease due to nasolacrimal duct obstruction or canaliculitis, who were treated between October 2013 and December 2020 at 3 hospitals were included. Patient background, anterior ocular findings, organisms in secretion, and time course during treatment were retrospectively analyzed. The corneal findings were peripheral ulcers (5 cases), phlyctenular keratitis (1 case), and paracentral perforation with slight cellular infiltration (2 cases). All cases were suspected as autoimmune disease-related-corneal ulcers because of the pathogenic region and clinical appearance and later diagnosed as corneal disorders derived from nasolacrimal duct obstruction or canaliculitis. The autoimmune disease-like appearance and purulent secretion connecting the punctum with/without swelling were characteristic. The most common microorganism detected in the purulent secretions was Streptococcus spp.. The resolution of corneal lesions needed steroid eye drops with antibiotic eye drops. Two patients required a superficial corneal transplantation. The extraction of nasolacrimal calculus, punctal tube insertion, or dacryocystorhinostomy was necessary for complete healing of ocular surface disease. Conclusions and importance: Nasolacrimal duct diseases cause corneal disorders without bacterial colonization and growth. When corneal ulcers resemble autoimmune disease in shape and are not accompanied by systemic disease, attention should be paid to nasolacrimal duct obstruction or canaliculitis.

16S rRNA nanopore sequencing for the diagnosis of ocular infection: a feasibility study.

OBJECTIVE: We conducted a feasibility study to verify the effectiveness of 16S ribosomal RNA (rRNA) gene analysis using the nanopore sequencer MinION for identifying causative bacteria in several types of ocular infections. METHODS AND ANALYSIS: Four cases of corneal ulcers, one case of endophthalmitis and one case of a conjunctival abscess were included in this study. DNA was extracted from corneal scraping, vitreous samples and secretions from the conjunctival abscess. We conducted 16S rRNA gene amplicon sequencing using MinION and metagenomic DNA analysis. The efficacy of bacterial identification was verified by comparing the conventional culture method with smear observations. RESULTS: 16S rRNA gene sequencing analysis with MinION identified the causative organisms promptly with high accuracy in approximately 4 hours, from ophthalmic specimens. The results of the conventional culture method and 16S rRNA gene sequencing were consistent in all cases. In four of the six cases, a greater variety of organisms was found in the 16S rRNA gene analysis than in bacterial culture. CONCLUSION: Using our workflow, 16S rRNA gene analysis using MinION enabled rapid and accurate identification possible in various kinds of bacterial ocular infections.

Successful identification of Granulicatella adiacens in postoperative acute infectious endophthalmitis using a bacterial 16S ribosomal RNA gene-sequencing platform with MinION™: A case report.

Purpose: To evaluate the efficacy of identifying the bacteria by aqueous sampling and vitreous sampling in postoperative infectious endophthalmitis using 16S ribosomal RNA (rRNA) gene analysis with a nanopore sequencer (MinION™). Observation: A 55-year-old woman who underwent cataract surgery at an ophthalmology clinic 18 days ago was referred to our hospital for suspected endophthalmitis. She had light perception visual acuity in her right eye; however, the eye was severely inflamed, with a hypopyon and a fibrinous membrane in the anterior chamber. The fundus was not visible because of vitreous opacity on a B-scan image. Based on the diagnosis of postoperative acute infectious endophthalmitis, we performed a vitrectomy, intraocular lens extraction, and silicone oil tamponade. On postoperative day 14, the inflammation resolved. An aqueous sample was collected before surgical treatment, and the vitreous sample was collected during the operation. Both samples underwent 16S rRNA gene analysis with a nanopore sequencer MinION™ to identify the causative organism. Conclusions and Importance: In the aqueous humor, Granulicatella adiacens and Cutibacterium acnes were identified before the operation, while only Granulicatella adiacens was detected in the vitreous sample after the operation. Although the aqueous humor sample might contain commensal bacteria, it could provide a predictable result before the operation. It can also provide a substitute for a vitreous sample to allow earlier identification of the causative organism.

Multicenter prospective observational study of fungal keratitis in Japan: analyses of culture-positive cases.

PURPOSE: To investigate the clinical characteristics and causative fungi in patients with fungal keratitis in Japan, and to determine factors related to the prognosis. STUDY DESIGN: Multicenter prospective observational study. METHODS: Eligible patients were enrolled from November 2011 to October 2013 at the 1st stage and from April 2015 to March 2016 at the 2nd stage. The corneal foci were scraped, and the scrapings were cultured in potato dextrose agar. The isolated fungi were identified by gene analyses. Data were collected from the clinical records and statistically analyzed by Cox and logistic regression analyses. RESULTS: Ninety-four fungal strains were isolated from 93 cases, including 42 yeast-like fungi and 52 filamentous fungi. The fungi affected the deep layers of the cornea in 23 cases (24.7%) and the peripheral cornea in 29 cases (31.2%). The incidences of lid swelling/redness, ciliary injection, anterior chamber cells/flare, anterior chamber fibrin, and hyphate ulcer in cases of filamentous fungi were significantly higher than in yeast-like fungi. No history of topical steroids, absence of a main lesion in the peripheral cornea, and best-corrected visual acuity (BCVA) of more than 0.04 at the first visit were related to a shorter healing time. No history of ocular surgery, absence of lesion at one-third deep stromal layer and BCVA of more than 0.04 at the first visit were correlated with BCVA at 3 months after the initial examination. CONCLUSION: Fungal keratitis is caused by various species of fungi and can become refractory due to poor prognosis factors.

Multicenter prospective observational study of fungal keratitis in Japan: analyses of in vitro susceptibility tests for combinations of drugs.

PURPOSE: To determine the effects of a combination of two antifungal drugs against causative fungi of fungal keratitis in Japan. STUDY DESIGN: Multicenter prospective observational study. METHODS: Eighteen isolates of yeast-like fungi and 22 isolates of filamentous fungi collected by the Multicenter Prospective Observational Study of Fungal Keratitis in Japan were studied. Specially manufactured minimum inhibitory concentration (MIC) measurement plates were used to test the effectiveness of 10 combinations of two antifungal drugs against the isolates. The combinations were pimaricin (PMR) + voriconazole (VRCZ), PMR + fluconazole (FLCZ), PMR + miconazole (MCZ), PMR + micafungin (MCFG), VRCZ + FLCZ, VRCZ + MCZ, VRCZ + MCFG, VRCZ + amphotericin-B (AMPH-B), MCZ + FLCZ, and MCZ + MCFG. The checkerboard microdilution method was used, and the fractional inhibitory concentration (FIC) index was calculated based on the guidelines of The Clinical & Laboratory Standards Institute (CLSI). RESULTS: In yeast-like fungi, additive effects were observed between PMR and MCFG in 77.8% of the isolates, and they were also observed between the azoles. Synergistic effects were observed on 11.1% of the isolates for MCZ and FLCZ. On the other hand, antagonistic effects were present between PMR and azoles with 88.9% between PMR and VRCZ, 72.2% between PMR and FLCZ, and 94.4% between PMR and MCZ. In filamentous fungi, additive effects were observed between PMR and MCFG in 40.9% of the isolates, and between VRCZ and MCZ in 40.9% of the isolates. Antagonistic effects were observed for PMR and the azoles. CONCLUSIONS: The combination of drugs prescribed for fungal keratitis incurs a possibility of synergistic, additive, indifferent, or antagonistic effects, depending on drug combinations and fungal strains.

Topical Corticosteroids for Infectious Keratitis Before Culture-Proven Diagnosis.

PURPOSE: In clinical practice we sometimes encounter patients with severe corneal ulcers who have been treated with topical corticosteroids. This study reviewed the clinical features and visual outcomes of these patients and investigated the background of the prescription of topical corticosteroids. PATIENTS AND METHODS: The medical records of patients who visited the Cornea Service at Fujita Health University Bantane Hospital and were treated for infectious keratitis from April 2016 to March 2020 were retrospectively reviewed. Patients treated with topical corticosteroids before a culture-proven diagnosis were studied in terms of demographics, best-corrected visual acuity at arrival and at last visit, the clinical course after visit, ocular history, and combination therapy by the previous ophthalmologist. RESULTS: Out of the 200 eyes of 197 patients with infectious keratitis, 14 eyes of 14 patients were treated with topical corticosteroids before a culture-proven diagnosis. All 14 patients were referred, as they had severe keratitis that could not be cured with topical antibiotics and corticosteroids. Based on the culture results, we diagnosed Acanthamoeba keratitis (AK) in six patients, fungal keratitis (FK) in two patients, bacterial keratitis (including a suspected case) in two patients, and unknown cause in four patients. Two patients with AK, FK, and unknown keratitis had unfortunate clinical courses and poor visual outcomes. From the information in the referral letters, at least six of the 14 patients were treated with either acyclovir ocular ointment or valaciclovir tablets, along with topical corticosteroids. CONCLUSION: Application of topical corticosteroids for keratitis that does not respond to empirical antibiotic therapy is harmful since AK or FK is likely involved in these topical antibiotic-resistant cases. Microbiological evidence, as well as a differential diagnosis of herpetic stromal keratitis, is needed when prescribing topical corticosteroid for the treatment of suspected infectious keratitis.

Synthetic rubber sheet to manage exposure keratopathy.

PURPOSE: Exposure keratopathy often progresses even with conventional various treatments and needs plastic surgery. However, plastic surgery of eye lid is often difficult in cases with poor general condition by cerebrovascular disorders. We will propose a novel method using synthetic rubber sheet to manage the exposure keratopathy under poor general conditions. OBSERVATIONS: We treated with synthetic rubber sheet on 9 eyes of 9 patients who suffered from refractory exposure keratopathy due to cerebrovascular disorders. Sheets cut from sterile surgical gloves made of synthetic rubber (SR sheets) were placed directly onto the ocular surface with antibiotic ointment and fixed with gauze and tape. Severity of the exposure keratopathy was scored before and after the application of SR sheet. Covered ocular surface with SR sheet could keep an adequate moist environment and exposure keratopathy was improved in all the cases with no adverse effects during an average observation period of 166.2 days. CONCLUSIONS AND IMPORTANCE: Sterile synthetic rubber sheet cut from surgical gloves is an effective, safe, easy, and economical material to maintain better condition of ocular surface and especially useful for refractory exposure keratopathy under poor general condition.

Case of bilateral Mooren's ulcers following filtering surgery using EX-PRESS glaucoma filtering devices.

PURPOSE: To report our findings in a case of bilateral Mooren's ulcer that developed after filtering surgeries using the EX-PRESS glaucoma filtering device (EX-PRESS surgery). PATIENTS AND METHODS: A 71-year-old Japanese man with primary open angle glaucoma underwent EX-PRESS surgery first in his left eye and 1 month later in his right eye. He developed Mooren's ulcer in his right eye at 7 months and in his left eye at 10 months after the initial EX-PRESS surgery. Systemic examinations showed no collagen vascular disease, and he did not have a history of bacterial or viral infections. He was not allergic to metallic materials. Before the EX-PRESS surgery, he had underdone cataract surgery combined with trabeculotomy in both eyes, and a reoperation of trabeculotomy in his left eye. He had not developed Mooren's ulcer after these surgeries. The Mooren's ulcer after the EX-PRESS surgery was treated with oral prednisolone (30 mg tapering) in combination with topical 0.1% betamethasone sodium. The ulcers were responsive and healed well in three months. CONCLUSIONS: The EX-PRESS devices were most likely the cause of the Mooren's ulcers considering that they were located close to the site of EX-PRESS insertion and no peripheral corneal ulcer developed after prior intraocular surgeries.

Decellularizing corneal stroma using N2 gas.

PURPOSE: To examine the efficacy of a novel method of decellularizing porcine corneal stroma using N(2) gas from liquid N(2) and the feasibility of using decellularized porcine corneal stroma in a corneal transplantation model in rabbits. METHODS: Porcine corneas were placed in a tube, and N(2) gas from liquid N(2) was poured into the tube to freeze the corneas and make the inside of the tube hypoxic. After fastening the cap firmly, the tube was kept at room temperature for seven days, and the porcine corneas were examined histologically. A porcine corneal stromal disk treated with the aforementioned method was inserted into a pocket of rabbit corneal stroma and observed for six months. RESULTS: Hoechst 33342 and hematoxylin and eosin staining both showed few cellular nuclei in the porcine corneal stroma incubated in N(2) gas for one week. A terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay showed many positively stained nuclei in the porcine corneal stroma incubated in N(2) gas for three days. The porcine corneal stroma that was decellularized and transplanted into a rabbit corneal stromal pocket remained clear for six months after transplantation. CONCLUSIONS: This method using N(2) gas decellularizes corneal stroma without reducing corneal transparency.

Graft rejection-like reactions in the early postoperative period after deep anterior lamellar keratoplasty for keratoconus: a retrospective study.

BACKGROUND: Deep anterior lamellar keratoplasty (DALK) is indicated to correct high astigmatism in patients with keratoconus (KC) and no evidence of Descemet's membrane rupture. However, some patients with KC experience graft rejection-like inflammatory reactions within 2 months (usually in the first week) after DALK. The aim of this study was to identify the characteristics and influencing factors of these reactions by reviewing the records of patients who underwent DALK or penetrating keratoplasty (PKP) for KC or other corneal problems. METHODS: We retrospectively reviewed the medical records of patients who underwent DALK for KC (DALK/KC), PKP for KC (PKP/KC), or DALK for other corneal problems (DALK/non-KC) at Ban Buntane Hotokukai Hospital between January 2006 and December 2015 and who were followed for more than 1 year. We collected data on the characteristics and incidence of severe inflammatory graft reactions in the early postoperative phase (ie, within 2 months after keratoplasty) and visual outcomes after these inflammatory reactions. RESULTS: Postoperative inflammatory reactions characterized by persistent epithelial defects, loose suture with infiltration, and vessel invasion occurred in seven of eleven DALK/KC patients, three of 50 DALK/non-KC patients, and none of five PKP/KC patients. These reactions were nonresponsive to topical steroids, and suture removal was required. Although a clear corneal graft in the pupillary area was obtained and best-corrected visual acuity was good after the resolution of inflammation, a risk of corneal astigmatism remained. CONCLUSION: The incidence of these reactions in the early postoperative period is high after DALK for KC. These reactions do not respond well to topical steroids, and suture removal may be required, which may cause high astigmatism after the inflammation subsides. Lamellar keratoplasty should be considered carefully for patients with KC.

Collagen fiber changes related to keratoconus with secondary corneal amyloidosis.

We describe the histological changes in the collagen fibers of a 50-year-old male who presented keratoconus with secondary corneal amyloidosis. Corneal tissue from the patient was obtained following a penetrating keratoplasty and was subjected to histochemical analysis using Masson's trichrome staining, Congo red staining, anti-lactoferrin antibody, and anti-transforming growth factor-beta-induced protein (TGFBIp) antibody. A Congo red-positive region was detected in the anterior half of the stroma in the center and inferior cornea. Although hemotoxylin and eosin staining revealed irregularity in the Congo red-positive region, other parts of the stroma did not show any abnormalities. Positive staining both by anti-TGFBIp and anti-lactoferrin antibodies was observed in the Congo red-positive region. Interestingly, all the layers of the corneal stroma, including the peripheral region, were positively stained by anti-TFGBIp antibody, even in the Congo red-negative area. Masson's trichrome staining also showed irregular staining throughout the corneal stroma, even outside of the Congo red-positive region. Additionally, Bowman's layer, which consists of collagen type IV, was damaged. TGFBIp was strongly expressed and Masson's trichrome staining was reduced throughout the entire keratoconic stroma. The constant qualitative changes in keratoconic collagen fibers, along with the observed abnormality in the Bowman's membrane, might point to the pathogenesis of secondary corneal amyloidosis in keratoconus.