Potential biomarker proteins for aspiration pneumonia detected by shotgun proteomics using buccal mucosa samples: a cross-sectional case-control study.

BACKGROUND: Aspiration pneumonia (AP), which is a major cause of death in the elderly, does present with typical symptoms in the early stages of onset, thus it is difficult to detect and treat at an early stage. In this study, we identified biomarkers that are useful for the detection of AP and focused on salivary proteins, which may be collected non-invasively. Because expectorating saliva is often difficult for elderly people, we collected salivary proteins from the buccal mucosa. METHODS: We collected samples from the buccal mucosa of six patients with AP and six control patients (no AP) in an acute-care hospital. Following protein precipitation using trichloroacetic acid and washing with acetone, the samples were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS/MS). We also determined the levels of cytokines and chemokines in non-precipitated samples from buccal mucosa. RESULTS: Comparative quantitative analysis of LC-MS/MS spectra revealed 55 highly (P values < 0.10) abundant proteins with high FDR confidence (q values < 0.01) and high coverage (> 50%) in the AP group compared with the control group. Among the 55 proteins, the protein abundances of four proteins (protein S100-A7A, eukaryotic translation initiation factor 1, Serpin B4, and peptidoglycan recognition protein 1) in the AP group showed a negative correlation with the time post-onset; these proteins are promising AP biomarker candidates. In addition, the abundance of C-reactive protein (CRP) in oral samples was highly correlated with serum CRP levels, suggesting that oral CRP levels may be used as a surrogate to predict serum CRP in AP patients. A multiplex cytokine/chemokine assay revealed that MCP-1 tended to be low, indicating unresponsiveness of MCP-1 and its downstream immune pathways in AP. CONCLUSION: Our findings suggest that oral salivary proteins, which are obtained non-invasively, can be utilized for the detection of AP.

A multilocus sequence typing method of Staphylococcus aureus DNAs in a sample from human skin.

The skin and mucous membranes are the primary sites of Staphylococcus aureus colonization, particularly those of health care personnel and patients in long-term care centers. We found that S. aureus colonized with a higher abundance ratio on skins which had recovered from pressure injury (PI) than on normal skins in our earlier research on the skin microbiota of bedridden patients. Multilocus sequence typing (MLST) is a useful tool for typing S. aureus isolated from clinical specimens. However, the MLST approach cannot be used in microbiota DNA owing to the contamination from other bacteria species. In this study, we developed a multiplex-nested PCR method to determine S. aureus MLST in samples collected from human skins. The seven pairs of forward and reverse primers were designed in the upstream and downstream regions, which were conserved specifically in S. aureus. The first amplifications of the seven pairs were conducted in a multiplex assay. The samples were diluted and applied to conventional PCR for MLST. We confirmed that the method amplified the seven allele sequences of S. aureus specifically in the presence of untargeted DNAs from human and other skin commensal bacteria. Using this assay, we succeeded in typing sequence types (STs) of S. aureus in the DNA samples derived from the skins healed from PI. Peaks obtained by Sanger sequencing showed that each sample contained one ST, which were mainly categorized into clonal complex 1 (CC1) or CC5. We propose that this culture-free approach may be used in detecting S. aureus in clinical specimens without isolation.

Specific Activation of Yamanaka Factors via HSF1 Signaling in the Early Stage of Zebrafish Optic Nerve Regeneration.

In contrast to the case in mammals, the fish optic nerve can spontaneously regenerate and visual function can be fully restored 3-4 months after optic nerve injury (ONI). However, the regenerative mechanism behind this has remained unknown. This long process is reminiscent of the normal development of the visual system from immature neural cells to mature neurons. Here, we focused on the expression of three Yamanaka factors (Oct4, Sox2, and Klf4: OSK), which are well-known inducers of induced pluripotent stem (iPS) cells in the zebrafish retina after ONI. mRNA expression of OSK was rapidly induced in the retinal ganglion cells (RGCs) 1-3 h after ONI. Heat shock factor 1 (HSF1) mRNA was most rapidly induced in the RGCs at 0.5 h. The activation of OSK mRNA was completely suppressed by the intraocular injection of HSF1 morpholino prior to ONI. Furthermore, the chromatin immunoprecipitation assay showed the enrichment of OSK genomic DNA bound to HSF1. The present study clearly showed that the rapid activation of Yamanaka factors in the zebrafish retina was regulated by HSF1, and this sequential activation of HSF1 and OSK might provide a key to unlocking the regenerative mechanism of injured RGCs in fish.

Relationship between healing status and microbial dissimilarity in wound and peri-wound skin in pressure injuries.

AIM: Wound infection is the most serious cause of delayed healing for patients with pressure injuries. The wound microbiota, which plays a crucial role in delayed healing, forms by bacterial dissemination from the peri-wound skin. To manage the bioburden, wound and peri-wound skin care has been implemented; however, how the microbiota at these sites contribute to delayed healing is unclear. Therefore, we investigated the relationship between healing status and microbial dissimilarity in wound and peri-wound skin. METHODS: A prospective cohort study was conducted at a long-term care hospital. The outcome was healing status assessed using the DESIGN-R® tool, a wound assessment tool to monitor the wound healing process. Bacterial DNA was extracted from the wound and peri-wound swabs, and microbiota composition was analyzed using 16S rRNA gene analysis. To evaluate microbial similarity, the weighted UniFrac dissimilarity index between wound and peri-wound microbiota was calculated. RESULTS: Twenty-two pressure injuries (7 deep and 15 superficial wounds) were included in the study. For deep wounds, the predominant bacteria in wound and peri-wound skin were the same in the healing wounds, whereas they were different in all cases of hard-to-heal wounds. Analysis based on the weighted UniFrac dissimilarity index, there was no significant difference for healing wounds (p = 0.639), while a significant difference was found for hard-to-heal wounds (p = 0.047). CONCLUSIONS: Delayed healing is possibly associated with formation of wound microbiota that is different in composition from that of the skin commensal microbiota. This study provides a new perspective for assessing wound bioburden.

Increased temperature at the healed area detected by thermography predicts recurrent pressure ulcers.

Preventing recurrent pressure ulcers is an important challenge in healthcare. One of the reasons for the high rate of recurrent pressure ulcers is the lack of assessment methods for their early detection. Therefore, this study aimed to determine the thermographic characteristics of the healed area and to consider the predictive validity of thermographic images for recurrent pressure ulcers within a 2-week period. This observational study was conducted at a long-term care facility in Japan between July 2017 and February 2019 among patients whose pressure ulcers had healed. Thermographic images of the healed area were recorded once a week until recurrence or until the end of the study. We enrolled 30 participants, among whom 8 developed recurrent pressure ulcers. The generalised estimation equation revealed that the thermographic finding of increased temperature at the healed area compared to that of the surrounding skin was significantly associated with recurrent pressure ulcers (odds ratio: 101.13, 95% confidence interval: 3.60-2840.77, p = .007); the sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio and negative likelihood ratio for recurrent pressure ulcers within 2 weeks were 0.80, 0.94, 0.62, 0.97, 12.9 and 0.2, respectively. Our thermographic findings revealed that the temperature of the healed area was higher than that of the surrounding skin; this could be a useful predictor of pressure ulcer recurrence within 2 weeks, even in the absence of macroscopic changes.

Amplicon-based skin microbiome profiles collected by tape stripping with different adhesive film dressings: a comparative study.

BACKGROUND: Medical film dressings have been used to obtain skin microbiota for skin microbiome studies, although their adhesive force may be so strong that the skin could be injured when applied to those who have fragile skin, such as older people. Several products with less adhesive force are available, although their applicability for skin microbiome studies remains unknown. This study aimed to test whether the dressings with less adhesive force could be used for amplicon-based skin microbiome studies. A set of three different film dressings, with acrylic, urethane, or silicone adhesive, was applied to the back skin of nine healthy young participants. The copy number of the 16S ribosomal RNA (rRNA) gene, microbial compositions, and alpha and beta diversity indices were analyzed by amplicon analysis of the 16S rRNA gene using next-generation sequencing and were compared among the three film dressings. RESULTS: The dressing with acrylic adhesive yielded the highest copy number of 16S rRNA genes, followed by that with urethane adhesive. The silicone-adhesive dressing yielded a significantly lower copy number of the 16S rRNA gene. The microbial composition of skin microbiota was similar among the three film dressings, although significant differences in the relative abundance of Pseudomonas species and alpha diversity indices were found in the silicone-adhesive dressing. The Bray-Curtis dissimilarity was significantly higher between the acrylic- and silicone-adhesive dressings than between the acrylic- and urethane-adhesive dressings. No adverse effects related to tape stripping were observed for any of the film dressings. CONCLUSION: We recommend dressings with acrylic or urethane adhesive for amplicon-based skin microbiome studies. An acrylic adhesive has an advantage in the yield of skin microbiota, and a urethane adhesive should be chosen when applied to fragile skin. The adhesive force of the dressing with silicone adhesive was too weak to be used for collecting skin microbiota.

Effects of skin moisturizer on the skin barrier dysfunction model: An evaluation of the heel via tape-stripping in healthy, young adults.

BACKGROUND: This study seeks to establish the skin barrier dysfunction model at the heel via tape-stripping (TS) by evaluating the skin moisturizing effects. MATERIALS AND METHODS: Nineteen young, female participated in the study. A sequence of TS was performed at the heel and trans-epidermal water loss (TEWL), stratum corneum (SC) hydration, and surface pH were measured. Following TS, the subjects were divided into three groups: moisturizer, emollient, and overcoat. These agents were applied daily at night, and the skin parameters were measured the next morning for a week. RESULTS: The TEWL value of TS immediately and 5 min after TS were significantly higher than what was obtained before TS, while the SC hydration after TS was significantly lower than what was obtained before TS. However, there were no significant differences in the skin parameters among the three agents on day 7 after application, with the two-way ANOVA showing no interaction among the agents and number of days. CONCLUSION: The skin barrier dysfunction model at the heel was established by TS in healthy, young adults. However, the physiological function of the skin at the heel did not change drastically and showed no differences even after continuous application for 7 days.

Development of recurrent pressure ulcers, risk factors in older patients: a prospective observational study.

OBJECTIVE: Prevention of recurrent pressure ulcers (PU) is one of the most important challenges in wound care, furthermore, the risk factors for recurrent PUs are still not fully understood. This study aimed to explore the risk factors for recurrent PU development within two weeks, including biophysical skin properties, pro-inflammatory cytokine (tumour necrosis factor [TNF]-α) levels and bacterial species, in older patients. METHOD: This prospective study was conducted in a long-term care facility with patients whose PU had healed within two months. Biophysical skin properties were evaluated by stratum corneum hydration, pH, sebum content and transepidermal water loss. TNF-α level was measured using skin blotting. Skin bacteria were collected using tape stripping and determined by species-specific gene amplification. These parameters, along with Braden scale and interface pressure, were evaluated every two weeks for a total period of eight weeks. A penalised generalised estimating equation analysis was used to determine the risk factors for recurrent PUs. RESULTS: In total, 20 patients were included in this study, with 57 observations. Of these, recurrent PU was seen in eight observations. Elevation of pH (p=0.049; odds ratio [OR] per 1 unit=3.91, 95% confidence interval [CI]:1.01-15.15), presence of Acinetobacter spp. (p=0.039; OR versus culture-negative=6.28, 95%CI:1.10-35.86) and higher interface pressure (p=0.008; OR per 1 mmHg=1.06, 95%CI:1.01-1.10) on the healed PU were significantly related to the development of recurrent PU. CONCLUSION: Higher pH, existence of Acinetobacter spp. and higher interface pressure on the site of the healed PU were associated with the development of recurrent PUs in older patients undergoing conservative treatments.

A novel activation mechanism of cellular Factor XIII in zebrafish retina after optic nerve injury.

Cellular Factor XIII (cFXIII) mRNA is rapidly upregulated in the fish retina after optic nerve injury (ONI). Here, we investigated the molecular mechanism of cFXIII gene activation using genetic information from the A-subunit of cFXIII (cFXIII-A). Real-time PCR that amplified the active site (exons 7-8) of cFXIII-A showed increased cFXIII-A mRNA in the retina after ONI, whereas the PCR that amplified the activation peptide (exons 1-2) showed no change. RT-PCR analysis that amplified exons 1-8 showed two bands, a faint long band in the control retina and a dense short band in the injured retina. Therefore, we conclude that activated cFXIII-A mRNA after ONI is shorter than that of the control retina. Western blot analysis also confirmed an active form of 65 kDa cFXIII-A protein in the injured retina compared to the control 84 kDa protein. 5'-RACE analysis using injured retina revealed that the short cFXIII-A mRNA lacked exons 1, 2 and part of exon 3. Exon 3 has two sites of heat shock factor 1 (HSF-1) binding consensus sequence. Intraocular injection of HSF inhibitor suppressed the expression of cFXIII-A mRNA in the retina 1 day after ONI to 40% of levels normally seen after ONI. Chromatin immunoprecipitation provides direct evidence of enrichment of cFXIII-A genomic DNA bound with HSF-1. The present data indicate that rapid HSF-1 binding to the cFXIII-A gene results in cleavage of activation peptide and an active form of short cFXIII-A mRNA and protein in the zebrafish retina after ONI without thrombin.

Comparison of ceramide retention in the stratum corneum between dry skin and normal skin using animal model with fluorescent imaging method.

BACKGROUND/PURPOSE: Skin care via moisturization compensates for the lack of skin barrier function. However, moisturizer application methods are not clearly decided. Here, we focused on and examined the retention of externally applied ceramide in the stratum corneum (SC) using fluorescent imaging method. This study aimed to compare ceramide retention in the SC between normal skin and dry skin using an animal model. METHODS: Nine-week-old Sprague-Dawley rats were divided into two groups: normal skin and dry skin model. The dry skin model group was treated with acetone-diethyl ether solution. A fluorescently labeled ceramide solution was prepared and applied to rats' back skin. Skin samples were taken at 0 minute and 12 hours after ceramide application. Fluorescently labeled ceramide was evaluated and observed under a microscope. RESULTS: The intensity of externally applied ceramide in the normal skin group showed no significant change from 0 minute to 12 hours after application. In contrast, in the dry skin model group, the intensity of externally applied ceramide increased significantly from 0 minute to 12 hours after application. CONCLUSION: Our findings demonstrate that the externally applied ceramide penetrated the SC of dry skin more than that of normal skin.

Comparison of wiping methods for the removal of cleaning agent residue from hair follicles.

PURPOSE: This study aimed to clarify the influence of different wiping methods on cleaning agent residue in hair follicles when using skin cleaning agents that are "removable-by-wiping." METHODS: A total of 18 male volunteers were recruited and a cleaning agent containing 10% fluorescein sodium salt (cleaning agent) was used in this study. After gentle washing, the cleaning agent was removed via the washing method (control) or three distinct wiping methods (experiment). Hairs were obtained from the measurement area. The fluorescence intensities of the residual cleaning agent on the hair root and hair bulb were then evaluated after normalizing for autofluorescence from the hair. Fluorescence intensity was used to estimate the amount of cleaning agent residue. RESULTS: No significant differences were found among cleaning agent removal methods (hair root: P = 0.67, hair bulb: P = 0.62) with respect to the amount of residual cleaning agent on hair. CONCLUSION: Cleaning agent residual index did not differ according to removal method. Cleaning agent remained in the hair follicle (hair roots, hair bulbs) despite removal from the skin by washing or wiping. Further studies are required to apply "removable-by-wiping" cleaning agents for people with vulnerable skin.

Alternative Splicing for Activation of Coagulation Factor XIII-A in the Fish Retina After Optic Nerve Injury.

Factor XIII-A (FXIII-A), which has become known as cellular transglutaminase, plays important roles in mediating cross-linking reactions in various tissues. FXIII-A acts as one of the regeneration molecules in the fish retina and optic nerve after optic nerve injury and becomes activated at the site of injury within a few hours. Previous research has shown that activated FXIII-A induces neurite outgrowth from injured retinal ganglion cells and supports elongation of the regenerating optic nerve. However, the activation mechanism of FXIII-A remains unknown. Furthermore, the injured tissues do not express thrombin, a known activator of plasma FXIII. Here, we investigated the mRNA expression of FXIII-A based on two different regions, one encoding the activation peptide and the other encoding the enzymatic active site. We found that expression of the region encoding the activation peptide was markedly suppressed compared with the region encoding the active site. An overexpression study with a short-type FXIII-A cDNA lacking the activation peptide revealed induction of long neurite outgrowth in fish retinal explant cultures compared with full-length FXIII-A cDNA. The present findings suggest that alternative splicing may occur in the FXIII-A gene, resulting in deletion of the region encoding the activation peptide and thus allowing direct production of activated FXIII-A protein in the fish retina and optic nerve after optic nerve injury.

RANKL, Ephrin-Eph and Wnt10b are key intercellular communication molecules regulating bone remodeling in autologous transplanted goldfish scales.

This study aimed to investigate the precise data of gene expression, functions, and chronological relationships amongst communication molecules involved in the bone remodeling process with an in vivo model using autologous transplanted scales of goldfish. Autotransplantation of methanol-fixed cell-free scales triggers scale resorption and regeneration, as well as helps elucidate the process of bone remodeling. We investigated osteoclastic markers, osteoblastic markers, and gene expressions of communicating molecules (RANKL, ephrinB2, EphB4, EphA4, Wnt10b) by qPCR, in situ hybridization for Wnt10b, and immunohistochemistry for EphrinB2 and EphA4 proteins to elucidate the bone remodeling process. Furthermore, functional inhibition experiments for the signaling of ephrinB2/Eph, ephrin/EphA4, and Wnt10b using specific antibodies, revealed that these proteins are involved in key signaling pathways promoting normal bone remodeling. Our data suggests that the remodeling process comprises of two successive phases. In the first absorption phase, differentiation of osteoclast progenitors by RANKL is followed by the bone absorption by mature, active osteoclasts, with the simultaneous induction of osteoblast progenitors by multinucleated osteoclast-derived Wnt10b, and proliferation of osteoblast precursors by ehprinB2/EphB4 signaling. Subsequently, during the second formation phase, termination of bone resorption by synergistic cooperation occurs, with downregulation of RANKL expression in activated osteoblasts and Ephrin/EphA4-mediated mutual inhibition between neighboring multinucleated osteoclasts, along with simultaneous activation of osteoblasts via forward and reverse EphrinB2/EphB4 signaling between neighboring osteoblasts. In addition, the present study shows that autologous transplantation of methanol-fixed cell-free scale is an ideal in vivo model to study bone remodeling.

A novel function of neuroglobin for neuroregeneration in mice after optic nerve injury.

Neuroglobin (Ngb) is a recently discovered heme protein in the vertebrate brain that can bind to oxygen molecules. Mammalian Ngb plays a crucial role in neuroprotection under conditions of oxidative stress. To investigate other potential functions of Ngb, we investigated the mouse retinal Ngb system following optic nerve injury. In the retina of control mice, Ngb immunoreactivity was limited to the retinal ganglion cell (RGC) layer, and this immunoreactivity rapidly decreased to less than 50% of the control level 5 days after optic nerve injury. On the basis of this decrease, we designed in vivo experiments with enhanced expression of Ngb using adult mouse retina. The enhanced expression of Ngb was achieved by injecting chimeric human Ngb protein, which included the cell membrane-penetrating module of fish Ngb. One-day pretreatment with chimeric Ngb increased immunoreactivity levels of Ngb two-fold in mouse RGCs and increased the number of surviving RGCs three-fold by 14 days after optic nerve injury compared with vehicle controls. Furthermore, in the mouse retinas showing enhanced Ngb expression, several regenerating central optic axons exhibited outgrowth and were found to pass through the nerve crush site 14 days after nerve injury. No such regenerating optic axons were observed in the control mouse optic nerve during the same time frame. The data obtained from in vivo experiments strongly indicate that mammalian Ngb has neuroprotective and neuroregenerative properties.

Effects of hyperglycemia on bone metabolism and bone matrix in goldfish scales.

Increased risk of fracture associated with type 2 diabetes has been a topic of recent concern. Fracture risk is related to a decrease in bone strength, which can be affected by bone metabolism and the quality of the bone. To investigate the cause of the increased fracture rate in patients with diabetes through analyses of bone metabolism and bone matrix protein properties, we used goldfish scales as a bone model for hyperglycemia. Using the scales of seven alloxan-treated and seven vehicle-treated control goldfish, we assessed bone metabolism by analyzing the activity of marker enzymes and mRNA expression of marker genes, and we measured the change in molecular weight of scale matrix proteins with SDS-PAGE. After only a 2-week exposure to hyperglycemia, the molecular weight of α- and ß-fractions of bone matrix collagen proteins changed incrementally in the regenerating scales of hyperglycemic goldfish compared with those of euglycemic goldfish. In addition, the relative ratio of the γ-fraction significantly increased, and a δ-fraction appeared after adding glyceraldehyde-a candidate for the formation of advanced glycation end products in diabetes-to isolated type 1 collagen in vitro. The enzymatic activity and mRNA expression of osteoblast and osteoclast markers were not significantly different between hyperglycemic and euglycemic goldfish scales. These results indicate that hyperglycemia is likely to affect bone quality through glycation of matrix collagen from an early stage of hyperglycemia. Therefore, non-enzymatic glycation of collagen fibers in bone matrix may lead to the deterioration of bone quality from the onset of diabetes.

Relationship between maceration and wound healing on diabetic foot ulcers in Indonesia: a prospective study.

The aim of this study was to clarify the relationship between maceration and wound healing. A prospective longitudinal design was used in this study. The wound condition determined the type of dressings used and the dressing change frequency. A total of 62 participants with diabetic foot ulcers (70 wounds) were divided into two groups: non-macerated (n = 52) and macerated wounds (n = 18). Each group was evaluated weekly using the Bates-Jensen Wound Assessment Tool, with follow-ups until week 4. The Mann-Whitney U test showed that the changes in the wound area in week 1 were faster in the non-macerated group than the macerated group (P = 0·02). The Pearson correlation analysis showed a moderate correlation between maceration and wound healing from enrolment until week 4 (P = 0·002). After week 4, the Kaplan-Meier analysis showed that the non-macerated wounds healed significantly faster than the macerated wounds (log-rank test = 19·378, P = 0·000). The Cox regression analysis confirmed that maceration was a significant and independent predictor of wound healing in this study (adjusted hazard ratio, 0·324; 95% CI, 0·131-0·799; P = 0·014). The results of this study demonstrated that there is a relationship between maceration and wound healing. Changes in the wound area can help predict the healing of wounds with maceration in clinical settings.

Evaluation of validity of the new diabetic foot ulcer assessment scale in Indonesia.

We developed a new assessment tool for diabetic foot ulcers because no such tool specifically for diabetic foot ulcer exists. The diabetic foot ulcer assessment scale (DFUAS) has 11 domain items. The minimum and maximum scores on this scale are 0 and 98, respectively; higher scores indicate more severe wounds. The aim of this study was to evaluate the concurrent validity, construct validity and predictive validity of DFUAS in Indonesia. A prospective cohort study was conducted on patients with diabetic foot ulcer at Kitamura wound clinic in Indonesia. A total of 62 patients with 70 diabetic foot ulcers were assessed with DFUAS tool, Bates-Jensen wound assessment tool (BWAT), and pressure ulcer scale for healing (PUSH). Concurrent validity was determined by correlation of the DFUAS total score with the external criterion (BWAT, PUSH, and wound surface area). A comparison between the total DFUAS score and chronic wound status was made to determine construct validity. We also analyzed 41 wounds that were followed for 4 weeks to evaluate predictive validity. The correlation coefficient total scores of the DFUAS against the BWAT, PUSH, and wound surface area were 0.92, 0.87, and 0.82, respectively. The comparison of the total DFUAS score with chronic wound status was p < 0.001. The predictive validity test indicated that a DFUAS cutoff score of 12 produced the best balance of sensitivity, specificity, positive predictive value, and negative predictive value (89%, 71%, 86%, and 77%, respectively). In conclusion, the newly developed DFUAS is a valid tool for assessing diabetic foot ulcers.

Geranylgeranylacetone Suppresses N-Methyl-N-nitrosourea-Induced Photoreceptor Cell Loss in Mice.

Retinitis pigmentosa is a disease characterized by the loss of photoreceptor cells. The N-methyl-N-nitrosourea (MNU)-induced retinal degeneration model is widely used to study the mechanism of these retinal degenerative disorders because of its selective photoreceptor cell death. As for the cell death mechanism of MNU, calcium-calpain activation and lipid peroxidation processes are involved in the initiation of this cell death. Although such molecular mechanisms of the MNU-induced cell death have been described, the total image of the cell death is still obscure. Heat shock protein 70 (HSP70) has been shown to function as a chaperon molecule to protect cells against environmental and physiological stresses. In this study, we investigated the effect of geranylgeranylacetone (GGA), an accylic polyisoprenoid, on MNU-induced photoreceptor cell loss. HSP70 induction by GGA was effective against MNU-induced photoreceptor cell loss as a result of its ability to prevent HSP70 degradation. The data indicate that GGA may help to suppress the onset and progression of retinitis pigmentosa.

Nitric Oxide Synthase Activation as a Trigger of N-methyl-N-nitrosourea-Induced Photoreceptor Cell Death.

Retinal degeneration (RD) such as retinitis pigmentosa and age-related macular degeneration are major causes of blindness in adulthood. As one of the model for RD, intraperitoneal injection of N-methyl-N-nitrosourea (MNU) is widely used because of its selective photoreceptor cell death. It has been reported that MNU increases intracellular calcium ions in the retina and induces photoreceptor cell death. Although calcium ion influx triggers the neuronal nitric oxide synthase (nNOS) activation, the role of nNOS on photoreceptor cell death by MNU has not been reported yet. In this study, we investigated the contribution of nNOS on photoreceptor cell death induced by MNU in mice. MNU significantly increased NOS activation at 3 day after treatment. Then, we evaluated the effect of nNOS specific inhibitor, ethyl[4-(trifluoromethyl) phenyl]carbamimidothioate (ETPI) on the MNU-induced photoreceptor cell death. At 3 days, ETPI clearly inhibited the MNU-induced cell death in the ONL. These data indicate that nNOS is a key molecule for pathogenesis of MNU-induced photoreceptor cell death.

Cell Fate of Müller Cells During Photoreceptor Regeneration in an N-Methyl-N-nitrosourea-Induced Retinal Degeneration Model of Zebrafish.

Zebrafish can regenerate several organs such as the tail fin, heart, central nervous system, and photoreceptors. Very recently, a study has demonstrated the photoreceptor regeneration in the alkylating agent N-methyl-N-nitrosourea (MNU)-induced retinal degeneration (RD) zebrafish model, in which whole photoreceptors are lost within a week after MNU treatment and then regenerated within a month. The research has also shown massive proliferation of Müller cells within a week. To address the question of whether proliferating Müller cells are the source of regenerating photoreceptors, which remains unknown in the MNU-induced zebrafish RD model, we employed a BrdU pulse-chase technique to label the proliferating cells within a week after MNU treatment. As a result of the BrdU pulse-chase technique, a number of BrdU(+) cells were observed in the outer nuclear layer as well as the inner nuclear layer. This implies that regenerating photoreceptors are derived from proliferating Müller cells in the zebrafish MNU-induced RD model.