Better detection of Demodex mites by Löffler's alkaline methylene blue staining in patients with blepharitis.

PURPOSE: To determine whether the Löffler's alkaline methylene blue staining method is better than no staining in detecting Demodex mites in the eyelashes of patients with blepharitis. MATERIALS AND METHODS: Eyelashes were collected from 22 patients with blepharitis. The mean age of the patients was 82.5±6.2 years (± SD) with a range from 71 to 93 years. Eyelashes were epilated by forceps and placed individually on microscope slides. The number of Demodex mites was determined by conventional optical microscopy before and immediately after the addition of the methylene blue staining solution. RESULTS: The mean Demodex count before the addition of the methylene blue solution was 2.9±2.9, and it was 4.4±3.9 after the addition of the methylene blue solution (P<0.01, Wilcoxon test). CONCLUSION: The methylene blue staining method is a simple and useful method in detecting the presence and quantifying the number of Demodex mites. We recommend the methylene blue staining method not only for the diagnosis of the presence of Demodex mites but also to evaluate the therapeutic effects of medications to eliminate the mite infestation.

Rapid alkaline methylene blue supravital staining for assessment of anterior segment infections.

PURPOSE: To present the Löffler's alkaline methylene blue technique of staining eye discharges in eyes with anterior segment infections. METHOD: The Löffler's alkaline methylene blue staining method is a simple staining technique that can be used to differentiate bacterial, viral, and fungal infections. It is a cationic dye that stains cells blue because the positively charged dye is attracted to negatively charged particles such as polyphosphates, DNAs, and RNAs. Specimens collected from patients by swabbing are smeared onto microscope slides and the methylene blue solution is dropped on the slide. The slide is covered with a glass cover slip and examined under a microscope. The entire time from the collection to the viewing is about 30 seconds. RESULTS: Histopathological images of the conjunctival epithelial cells and neutrophils in eye discharges were dyed blue and the nuclei were stained more intensely blue. Bacterial infections consisted mainly of neutrophils, and viral infections consisted mainly of lymphocytes. CONCLUSIONS: Löffler's alkaline methylene blue staining can be done in about 30 seconds for diagnosis. Even though this is a one color stain, it is possible to infer the cause of the infection by detection of the absence of bacteria and/or fungi in context of the differential distribution of neutrophils and lymphocytes.

Linezolid-associated optic neuropathy in a patient with ocular sarcoidosis.

BACKGROUND: We describe a case of bilateral linezolid-associated optic neuropathy in a patient with ocular sarcoidosis. CASE: A 70-year-old woman with sarcoidosis noted foggy vision in both eyes. Best-corrected visual acuity was 0.5 in the right eye and 0.9 in the left. No abnormality other than slight optic disc hyperemia was visible in either eye. A central scotoma in both eyes and enlargement of the blind spot in the right eye were detected by Goldmann perimetry examination, and magnetic resonance imaging demonstrated an edematous optic nerve in the right eye. Therefore, retrobulbar optic neuritis resulting from sarcoidosis was initially suspected. Sub-Tenon's capsule injection of triamcinolone acetonide along with steroid pulse therapy was given; however, best-corrected visual acuity worsened to 0.06 in the right eye and 0.08 in the left. Pulse therapy was discontinued on day 1, and the possibility of linezolid-associated optic neuropathy was speculated because linezolid had been given for methicillin-resistant Staphylococcus aureus osteomyelitis 2 years before by an orthopedist. After discontinuation of linezolid, best-corrected visual acuity improved to 0.8 in the right eye and 0.9 in the left, and the optic disc hyperemia in both eyes disappeared. CONCLUSION: Our findings demonstrate that it is important for ophthalmologists as well as physicians and orthopedists to consider the possibility of optic neuropathy caused by long-term use of linezolid.

[Serious loss of vision in bilateral anterior ischemic optic neuropathy caused by interferon].

BACKGROUND: We treated a patient with anterior ischemic optic neuropathy caused by peginterferon. CASE: Upon medical examination of the eyes before starting interferon therapy for chronic hepatitis C at Saiseikai Izuo hospital, a 64-year-old man showed corrected visual acuity of 0.9 in the right eye and 1.0 in the left. No abnormality was visible in either eye except for mild cataracts. Six weeks after combination therapy with peginterferon alpha-2b and ribavirin was started, corrected visual acuity was found to have decreased in the right eye, and swelling of the optic nervehead in both eyes was evident. Bilateral anterior ischemic optic neuropathy caused by interferon therapy was diagnosed. Combination therapy with peginterferon alpha-2 b and ribavirin was discontinued, and administration of prednisolone was started at a dose of 60 mg. However, visual acuity declined in both eyes and the visual field defects worsend. At the most recent examination, visual acuity was 1.0 in the right eye and 0.01 in the left. The visual field included only the temporal periphery in the left eye, and part of the central and upper temporal periphery in the right. CONCLUSION: Since the outcome of visual acuity and visual fields in anterior ischemic optic neuropathy caused by interferon can be poor, an effective therapy for this complication needs to be developed.

Mecamylamine suppresses Basal and nicotine-stimulated choroidal neovascularization.

PURPOSE: Nicotinic acetylcholine receptors (nAChR) are best known for their role in neurotransmission, but they have recently been demonstrated on vascular endothelial cells. Acetylcholine is their endogenous ligand, but they are also stimulated by nicotine. By stimulating nAChR, nicotine promotes tumor angiogenesis as well as atherosclerotic plaque neovascularization. In this study, the authors investigated the role of nAChR in the pathogenesis of choroidal neovascularization (CNV). METHODS: The effect of the nonselective nAChR antagonist mecamylamine was tested on human retinal and choroidal endothelial cells in vitro and in a murine model of CNV. RESULTS: Several nAChR isoforms were identified in retinal and choroidal microvascular endothelial cells, and the ability of these cells to form tubules when grown in growth factor-reduced basement membrane matrix and supplemented with VEGF was suppressed by the nAChR antagonist mecamylamine. Supplementation of the drinking water of mice with nicotine increased the size of CNV lesions at Bruch membrane rupture sites, an effect that was blocked by subcutaneous administration of mecamylamine (50 mg/kg/d) by an osmotic pump. In the absence of nicotine, CNV formation was suppressed by the infusion of 50 mg/kg/d mecamylamine or by topical application 0.1 or 1% mecamylamine to the cornea. CONCLUSIONS: These data suggest that endogenous activation of nAChR promotes CNV and that activation of nAChR by nicotine may contribute to the increased incidence of CNV seen in smokers with age-related macular degeneration (AMD). Topically administered mecamylamine could provide an appealing new treatment approach for CNV.

[Case with metastasis of a squamous esophageal cancer of the iris with resemblance to hypopyon].

BACKGROUND: Metastasis of a malignant tumor to the iris is rare. We treated a patient with such a metastasis from esophageal cancer. CASE: A 58-year-old man who had had an operation for squamous esophageal cancer complained of conjunctival injection affecting the left eye. On examination, visual acuity in both eyes was 1.2, and intraocular pressure (IOP) in both eyes was 18 mmHg. A grayish tumor with irregular contours was found on the surface of the iris of the left eye at 2 o'clock, and cottonlike material was pooled in the anterior chamber. No metastases elsewhere in the body were clinically evident. After IOP rose to 34 mmHg accompanied by ocular pain, we performed a peripheral iridectomy for diagnosis. Pathologic findings indicated squamous esophageal cancer metastatic to the iris. Metastases to lung and liver were found by computed tomography shortly after hospitalization. Radiotherapy 40 Gy was applied to the iris tumor. IOP then fell, and ocular pain disappeared. CONCLUSION: Metastasis of a squamous esophageal cancer to the iris can resemble a hypopyon. Radiotherapy was effective in this patient.

The SDF-1/CXCR4 ligand/receptor pair is an important contributor to several types of ocular neovascularization.

Hypoxia causes increased expression of several proteins that have the potential to promote neovascularization. Vascular endothelial growth factor (VEGF) is up-regulated by hypoxia in the retina and plays a central role in the development of several types of ocular neovascularization, but the effects of other hypoxia-regulated proteins are less clear. Stromal-derived factor-1 (SDF-1) and its receptor, CXCR4, have hypoxia response elements in the promoter regions of their genes and are increased in hypoxic liver and heart. In this study, we found that SDF-1 and CXCR4 are increased in hypoxic retina, with SDF-1 localized in glial cells primarily near the surface of the retina and CXCR4 localized in bone marrow-derived cells. Glial cells also expressed CXCR4, which suggested the possibility of autocrine stimulation, but influx of bone marrow-derived cells is the major source of increased levels of CXCR4. High levels of VEGF in the retina in the absence of hypoxia also increased levels of Cxcr4 and Sdf1 mRNA. CXCR4 antagonists reduced influx of bone marrow-derived cells into ischemic retina and strongly suppressed retinal neovascularization, VEGF-induced subretinal neovascularization, and choroidal neovascularization. These data suggest that SDF-1 and CXCR4 contribute to the involvement of bone marrow-derived cells and collaborate with VEGF in the development of several types of ocular neovascularization. They provide new targets for therapeutic intervention that may help to bolster and supplement effects obtained with VEGF antagonists.

[Vitrectomy for proliferative diabetic retinopathy].

PURPOSE: We reviewed the outcome of vitrectomy for proliferative diabetic retinopathy (DR) and evaluated factors affecting the final visual outcome. METHODS: We performed primary vitreous surgery for proliferative DR in 148 eyes of 118 cases in three years from July 1999 to August 2002. All cases were followed for at least 3 months. We excluded vitreous surgery for diabetic maculopathy. Ages ranged from 24 to 80 (mean 57) years. Average postoperative follow-up period was 15 months. We evaluated the stage of DR by the new Fukuda classification. RESULT: Preoperative classification consisted of BIV (54 eyes, 36%), BV (94 eyes, 64%), and BV + VI (36 eyes). Final visual acuity was improved by 2 lines or more in 102 eyes (69%), remained unchanged in 28 eyes (19%), and decreased by two lines or more in 18 eyes (12%). There was a statistical correlation between preoperative visual acuity and final visual acuity. Earlier stages of DR had better visual outcome. Compared to the surgical outcome in the 1990s, the percentage of worsened eyes decreased. CONCLUSION: Vitrectomy for proliferative DR may be beneficial if performed in the earlier stages of DR or if the patient has better visual acuity before vitrectomy.

Functional rescue of N-methyl-N-nitrosourea-induced retinopathy by nicotinamide in Sprague-Dawley rats.

PURPOSE: A single intraperitoneal injection of 60 mg/kg body weight of N-methyl-N-nitrosourea (MNU) into rats results in retinal degeneration over a 7-day period in all treated animals. The purpose of this study was to determine whether nicotinamide (NAM) can lead to a functional rescue of the MNU-induced retinopathy. METHODS: NAM, a water-soluble B-group vitamin (vitamin B( 3)), was administered immediately after MNU injection, and retinas were examined morphologically and functionally. RESULTS: Morphologically, 1000 mg/kg NAM completely suppressed and 50 mg/kg NAM partially suppressed the photoreceptor cell loss. Functionally, scotopic and photopic electroretinographic (ERG) recordings showed that both rod and cone photoreceptor cells were well protected from MNU damage by 1000 mg/kg NAM and partially protected by 50 mg/kg NAM. CONCLUSIONS: NAM can protect photoreceptor cells from MNU-induced retinopathy both structurally and functionally.

Dietary docosahexaenoic acid protects against N-methyl-N-nitrosourea-induced retinal degeneration in rats.

The effect of dietary intake of specific types of fatty acids on retinal degeneration due to N-methyl-N-nitrosourea (MNU)-induced photoreceptor cell apoptosis was evaluated. Fifty-day-old female Sprague-Dawley rats were given a single intraperitoneal injection of 50 mg kg(-1) body weight of MNU, and were then switched to one of five different diets containing the following fatty acids at the following weight percentages: 10% linoleic acid (LA); 9.5% palmitic acid (PA) and 0.5% LA; 9.5% eicosapentaenoic acid (EPA) and 0.5% LA; 4.75% EPA, 4.75% docosahexaenoic acid (DHA) and 0.5% LA; or 9.5% DHA and 0.5% LA. When rats developed MNU-induced mammary tumors with a diameter of > or =1 cm, or at the termination of the experiment (20 weeks after MNU injection), retinal tissue samples were obtained and examined. Incidence and severity of retinal damage were compared by histologic examination. MNU-induced retinal degeneration was prevented in rats fed the diet containing 9.5% DHA (4.75% DHA was less effective), whereas it was accelerated in rats fed the 10% LA diet. Over the course of the 20-week experimental period, the fatty acid composition of serum reflected differences in dietary fatty acids. The present results indicate that a diet containing 9.5% DHA can counteract MNU retinotoxicity in the rat retina. DHA may play a role in protection against MNU-induced photoreceptor cell apoptosis in the rat retina.

p63 expression in normal human epidermis and epidermal appendages and their tumors.

BACKGROUND: p63, a member of the p53 gene family, is expressed in basal cells of several different organs. METHODS: The immunoreactivity of p63 was examined in normal human epidermis and epidermal appendages and their tumors, and compared with proliferative activity as evaluated by Ki-67. RESULTS: In normal skin, p63 expression was seen in basal/suprabasal cells of the epidermis, outer root sheath and hair matrix cells of the hair follicle, seboblast situated in the outermost layer of sebaceous glands, and outer layer cells of the ductal portion and myoepithelial cells of the secretory portion of the sweat glands. p63 expression was confined to the cells forming a continuous basal rim along the normal epithelial structure. In tumors, p63 expression resembled that in normal tissue in that tumor components originating from p63-positive cells were constantly positive for p63. In normal and tumor tissues, not all p63-positive cells were positive for Ki-67. CONCLUSIONS: p63 expression may be a marker of basal/progenitor cells in tumors of epidermis and epidermal appendages, and may be a diagnostic marker of these tumors.

Rapid induction of cataract by a single intraperitoneal administration of N-methyl-N-nitrosourea in 15-day-old Sprague-Dawley (Jcl: SD) rats.

Cataract was induced by a single intraperitoneal injection of N-methyl-N-nitrosourea (MNU) in 15-day-old Sprague-Dawley (Jcl: SD) rats. The threshold dose of MNU required for cataract induction in a 3-4 week time period was 70 mg/kg; 60 mg/kg was ineffective. Males and females were both equally affected. Mature cataract as confirmed histologically by degeneration, swelling, vacuolation, liquefaction of the lens fibers, and formation of Morgagni-like water vacuoles was seen in 80% (8/10), 70% (7/10) and 90% (9/10) of 100, 80 and 70 mg/kg MNU-treated rats, respectively, 4 weeks after dosing (43 days of age). At this time point, lens epithelial apoptosis was only rarely seen, but proliferating cell nuclear antigen (PCNA) labeled atypical nuclei with vacuolated lens fibers and macrophage migration were present within the injured lens. Cataracts were the only lesions induced by MNU and there were no other intra- or extraocular lesions seen. The dosing and timing schedule for the MNU administration in rats used in the present study is effective in rapidly causing cataract to occur.

Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina.

It has recently been shown that bone marrow cells can differentiate into various lineage cells including neural cells in vitro and in vivo. We therefore examined whether bone marrow stem cells can differentiate into retinal neural cells in adult rats. PKH-67-labeled stem cell-enriched bone marrow cells (BMCs) were injected into the vitreous space of eyes in which the retinas had been mechanically injured using a hooked needle. Two weeks after the injection of these cells, immunohistochemical examinations were carried out. The stem cell-enriched BMCs had been incorporated and had differentiated into retinal neural cells in the injured retina. The stem cell-enriched BMCs had accumulated mainly in the outer nuclear layer around the injured sites. The incorporated cells expressed glial fibrillary acidic protein, calbindin, rhodopsin, and vimentin. These results raise the possibility that stem cell-enriched BMCs have the ability to differentiate into retinal neural cells, and that the injection of stem cell-enriched BMCs into the retina would help repair damaged retinal cells.

Nicotinamide prevents N-methyl-N-nitrosourea-induced photoreceptor cell apoptosis in Sprague-Dawley rats and C57BL mice.

In previous studies, it was found that a single systemic administration of N-methyl-N-nitrosourea (MNU) to rats and mice resulted in the retinal degeneration in all treated animals over a 7 day period. Retinal degeneration was due to photoreceptor cell apoptosis that was identical to the apoptosis seen in human retinitis pigmentosa (RP). In the present study, nicotinamide (NAM), a water-soluble B-group vitamin (vitamin B(3)), suppressed photoreceptor cell loss in a dose-dependent manner when administered immediately after MNU treatment. In rats, a dose of NAM >or=25 mg kg(-1) completely suppressed photoreceptor cell loss, and 10 mg kg(-1) partially suppressed photoreceptor cell loss. In mice, doses of 1000 and >or=100 mg kg(-1) were needed for complete and partial suppression, respectively. Thus, rats were more responsive to NAM than mice. The retinoprotective effect of 1000 mg kg(-1) NAM lasted throughout the long-term (35 days) observation period, with no apparent toxicity. Also, in rats, 1000 mg kg(-1) NAM completely suppressed photoreceptor cell loss when administered up to 4 hr after MNU treatment, and partially suppressed photoreceptor cell loss when administered 6 hr after MNU treatment. In mice, administration of NAM 2-6 hr after MNU resulted in partial suppression. NAM did not reduce levels of 7-methyldeoxyguanosine DNA adduct, but did reduce photoreceptor cell apoptosis. Although the mechanism of action underlying this retinoprotection remains to be clarified, NAM may be a potential therapeutic agent for the treatment of retinal degeneration.

Caspase-3 inhibitor transiently delays inherited retinal degeneration in C3H mice carrying the rd gene.

BACKGROUND: The effect of a caspase-3 inhibitor on retinal degeneration in C3H mice carrying the rd gene, a mutation of a rod-specific phosphodiesterase, was investigated. METHODS: A quantity of 2 mg/kg of Ac-DEVD-CHO, as inhibitor, was injected intraperitoneally every other day from 8 days of age, and retinal damage was compared with that in saline-treated C3H mice at 13 days (1 day after the third treatment) and 17 days of age (1 day after the fifth treatment). Retina of ICR mice not carrying rd gene was also evaluated under the same protocol. The efficacy of Ac-DEVD-CHO was evaluated based on total retinal thickness and outer retinal thickness (thickness of outer nuclear layer and photoreceptor layer). An apoptotic index and a cell proliferation index for the photoreceptor cells, at 13 days of age, were calculated based on terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling (TUNEL) and proliferating cell nuclear antigen (PCNA) labeling, respectively. RESULTS: At 13 days of age, total and outer retinal thickness in saline-treated C3H mice were 140.3 microm and 37.5 microm, compared with 160.4 microm and 49.5 microm, respectively, in Ac-DEVD-CHO-treated C3H mice ( P<0.01, respectively). In ICR mice, total and outer retinal thickness were 182.1 microm and 90.9 microm, respectively, in saline-treated mice and 183.8 microm and 89.6 microm in Ac-DEVD-CHO-treated mice (not significant). At this time, the TUNEL index was 23.52 cells/10(4) microm (2) of outer nuclear layer in saline-treated C3H mice; Ac-DEVD-CHO treatment significantly reduced this value to 18.73 cells/10(4) microm(2) ( P<0.05). The TUNEL index in saline- and Ac-DEVD-CHO-treated ICR mice was 0.59 cells/10(4) microm(2) and 0.80 cells/10(4) microm(2), respectively (not significant); Ac-DEVD-CHO treatment had no influence on normally developing retina. The PCNA index was not affected by Ac-DEVD-CHO-treatment. However, at 17 days of age, Ac-DEVD-CHO treatment did not ameliorate retinal degeneration. CONCLUSIONS: The caspase-3 inhibitor was transiently effective in delaying retinal degeneration through inhibition of the apoptosis of photoreceptor cells in rd gene-carrying mice. The use of caspase-3 inhibitors may have therapeutic applications in the treatment of human retinal degeneration.

Morphologic characteristics of retinal degeneration induced by sodium iodate in mice.

PURPOSE: Retinal degeneration induced by sodium iodate (NaIO( 3)) in mice was evaluated morphologically. METHODS: Male and female ICR and C57BL mice were intraperitoneally administered 100 mg/kg NaIO(3) at 7 weeks of age, and were killed 6, 12, 24 hrs, and 3, 7 and 28 days after the treatment. Retinas were examined histologically, ultrastructurally, immunohistochemically, and by the TUNEL method. RESULTS: Retinal degeneration was evoked in all NaIO(3)-treated mice. The primary site of damage appeared in the retinal pigment epithelial (RPE) cells followed by photoreceptor cell degeneration. Initially, the RPE cells showed necrosis starting 6 hrs post-NaIO(3), followed by photoreceptor outer segment disruption and photoreceptor cell apoptosis at 24 hrs; photoreceptor cell apoptosis peaked at day 3 and was completed by day 7. At day 3, Müller cell proliferation, macrophage migration within the retina, and regeneration of damaged RPE cells occurred. Finally at day 7 and day 28, the retina showed a mosaic pattern of relatively normal retina and areas lacking RPE cells and photoreceptor cells. CONCLUSIONS: RPE cell necrosis followed by photoreceptor cell apoptosis and the resulting mosaic pattern of the retina phenotypically resembles gyrate atrophy of the choroid and retina.