Phototoxicity of indocyanine green and brilliant blue G under continuous fluorescent illumination on cultured human retinal pigment epithelial cells,”

PURPOSE. We compared the phototoxicity of indocyanine green (ICG) and Brilliant Blue G (BBG) in cultured RPE cells under fluorescent lamp illumination imitating ambient light. METHODS. Cultured human RPE line cells were stained with ICG or BBG solution at concentrations of clinical use, and cultured in a colorless medium for 24 hours in the dark or under illumination from a fluorescent lamp. After culture, cell morphology and TUNEL-positive apoptotic cells were observed. Cell viability and cell death rate were evaluated. Absorption spectral changes of BBG before and after incubation were measured. RESULTS. ICG-stained cells cultured under illumination changed to an oval morphology with increased number of apoptotic cells, whereas ICG-stained cells cultured in the dark, and BBGstained cells cultured under illumination and dark conditions maintained a flat morphology without increase in apoptotic cells. Cell viability decreased and cell death rate increased only in cells stained by ICG followed by culture under illumination. Staining cells with ICG at one-tenth concentration of clinical usage induced no cytotoxicity after culture under illumination. Approximately 30% of total BBG retained in the stained cells was released into the culture supernatant after incubation for 24 hours. The absorption spectrum of BBG did not change after fluorescent light irradiation. CONCLUSIONS. Illumination with a fluorescent lamp caused cell death via apoptosis in ICG-exposed, but not in BBG-exposed cultured RPE cells. BBG may be a safer dye than ICG because of low light-induced cytotoxicity and rapid elution from stained cells. (Invest Ophthalmol Vis Sci. 2012;53:7389-7394) DOI: 10.1167/iovs.12-10754 P eeling of the internal limiting membrane (ILM) is performed in vitrectomy for macular hole, 1-4 epiretinal membrane, 12-20 Moreover, a metaanalysis indicates that treatment of macular hole with ICG results in worse functional outcomes than treatment without ICG. 21-23 Thus, intravitreal ICG injection remains controversial. Use of safer dyes other than ICG for ILM staining and the conditions of clinical ICG administration have been investigated. Recently, Brilliant Blue G (BBG) has been reported to be a useful and safe dye for visualization of ILM. 13 In the present study, we used RPE cells because RPE is in direct contact with the vital dye at a high concentration in macular hole surgery with ILM peeling, and RPE injury is considered to affect visual prognosis. We compared phototoxicity of ICG and BBG retained in cultured RPE cells under ambient light illumination. Under visible light irradiation, we found that illumination equivalent to conventional fluorescence lamp light induced cytotoxicity in ICG-stained RPE cells but not in BBG-stained cells. MATERIALS AND METHODS Cells and Culture Medium The human RPE cell line ARPE-19 (American Type Culture Collection, Manassas, VA) was cultured in 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium (DMEM/F12; Sigma-Aldrich, Poole, UK) supplemented with 100 U/mL penicillin, 100 lg/mL streptomycin and 10% fetal bovine serum (FBS; JRH Bioscience, Lenexa, KS) at 378C in a humidified atmosphere of 5% CO 2 and 95% air. From th

Transgenic Monkey Model of the Polyglutamine Diseases Recapitulating Progressive Neurological Symptoms

Age-associated neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and the polyglutamine (polyQ) diseases, are becoming prevalent as a consequence of elongation of the human lifespan. Although various rodent models have been developed to study and overcome these diseases, they have limitations in their translational research utility owing to differences from humans in brain structure and function and in drug metabolism. Here, we generated a transgenic marmoset model of the polyQ diseases, showing progressive neurological symptoms including motor impairment. Seven transgenic marmosets were produced by lentiviral introduction of the human ataxin 3 gene with 120 CAG repeats encoding an expanded polyQ stretch. Although all offspring showed no neurological symptoms at birth, three marmosets with higher transgene expression developed neurological symptoms of varying degrees at 3–4 months after birth, followed by gradual decreases in body weight gain, spontaneous activity, and grip strength, indicating time-dependent disease progression. Pathological examinations revealed neurodegeneration and intranuclear polyQ protein inclusions accompanied by gliosis, which recapitulate the neuropathological features of polyQ disease patients. Consistent with neuronal loss in the cerebellum, brain MRI analyses in one living symptomatic marmoset detected enlargement of the fourth ventricle, which suggests cerebellar atrophy. Notably, successful germline transgene transmission was confirmed in the second-generation offspring derived from the symptomatic transgenic marmoset gamete. Because the accumulation of abnormal proteins is a shared pathomechanism among various neurodegenerative diseases, we suggest that this new marmoset model will contribute toward elucidating the pathomechanisms of and developing clinically applicable therapies for neurodegenerative diseases.ArticleeNeuro.4(2):e0250(2017)journal articl

Molecular imaging of aberrant crypt foci in the human colon targeting glutathione S-transferase P1-1

Aberrant crypt foci (ACF), the earliest precursor lesion of colorectal cancers (CRCs), are a good surrogate marker for CRC risk stratification and chemoprevention. However, the conventional ACF detection method with dye-spraying by magnifying colonoscopy is labor- and skill-intensive. We sought to identify rat and human ACF using a fluorescent imaging technique that targets a molecule specific for ACF. We found that glutathione S-transferase (GST) P1-1 was overexpressed in ACF tissues in a screening experiment. We then synthesized the fluorogenic probe, DNAT-Me, which is fluorescently quenched but is activated by GSTP1-1. A CRC cell line incubated with DNAT-Me showed strong fluorescence in the cytosol. Fluorescence intensities correlated significantly with GST activities in cancer cell lines. When we sprayed DNAT-Me onto colorectal mucosa excised from azoxymethane-treated rats and surgically resected from CRC patients, ACF with strong fluorescent signals were clearly observed. The ACF number determined by postoperative DNAT-Me imaging was almost identical to that determined by preoperative methylene blue staining. The signal-to-noise ratio for ACF in DNAT-Me images was significantly higher than that in methylene blue staining. Thus, we sensitively visualized ACF on rat and human colorectal mucosa by using a GST-activated fluorogenic probe without dye-spraying and magnifying colonoscopy

児童養護施設における被虐待児への心理的ケアに関する研究 (1)

Mental health careo for abused chiledren in orphanages has been understandeveloped in the field of both social welfare and clinical psychology. This article reviews psychotherapy for abused children in an orphanage and constructs up a model of effective mental care. Currently it is estimated that more than 50% of children in orphanages experience child abuse, the rate of which has dramaticaly increased year by year. In this regard, the orphanage is forced not only to foster children but also to provide mental health care for aubsed children. The common psychotherapy for abused children so far is play therapy, basically performed by clinical psychologists. Environment therapy from a psychological point of view has also been introduced

Separated Transcriptomes of Male Gametophyte and Tapetum in Rice: Validity of a Laser Microdissection (LM) Microarray

In flowering plants, the male gametophyte, the pollen, develops in the anther. Complex patterns of gene expression in both the gametophytic and sporophytic tissues of the anther regulate this process. The gene expression profiles of the microspore/pollen and the sporophytic tapetum are of particular interest. In this study, a microarray technique combined with laser microdissection (44K LM-microarray) was developed and used to characterize separately the transcriptomes of the microspore/pollen and tapetum in rice. Expression profiles of 11 known tapetum specific-genes were consistent with previous reports. Based on their spatial and temporal expression patterns, 140 genes which had been previously defined as anther specific were further classified as male gametophyte specific (71 genes, 51%), tapetum-specific (seven genes, 5%) or expressed in both male gametophyte and tapetum (62 genes, 44%). These results indicate that the 44K LM-microarray is a reliable tool to analyze the gene expression profiles of two important cell types in the anther, the microspore/pollen and tapetum

Definite Infective Endocarditis: Clinical and Microbiological Features of 155 Episodes in One Japanese University Hospital

To evaluate the epidemiology, clinical features, and microbiological features (including antibiotic susceptibility) of infective endocarditis (IE) at Kitasato University Hospital, Japan. Methods: We retrospectively analyzed 153 patients (155 episodes) with definite IE according to the Duke criteria, who presented over a 17-year period. The minimum inhibitory concentrations of antibiotics for cultured causative microorganisms were also examined. Results: Viridans group streptococci were the most common pathogens (36.8%, 57 episodes), followed by Staphylococcus aureus [21.3%, 33 episodes, including 10 episodes due to methicillin-resistant S. aureus (MRSA)]. Thirty-nine of the 40 strains of viridans streptococci were fully susceptible to penicillin. Comparison of IE due to methicillin-sensitive S. aureus (MSSA) and MRSA showed that the latter had a higher mortality rate (34.8%, 8/23 vs. 70.0%, 7/10). Compared with MSSA, IE caused by MRSA was significantly more likely to be related to nosocomial infection (10/10, p < 0.001), hemodialysis (4/10, 40.0%, p = 0.005), and surgery or intravascular catheter insertion (8/10, 80.0%, p = 0.007). There was a significantly higher mortality rate in non-operated (15/43, 34.9%) than in operated (2/21, 9.5%) (p < 0.001) elderly patients. In 92/155 episodes (59.4%), antibiotics were given before blood cultures were obtained. Culture-negative IE occurred in 20.7% (19/92) of patients on antibiotics versus 6.3% (4/63) of those not on antibiotics (p = 0.02). Of 155 episodes of IE, 34 (21.9%) were fatal and staphylococcal had significantly higher mortality than streptococcal IE [(19/40, 47.5%) vs. (7/72, 9.7%); p < 0.001]. Conclusion: The most frequently isolated pathogens were viridans group streptococci, which differed from other recent studies. In the present study, no penicillin-resistant strains were detected and there was a higher mortality rate for IE caused by MRSA than MSSA. IE should be considered in MRSA patients with the following risk factors: nosocomial infection, hemodialysis, and surgery or intravascular catheter insertion