RNA-seq analysis of human trabecular endothelial cells after treatment with timolol maleate.

PURPOSE: Timolol maleate (timolol), a ß-receptor blocker, reduces intraocular pressure by decreasing aqueous humor production. Timolol reportedly also protects ganglion cells, decreases aqueous humor outflow facility, and destroys the extracellular matrix in the trabecular meshwork. In this study, we investigated the effects of timolol on cultured human trabecular endothelial cells purchased from ScienCell using next-generation sequencing. STUDY DESIGN: Experimental investigation. METHODS: Total ribonucleic acid (RNA) was extracted after 24 h. More than 100 million RNAs in control and timolol-treated group were sequenced using a next-generation sequencer. The expression of 55,778 RNAs was analyzed. RESULTS: A total of 2,105 genes were significantly upregulated and 2,125 genes were downregulated, after the addition of timolol. VGF nerve growth factor inducible (VGF) (388-fold) had the maximum increase in expression, followed by amphiregulin (333-fold), a member of the epidermal growth factor family. Moreover, the expression of extracellular matrix-degrading enzymes, matrix metalloproteinases (MMPs) 1, 2, 3, 10, 12, and 14, increased. CONCLUSION: Timolol exerts various effects on human trabecular endothelial cells. The increase in MMP expression may contribute to the decrease in the aqueous humor outflow facility.

Analysis of treatment protocols using azithromycin eye drops for bacterial blepharitis: second report-bacteriological investigation.

PURPOSE: To gain new insights into the etiology of blepharitis, we investigated the causative bacteria in patients with blepharitis and the effects of 1% azithromycin ophthalmic solution. STUDY DESIGN: A multicenter, prospective observational study. METHODS: After the subjects were diagnosed as having blepharitis they were administered 1% azithromycin ophthalmic solution for up to 14 days. Bacterial cultures and smear microscopic examinations of the eyelid margin were conducted at the initial visit, after administering eye drops, and 1 month after the end of eye drop administration. The minimum inhibitory concentrations (MICs) of azithromycin were measured. RESULTS: At the initial visit, the bacterial morphology determined by smear microscopic examinations coincided with that of strains isolated by culture taken from 22 of 45 patients. All detected bacteria were gram-positive, and Corynebacterium spp., Cutibacterium acnes, Staphylococcus epidermidis, Streptococcus spp., and Enterococcus faecalis were isolated most commonly. Compared with the initial visit the number of isolated strains per eye decreased significantly at 7 days after the start of eye drop administration and 1 month after the end of eye drop administration. The azithromycin MICs were temporarily increased after the start of eye drops but then decreased. CONCLUSION: Our study suggests that in blepharitis pathogenicity is characterized by increased strain numbers and amounts of indigenous bacteria. Administering a 1% azithromycin ophthalmic solution suppresses the number of bacterial strains within 1 month after the end of eye drop administration without increasing drug resistance.

Risk Factors for Acetaminophen-induced Liver Injury: A Single-center Study from Japan.

PURPOSE: Acetaminophen has been increasingly used for the treatment of cancer-related pain in Japan since the revision of the package insert on January 21, 2011. However, high-dose acetaminophen may cause liver injury. The objectives of this study were to investigate the prevalence of liver injury in patients receiving acetaminophen and to identify the risk factors. METHODS: The subjects were patients who were treated with acetaminophen ≥1500 mg/d for ≥4 weeks at Ehime University Hospital between April 2011 and December 2014. Drug-induced liver injury was evaluated by alanine aminotransferase and alkaline phosphatase levels, Naranjo score, and Child-Pugh classification. FINDINGS: A total of 287 of 562 patients were treated for 4 weeks with acetaminophen ≥1500 mg/d. Twenty of 102 patients analyzed had drug-induced liver injury. Multivariate analysis was performed with variables from the results of univariate analysis (sex, age ≥70 years, abnormal alanine aminotransferase and alkaline phosphatase levels, and serious liver disease), and age ≥70 years and serious liver disease were significant risk factors. IMPLICATIONS: The findings from the present observational, single-center study suggest that serious liver disease before administration is an independent risk factor for acetaminophen-induced liver injury.

Ciliary sulcus suture fixation of intraocular lens using an auxiliary device.

We developed an injector with a tip that matches the shape of the ciliary sulcus (Ciliary Sulcus Pad Injector) and is used in ciliary sulcus suture fixation of intraocular lenses. The injector is inserted through the corneal incisions and slid along the posterior surface of the iris to the opposite side. When the tip of injector is fitted in the ciliary sulcus, the plunger is pressed, causing the needle to pierce the ciliary sulcus. A suture is inserted in the ciliary sulcus without direct observation. In 91.4% of cases, the needle correctly pierced the ciliary sulcus in a single attempt. The difference between the preoperative expected refractive power and the postoperative spherical equivalent power was -0.63 diopter 6 months after surgery.

Anatomy of the ciliary sulcus and the optimum site of needle passage for intraocular lens suture fixation in the living eye.

PURPOSE: To determine the relationship of the anatomic position of tissue surrounding the ciliary sulcus using ultrasound biomicroscopy (UBM) for measurement of ciliary sulcus parameters and the surrounding tissue after intracapsular cataract extraction (ICCE) and determine the correct suture site for intraocular lens (IOL) suture fixation. SETTING: Sugiura Eye Clinic, Asahi General Hospital, Shizouka-ken, Japan. DESIGN: Prospective case series. METHODS: Using UBM, the angle of the ciliary sulcus and several areas of the tissue surrounding the ciliary sulcus were measured from 8 orientations in eyes after ICCE. These measurements and endoscopic observation of the ciliary sulcus indicated the optimum points of needle insertion and needle emergence on the sclera for IOL suture fixation. Endoscopic observation of the ciliary sulcus also showed its shape. RESULTS: The shape of the surrounding tissue of the ciliary sulcus was measured in 16 eyes after ICCE, and endoscopy was used to observe the ciliary sulcus during surgery in 150 eyes. For ab interno ciliary sulcus suture fixation, the correct point of needle emergence on the sclera was 2.5 mm from the posterior surgical limbus when a straight needle was used. For ab externo pars plana suture fixation, the correct point of needle insertion on the sclera was 3.0 mm from the posterior surgical limbus. Endoscopic observation showed that the lower surface of the ciliary sulcus was formed by the fusion of neighboring ciliary processes. CONCLUSION: Knowing the detailed shape of ciliary sulcus will allow for more precise IOL suture fixation.

Monocular Oculomotor Nerve Disorder Manifesting as Cranial Neuropathy in Systemic Lupus Erythematosus.

We herein report the case of a patient who developed peripheral neuropathy of the bilateral lower legs that later became complicated with isolated oculomotor nerve disorder and was finally diagnosed as systemic lupus erythematosus (SLE). Based on the findings for oculomotor nerve paralysis and contrast-enhanced magnetic resonance imaging findings for the oculomotor nerve in the prepontine cistern, the isolated oculomotor nerve disorder was considered to be a manifestation of peripheral neuropathy. This oculomotor nerve disorder may contribute to the diagnosis of SLE and can be effectively treated with steroid pulse therapy. Reports of SLE manifesting as isolated oculomotor nerve paralysis are rare.

Raman Microscopy: A Noninvasive Method to Visualize the Localizations of Biomolecules in the Cornea.

PURPOSE: In vivo and in situ visualization of biomolecules without pretreatment will be important for diagnosis and treatment of ocular disorders in the future. Recently, multiphoton microscopy, based on the nonlinear interactions between molecules and photons, has been applied to reveal the localizations of various molecules in tissues. We aimed to use multimodal multiphoton microscopy to visualize the localizations of specific biomolecules in rat corneas. METHODS: Multiphoton images of the corneas were obtained from nonlinear signals of coherent anti-Stokes Raman scattering, third-order sum frequency generation, and second-harmonic generation. RESULTS: The localizations of the adhesion complex-containing basement membrane and Bowman layer were clearly visible in the third-order sum frequency generation images. The fine structure of type I collagen was observed in the corneal stroma in the second-harmonic generation images. The localizations of lipids, proteins, and nucleic acids (DNA/RNA) was obtained in the coherent anti-Stokes Raman scattering images. CONCLUSIONS: Imaging technologies have progressed significantly and been applied in medical fields. Optical coherence tomography and confocal microscopy are widely used but do not provide information on the molecular structure of the cornea. By contrast, multiphoton microscopy provides information on the molecular structure of living tissues. Using this technique, we successfully visualized the localizations of various biomolecules including lipids, proteins, and nucleic acids in the cornea. We speculate that multiphoton microscopy will provide essential information on the physiological and pathological conditions of the cornea, as well as molecular localizations in tissues without pretreatment.

SHG-specificity of cellular Rootletin filaments enables naïve imaging with universal conservation.

Despite growing demand for truly naïve imaging, label-free observation of cilium-related structure remains challenging, and validation of the pertinent molecules is correspondingly difficult. In this study, in retinas and cultured cells, we distinctively visualized Rootletin filaments in rootlets in the second harmonic generation (SHG) channel, integrated in custom coherent nonlinear optical microscopy (CNOM) with a simple, compact, and ultra-broadband supercontinuum light source. This SHG signal was primarily detected on rootlets of connecting cilia in the retinal photoreceptor and was validated by colocalization with anti-Rootletin staining. Transfection of cells with Rootletin fragments revealed that the SHG signal can be ascribed to filaments assembled from the R234 domain, but not to cross-striations assembled from the R123 domain. Consistent with this, Rootletin-depleted cells lacked SHG signal expected as centrosome linker. As a proof of concept, we confirmed that similar fibrous SHG was observed even in unicellular ciliates. These findings have potential for broad applications in clinical diagnosis and biophysical experiments with various organisms.

In Vivo and In Vitro Feasibility Studies of Intraocular Use of Polyethylene Glycol-Based Synthetic Sealant to Close Retinal Breaks in Porcine and Rabbit Eyes.

PURPOSE: Absorbable polyethylene glycol-based synthetic sealant (PEG sealant) polymerizes under xenon illumination and forms a clear, flexible, and firmly adherent hydrogel. The intraocular biocompatibility of PEG sealant and efficacy for closing retinal breaks were evaluated. METHODS: In an in vitro study, retinal detachment with a tear was created in porcine eyecups after vitreous gel removal. Polyethylene glycol-based synthetic sealant was applied to cover the tear and polymerized with a 40-second application of xenon light. Retinal adhesion strength was tested by forcefully squirting balanced salt solution (BSS) onto the retinal tear. Polyethylene glycol-based synthetic sealant was soaked in the BSS, incubated at 37°C, and the pH measured periodically over 72 hours. In an in vivo study, PEG sealant was injected into the vitreous cavity of the left eyes of rabbits. Ophthalmologic examinations were performed and bilateral ERGs were recorded simultaneously before and 28 days after injection. The eyes were enucleated for histological evaluation. RESULTS: Adhesion of PEG sealant to the retina was good in BSS. A forceful squirt of BSS onto the retinal tear covered with PEG sealant did not detach the retina; the retinal tear without PEG sealant detached immediately. The pH of the BSS containing PEG sealant was between 7.2 and 8.2. No inflammatory reaction was observed in the eyes throughout 28 days of follow-up. The ERGs recorded before and after injection showed typical patterns. Histological examinations did not reveal any abnormality or inflammation. CONCLUSIONS: Polyethylene glycol-based synthetic sealant appeared to effectively seal retinal breaks and was not toxic to the eye.

Immunohistochemical localization of D-ß-aspartic acid-containing proteins in pterygium.

Biologically uncommon D-ß-aspartic acid (D-ß-Asp) residues have been reported to accumulate in organs affected by age-related disorders. In the present study, we investigated the localization of D-ß-Asp-containing proteins in cases of pterygium, one of the most prominent age-related ocular conditions. Immunohistochemical localization of D-ß-Asp-containing proteins was investigated in surgical specimens of pterygium from 20 patients and control specimens from 10 patients. Strong immunoreactivity to D-ß-Asp-containing proteins was observed in subepithelial elastotic lesions and surrounding collagenous lesions from all surgical specimens with pterygia. In contrast, no immunoreactivity to D-ß-Asp-containing proteins was seen in pterygium-free specimens. D-ß-Asp-containing proteins are produced in organs as they are affected by the aging process. In addition, conversion of L- to D-aspartyl residues is accelerated by ultraviolet (UV) irradiation. Since pterygia can form due to aging or UV exposure, it is reasonable to find D-ß-Asp-containing proteins in specimens with pterygia. Furthermore, since D-ß-Asp is a non-native amino acid, D-ß-Asp-containing proteins may be recognized as allogeneic antigens. Therefore, D-ß-Asp-containing proteins in pterygia may responsible for the fibrovascular changes seen in the disorder.

Multimodal and multiplex spectral imaging of rat cornea ex vivo using a white-light laser source.

We applied our multimodal nonlinear spectral imaging microscope to the measurement of rat cornea. We successfully obtained multiple nonlinear signals of coherent anti-Stokes Raman scattering (CARS), third-order sum frequency generation (TSFG), and second harmonic generation (SHG). Depending on the nonlinear optical processes, the cornea tissue was visualized with different image contrast mechanism simultaneously. Due to white-light laser excitation, multiplex CARS and TSFG spectra were obtained. Combined multimodal and spectral analysis clearly elucidated the layered structure of rat cornea with molecular structural information. This study indicates that our multimodal nonlinear spectral microscope is a promising bioimaging method for tissue study. Multimodal nonlinear spectral images of rat cornea at corneal epithelium and corneal stroma in the in-plane (XY) direction. With use of the combinational analysis of different nonlinear optical processes, detailed molecular structural information is available without staining or labelling.

Clinicopathological features of IgG4-related disease complicated with orbital involvement.

OBJECTIVE: IgG4-related disease (IgG4-RD) is characterized by IgG4-positive plasmacytic infiltration and fibrosis in various organs. Orbital involvement in IgG4-RD includes lacrimal glands, extra-ocular muscles, trigeminal nerve and other parts of the orbit. Immunohistochemical staining is used to diagnose IgG4-RD in patients with orbital inflammation. The purpose of this retrospective study was to clarify the clinicopathological features of IgG4-RD complicated with orbital involvement. METHODS: We examined the clinical features, pathological findings and response to treatment in nine patients with IgG4-RD who underwent orbital tissue biopsy between April 2010 and August 2012 at the University of Tsukuba Hospital. RESULTS: Among the nine patients, eight had dacryoadenitis, one had infraorbital nerve swelling, and another one had IgG4-related orbital inflammation. Involvement of other organs was identified in all patients, including involvement of the salivary glands, lymph nodes, lung, kidney and para-aorta. In all patients, biopsy samples from orbital tissues showed lymphoplasmacytic infiltration and fibrosis, and IgG4-positive/IgG-positive plasmacyte ratio of > 40%. All patients were treated with prednisolone (0.6 mg/kg/day) and responded well in early phase, although relapse was noted in two patients following tapering of prednisolone, evident by swelling of lacrimal glands. CONCLUSION: Patients with IgG4-RD complicated with orbital involvement often present with involvement of other organs. The histopathological findings of orbital tissue match the characteristic features of IgG4-RD. Corticosteroid is effective for orbital and systemic involvement in IgG4-RD.

The role of SIRT1 in ocular aging.

The sirtuins are a highly conserved family of nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylases that helps regulate the lifespan of diverse organisms. The human genome encodes seven different sirtuins (SIRT1-7), which share a common catalytic core domain but possess distinct N- and C-terminal extensions. Dysfunction of some sirtuins have been associated with age-related diseases, such as cancer, type II diabetes, obesity-associated metabolic diseases, neurodegeneration, and cardiac aging, as well as the response to environmental stress. SIRT1 is one of the targets of resveratrol, a polyphenolic SIRT1 activator that has been shown to increase the lifespan and to protect various organs against aging. A number of animal studies have been conducted to examine the role of sirtuins in ocular aging. Here we review current knowledge about SIRT1 and ocular aging. The available data indicate that SIRT1 is localized in the nucleus and cytoplasm of cells forming all normal ocular structures, including the cornea, lens, iris, ciliary body, and retina. Upregulation of SIRT1 has been shown to have an important protective effect against various ocular diseases, such as cataract, retinal degeneration, optic neuritis, and uveitis, in animal models. These results suggest that SIRT1 may provide protection against diseases related to oxidative stress-induced ocular damage, including cataract, age-related macular degeneration, and optic nerve degeneration in glaucoma patients.

Benzalkonium chloride accelerates the formation of the amyloid fibrils of corneal dystrophy-associated peptides.

Corneal dystrophies are genetic disorders resulting in progressive corneal clouding due to the deposition of amyloid fibrils derived from keratoepithelin, also called transforming growth factor ß-induced protein (TGFBI). The formation of amyloid fibrils is often accelerated by surfactants such as sodium dodecyl sulfate (SDS). Most eye drops contain benzalkonium chloride (BAC), a cationic surfactant, as a preservative substance. In the present study, we aimed to reveal the role of BAC in the amyloid fibrillation of keratoepithelin-derived peptides in vitro. We used three types of 22-residue synthetic peptides covering Leu110-Glu131 of the keratoepithelin sequence: an R-type peptide with wild-type R124, a C-type peptide with C124 associated with lattice corneal dystrophy type I, and a H-type peptide with H124 associated with granular corneal dystrophy type II. The time courses of spontaneous amyloid fibrillation and seed-dependent fibril elongation were monitored in the presence of various concentrations of BAC or SDS using thioflavin T fluorescence. BAC and SDS accelerated the fibrillation of all synthetic peptides in the absence and presence of seeds. Optimal acceleration occurred near the CMC, which suggests that the unstable and dynamic interactions of keratoepithelin peptides with amphipathic surfactants led to the formation of fibrils. These results suggest that eye drops containing BAC may deteriorate corneal dystrophies and that those without BAC are preferred especially for patients with corneal dystrophies.

Co-localisation of advanced glycation end products and D-ß-aspartic acid-containing proteins in gelatinous drop-like corneal dystrophy.

PURPOSE: Gelatinous drop-like corneal dystrophy (GDLD), also known as familial subepithelial corneal amyloidosis, is an autosomal recessive disorder that causes progressive corneal opacity due to accumulation of amyloid fibrils in the corneal stroma. Genetic analyses have revealed that a mutation in membrane component chromosome 1 surface marker 1 gene is responsible for GDLD. However, the mechanism of amyloid formation in the corneal stroma remains unclear. The present study attempted to reveal the role of advanced glycation end products (AGE) and d-amino acids in amyloid formation in GDLD. METHODS: Informed consent was obtained from five patients with GDLD, three patients with bullous keratopathy and three patients with interstitial keratitis and all the specimens were analysed. Localisation of amyloid fibrils was analysed using Congo-red and thioflavin T staining. In addition, the localisation of AGE (N(ε)-carboxy(methyl)-L-lysine, pyrraline and pentosidine) and D-ß-aspartic acid-containing proteins, a major form of d-amino acid-containing proteins, was analysed immunohistochemically. RESULTS: In all GDLD specimens, strong immunoreactivity to AGE and D-ß-aspartic acid-containing proteins was detected in the subepithelial amyloid-rich region. In contrast, amyloid fibrils, AGE, or D-amino acid-containing proteins were slightly detected in the corneal stroma of patients with bullous keratopathy and interstitial keratitis. CONCLUSIONS: Abnormally accumulated proteins rich in AGE and D-ß-aspartic acid co-localise in the amyloid lesions in GDLD. These results indicate that non-enzymatic post-translational modifications of proteins, including AGE formation and isomerisation of aspartyl residues, will be the cause as well as the result of amyloid fibril formations in GDLD.

Visualization of irrigation fluid flow and calculation of its velocity distribution in the anterior chamber by particle image velocimetry.

PURPOSE: To visualize irrigation fluid flow and calculate its velocity distribution in the anterior chamber during phacoemulsification by particle image velocimetry. METHODS: Porcine eyes were fixed in a glass chamber filled with balanced salt solution. An ultrasound handpiece was fixed to the glass chamber, and its tip was inserted into the anterior chamber through a corneal incision. Irrigation fluid was mixed with fluorescein-labeled liposomes as tracer particles. During phacoemulsification without ultrasound, a sheet-like Nd-YAG pulsed laser beam was emitted and moved from the iris plane to the top of the cornea continuously. Images of illuminated liposomes in the anterior chamber were captured at short intervals with a CCD camera, and the velocity distribution of irrigation fluid flow was calculated by particle image velocimetry. RESULTS: By particle image velocimetry, the flow velocity distribution could be calculated in any plane of the anterior chamber. Dynamic flow of the irrigation fluid, ejected from the tip of the ultrasound handpiece and returned to an aspiration port, was visualized clearly in the anterior chamber. The maximum flow velocity in the anterior chamber was 342 ± 131 mm/s. CONCLUSIONS: Particle image velocimetry enabled the visualization of irrigation fluid flow and quantification of its velocity distribution in different planes of the anterior chamber during cataract surgery. These data are essential for evaluating the safety and efficacy of new surgical settings and devices during phacoemulsification.

Birefringence measurement of cornea and anterior segment by office-based polarization-sensitive optical coherence tomography.

We present a case series of cornea and anterior segment disorders investigated by an office-based polarization-sensitive optical coherence tomography (PS-OCT). Blebs of glaucoma patients treated by trabeculectomy, and corneas of keratoconus and keratoplasty patients were measured by PS-OCT. Birefringence formations in trabeculectomy bleb were measured in 1 control eye and 3 eyes of trabeculectomy model rabbits. Polarization insensitive scattering OCT and the depth-resolved birefringence were measured simultaneously by PS-OCT. Abnormal birefringence was observed in keratoconus cases with advanced thinning and with a rupture of Descemet's membrane. The graft-host interface of the keratoplasty case showed abnormal birefringence. The appearance of abnormal birefringence in the cornea was likely to be an indication of cross-linking of collagen fibrils. The measurement of rabbit showed abnormal birefringence in the scarring eyes. Wide regions of strong birefringence were observed in the eyes of trabeculectomy patients who had high intraocular pressure. Visualization of scarring in bleb by PS-OCT may be useful for the planning of secondary surgery. PS-OCT showed promising for the study and diagnosis diseases related to abnormal fibrous tissues of the cornea and anterior eye segment.

D-Amino acids in aged proteins: analysis and biological relevance.

Homochirality is essential for life. L-Amino acids are exclusively used as substrates for the polymerization and formation of peptides and proteins in living systems. However, d-amino acids, which are enantiomers of L-amino acids, were recently detected in various living organisms in the form of free D-amino acids and D-amino acid residues in peptides and proteins. In particular, D-aspartyl (Asp) residues have been detected in various proteins from diverse tissues of elderly individuals. Here, we describe three important aspects of our research: (i) a method for detecting D-ß-Asp at specific sites in particular proteins, (ii) a likely spontaneous mechanism by which Asp residues in proteins invert and isomerize to the D-ß-form with age under physiological conditions, (iii) a discussion of factors that favor such a reaction.

UV B-irradiation enhances the racemization and isomerizaiton of aspartyl residues and production of Nε-carboxymethyl lysine (CML) in keratin of skin.

UV-B irradiation is one of the risk factors in age-related diseases. We have reported that biologically uncommon D-ß-Asp residues accumulate in proteins from sun-exposed elderly human skin. A previous study also reported that carboxymethyl lysine (CML; one of the advanced glycation end products (AGEs)) which is produced by the oxidation of glucose and peroxidation of lipid, also increases upon UV B irradiation. The formation of D-ß-Asp and CML were reported as the alteration of proteins in UV B irradiated skin, independently. In this study, in order to clarify the relationship between the formation of D-ß-Asp and CML, immunohistochemical analysis using anti-D-ß-Asp containing peptide antibodies and anti-CML antibodies was performed in UV B irradiated mice. Immunohistochemical analyses clearly indicated that an anti-D-ß-Asp containing peptide antibody and anti-CML antibody reacted at a common area in UV B irradiated skin. Western blot analyses of the proteins isolated from UV B irradiated skin demonstrated that proteins of 50-70 kDa were immunoreactive towards antibodies for both D-ß-Asp containing peptide and CML. These proteins were identified by proteomic analysis as members of the keratin families including keratin-1, keratin-6B, keratin-10, and keratin-14.