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.

Novel Types of Small RNA Exhibit Sequence- and Target-dependent Angiogenesis Suppression Without Activation of Toll-like Receptor 3 in an Age-related Macular Degeneration (AMD) Mouse Model.

RNA interference (RNAi) has become a powerful tool for suppressing gene expression in vitro and in vivo. A great deal of evidence has demonstrated the potential for the use of synthetic small interfering RNAs (siRNAs) as therapeutic agents. However, the application of siRNA to clinical medicine is still limited, mainly due to sequence-independent suppression of angiogenesis mediated by Toll-like receptor 3 (TLR3). Here, we describe novel types of synthetic RNA, named nkRNA and PnkRNA, that exhibit sequence-specific gene silencing through RNAi without activating TLRs or RIG-I-like receptor signaling. In addition, we confirmed the therapeutic effect for the novel types of RNA in an animal model of age-related macular degeneration (AMD) without retinal degeneration. These data indicate that nkRNA and PnkRNA are of great potential utility as therapies against blinding choroidal neovascularization due to AMD.

Unilateral corneal leukoplakia without limbal involvement.

PURPOSE: Leukoplakia is the term given to a white patch or plaque that is found mainly on the oral mucus membrane. It can occasionally be seen on the corneal surface. We report our clinical and histopathological findings in a case of unilateral corneal leukoplakia. METHODS: A 26-year-old woman was referred to our hospital because of a white patch on her right cornea that continued to expand. She first noticed the white patch when she was 20 years old, and the white patch had expanded to cover the pupillary area affecting her vision. After plastic surgery on both eyelids for bilateral entropion to alleviate the pain caused by the eyelashes rubbing the cornea, the white corneal patch decreased in size. Because of this reduction, we performed surgery to remove the patch with microforceps under topical anesthesia. The plaque was removed easily and completely, and submitted for histopathological examination. RESULTS: Histopathological examination showed that the specimen had characteristics of epidermis with a basal cell layer, spinous cell layer, granular cell layer, and horny layer with hyperkeratosis. She was diagnosed with leukoplakia of the corneal surface. The basic structure of the squamous cell layer was preserved, and there were no signs of metaplasia. Six months after the removal of the leukoplakia, no recurrence was seen and her corrected decimal visual acuity recovered to 1.0. CONCLUSION: Our case of unilateral corneal leukoplakia without limbal involvement was most likely caused by chronic irritation of the cornea by the eyelashes. Although it was totally removed with good recovery of vision, we continue to follow the patient because of the potential of malignant transformation.

Findings of secondary corneal amyloidosis with ultrahigh-resolution optical coherence tomography.

PURPOSE: To describe observations by ultrahigh-resolution optical coherence tomography (OCT) in a secondary corneal amyloidosis (SCA) patient with histological analysis of excised tissue. A unique finding under OCT of her fellow eye is also described. CASE: A 39-year-old female had suffered from trichiasis in both of her eyes for more than 30 years. Slit-lamp examination showed a milky-white soft mass on her left cornea and a linear opacity on the fellow cornea at the cilia-attached region. OCT demonstrated the presence of a mass region within a thin epithelial layer and no destruction of Bowman's layer in her left cornea. In the fellow cornea, which exhibited a linear opacity, a high-density spot in Bowman's layer was observed at the cilia-attached region covered by the epithelial layer, with normal thickness. Histological examination of the excised cornea showed that the mass was positive with both Congo red and antilactoferrin antibody. CONCLUSION: SCA, amyloid gradually accumulates above Bowman's layer, occupying the epithelial layer, with no destruction of Bowman's layer until the advanced stage. A high-density spot in Bowman's layer might be the first stage of SCA.

The clinical characteristics of fungal keratitis in eyes after Descemet's stripping and automated endothelial keratoplasty.

The purpose of this study was to describe the clinical characteristics of fungal keratitis caused by Candida albicans in an eye after Descemet stripping automated endothelial keratoplasty (DSAEK). A 72-year-old male with a history of three trabeculectomies, cataract surgery, and two DSAEK procedures developed a corneal ulcer in his right eye two years after his last DSAEK. Fungal keratitis was most likely related to the immunosuppressive conditions that occurred due to the previous operations, the continuous use of steroid eye drops, and the use of disposable soft contact lenses. A smear and culture from the ulcer detected Candida albicans. Slit-lamp examination showed the characteristic feature was the presence of interface infiltrates located between the host and the graft cornea and in the enlarged area around the ulcer. Two weeks after intense antimycotic treatments with voriconazole, miconazole, and natamycin, perforation of the cornea occurred and further therapeutic penetrating keratoplasty was required. Histological analysis revealed an accumulation of infiltrated cells and fibrotic tissue. The poor prognosis for fungal keratitis that occurs in eyes after undergoing DSAEK may be related to the rapid expansion of inflammatory cells through the interface between the host and the graft. In eyes that develop fungal keratitis after DSAEK, special attention should be paid to the possibility that perforation could occur in these patients.

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.

Intraoperative mydriasis by intracameral injection of mydriatic eye drops: in vivo efficacy and in vitro safety studies.

BACKGROUND: This study investigated the efficacy and safety of intracameral injection of commercially available eye drops containing 0.5% tropicamide and 0.5% phenylephrine hydrochloride (Mydrin-P, Santen Pharmaceutical, Osaka, Japan). DESIGN: In vitro experiment and prospective clinical study at a private hospital. PARTICIPANTS AND SAMPLES: Mydrin-P was applied to confluent cultured human corneal endothelial cells, and the cellular morphology was examined. Clinical study subjects were 65 eyes of 65 patients that underwent phaco-emulsification and aspiration with intraocular lens implantation and received intracameral injection of Mydrin-P for poor mydriasis after preoperative topical instillation of mydriatics (intraocular mydriasis group; with five subgroups based on cause: diabetes, pseudo-exfoliation, post-surgery, uveitis, unknown). Controls, comprising 39 eyes of 39 patients, were not injected with Mydrin-P. METHODS: The ratio of pupillary diameter to corneal diameter was determined before and after injection of Mydrin-P. Corneal endothelial density was measured preoperatively and 3 months and 1 year postoperatively. MAIN OUTCOME MEASURES: Pupillary diameter and corneal endothelial density. RESULTS: Human corneal endothelial cell morphology was unaltered after Mydrin-P injection. The mean ratio of the pupillary diameter to corneal diameter increased in the intraocular mydriasis group (before: 54.2 ± 4.8%, after: 58.4 ± 6.6%; P < 0.001) and in the diabetes and unknown subgroups. The corneal endothelial cell density reduction rate 3 months and 1 year after surgery was not significantly different between the intraocular mydriasis group and controls. CONCLUSION: Intracameral injection of Mydrin-P appears to be effective and safe for dilating the pupil in cases with poor mydriasis after preoperative instillation of mydriatics.

Detection of cytomegalovirus DNA from cytomegalovirus corneal endotheliitis after penetrating keratoplasty.

PURPOSE: To report the detection of cytomegalovirus (CMV) DNA in the cornea of a CMV endotheliitis patient after penetrating keratoplasty (PKP). CASE: A 71-year-old man without immunodeficiency developed corneal endotheliitis in the right eye. The patient had previously received PKP several times. Polymerase chain reaction (PCR) detected CMV-DNA in the aqueous humor in his affected eye, and we started administration of ganciclovir. There was resolution of the inflammation; however, bullous keratopathy was subsequently noted in the cornea. Additional PKP was performed with perioperative intravenous administration of ganciclovir. The failed graft obtained during the additional PKP was subjected to PCR analysis and histopathological examination. PCR analysis showed CMV-DNA in the failed graft. Little inflammatory change was noted in either the epithelial or stromal layers of the failed graft. With continued ganciclovir treatment, the graft remained clear and no recurrence or rejection occurred until 12 months after the last PKP. CONCLUSION: Our PCR analysis showed the presence of CMV-DNA within the cornea of the patient with corneal endotheliitis.

Karyotype changes in cultured human corneal endothelial cells.

PURPOSE: To examine karyotype changes in cultured human corneal endothelial cells (HCECs). METHODS: HCECs with Descemet's membrane were removed from 20 donors of various ages (range, 2-77 years; average, 43.7+/-26.4 years) and cultured on dishes coated with extracellular matrix produced by bovine corneal endothelial cells (BCECs). Karyotype changes were examined by G-band karyotyping of HCECs at the third passage from 12 donors and the fifth passage from 16 donors. The number of chromosomes was analyzed in 40-50 cells from the third and fifth passages of each HCEC preparation. A detailed karyotype analysis of 10-16 cells from the third and fifth passages of each HCEC preparation was performed. The frequency of aneuploid cells per case (the number of abnormal cells divided by the total number of cells examined at metaphase) was tested for correlation with age by Spearman's correlation analysis. RESULTS: At the third passage, five cases (41.7%) showed an almost normal karyotype, and five cases (41.7%) showed sex chromosome loss. One case (8.3%) showed chromosome 21 trisomy. At the fifth passage, five cases (31.3%) showed an almost normal karyotype, and four cases (25%) showed sex chromosome loss. Three cases (18.8%) showed chromosome 8 trisomy, and one case (6.3%) showed chromosome 21 trisomy. Donor age and the frequency of aneuploidy had a statistically significant correlation at the fifth passage (R=0.653, p=0.042). CONCLUSIONS: Donor age and the frequency of aneuploidy have a positive correlation in cultured HCECs at the fifth passage. Therefore, HCECs for clinical therapies should be obtained from donors as young as possible. Karyotyping cultured HCECs is crucial before clinical application.

Histologic evaluation of a cornea in a patient with apolipoprotein A-I deficiency.

PURPOSE: To report the histology of a cornea in a patient with apolipoprotein A-I (apoA-I) deficiency. METHODS: A cornea from a patient with apoA-I deficiency was obtained by keratoplasty and examined histologically. RESULTS: There were many small vesicles in the extracellular matrix throughout the corneal stroma. The vesicles were round or oval, and their diameters ranged from 200 nm to 2 microm. These histologic properties suggest that the small vesicles are lipid droplets. CONCLUSIONS: Myriad lipid droplets in the extracellular matrix in the corneal stroma induce diffuse corneal opacity in a patient with apoA-I deficiency.

Detection of Streptococcus species by polymerase chain reaction in infectious crystalline keratopathy.

PURPOSE: Investigation of a positive polymerase chain reaction (PCR) for Streptococcus species from an excised cornea of a patient with infectious crystalline keratopathy. METHODS: Samples microdissected from corneal intrastromal aggregates present in infectious crystalline keratopathy were subjected to PCR for 3 primer pairs respectively specific for Streptococcus, Staphylococcus, and several kinds of fungi. RESULTS: The PCR detected DNA from Streptococcus but not from Staphylococcus or fungi. Direct sequencing of the PCR products showed that the PCR products had the highest identity (99.8%) with Streptococcus mitis and Streptococcus gordoni, indicating that these species were the most probable causes of infectious crystalline keratopathy in this patient. CONCLUSION: Our findings suggest that PCR provides a good adjunct technique in the diagnosis of corneal infection.

Properties of corneas reconstructed with cultured human corneal endothelial cells and human corneal stroma.

PURPOSE: To examine the properties of corneas tissue-engineered with cultured human corneal endothelial cells (HCEC) and human corneal stroma. METHODS: Primary HCEC cultures were established from endothelial cell layer explants and propagated on culture dishes coated with bovine corneal endothelial extracellular matrix. A cell suspension of HCEC at the fifth passage was transferred onto human corneal stroma deprived of endothelial cells, and the corneas were gently centrifuged to enhance cell attachment. The cell density of the tissue-engineered corneas was examined after staining with alizarin red and trypan blue. The tissue-engineered corneas were histologically examined by light and electron microscopy. The pump function of the tissue-engineered corneas was measured using an Ussing chamber. RESULTS: The mean endothelial cell density of four tissue-engineered corneas was 2380 +/- 264 cells/mm(2) (mean +/- SD). HCEC on the tissue-engineered corneas had a morphology similar to HCEC in vivo. The pump function parameters of the tissue-engineered corneas were 55%-75% of those of normal corneas. CONCLUSIONS: HCEC on the tissue-engineered corneas have morphology and cellular density similar to HCEC in vivo, whereas the pump function of the tissue-engineered corneas was lower than in normal corneas.

Evaluation of posterior capsule opacification using a new posterior view method in rabbits: single-piece acrylic versus 3-piece acrylic intraocular lens.

PURPOSE: To introduce a new procedure for evaluating posterior capsule opacification (PCO) in rabbit eyes and to perform a comparative study of the single-piece and 3-piece acrylic intraocular lenses (IOLs) on PCO using the new evaluation method. SETTING: Jinshikai Medical Foundation, Nishi Eye Hospital, Osaka, Japan. METHODS: A single-piece or 3-piece acrylic IOL was implanted in 1 eye and the other in the contralateral eye of 5 rabbits. Three weeks after surgery, PCO was scored by Evaluation of Posterior Capsule Opacification (EPCO) in posterior view. Before the posterior view was photographed, the anterior capsule was removed from the whole optic area to eliminate disturbing anterior capsule opacification (ACO) for the PCO evaluation. RESULTS: Posterior capsule opacification could be well observed and viewed in the posterior view so that it could be scored by EPCO without confusion with ACO. The mean PCO score of the single-piece and 3-piece acrylic IOLs was 3.12 +/- 0.19 and 2.41 +/- 0.70, respectively (P < .05 and P = .03, respectively). CONCLUSION: The removal of ACO allowed scoring of PCO by EPCO in rabbit eyes. The single-piece acrylic IOL showed significantly more PCO than the 3-piece acrylic IOL at least 3 weeks after surgery in rabbits.