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SignificanceRaman spectroscopy is a well-established analytical method in the fields of chemistry, industry, biology, pharmaceutics, and medicine. Previous studies have investigated optical imaging and Raman spectroscopy for osteoarthritis (OA) diagnosis in weight-bearing joints such as hip and knee joints. However, to realize early diagnosis or a curable treatment, it is still challenging to understand the correlations with intrinsic factors or patients' background.AimTo elucidate the correlation between the Raman spectral features and pathological variations of human shoulder joint cartilage.ApproachOsteoarthritic cartilage specimens excised from the humeral heads of 14 patients who underwent shoulder arthroplasty were assessed by a confocal Raman microscope and histological staining. The Raman spectroscopic dataset of degenerative cartilage was further analyzed by principal component analysis and hierarchical cluster analysis.ResultsMultivariate association of the Raman spectral data generated three major clusters. The first cluster of patients shows a relatively high Raman intensity of collagen. The second cluster displays relatively low Raman intensities of proteoglycans (PGs) and glycosaminoglycans (GAGs), whereas the third cluster shows relatively high Raman intensities of PGs and GAGs. The reduced PGs and GAGs are typical changes in OA cartilage, which have been confirmed by safranin-O staining. In contrast, the increased Raman intensities of collagen, PGs, and GAGs may reflect the instability of the cartilage matrix structure in OA patients.ConclusionsThe results obtained confirm the correlation between the Raman spectral features and pathological variations of human shoulder joint cartilage. Unsupervised machine learning methods successfully yielded a clinically meaningful classification between the shoulder OA patients. This approach not only has potential to confirm severity of cartilage defects but also to determine the origin of an individual's OA by evaluating the cartilage quality.Raman spectroscopy is a well-established analytical method in the fields of chemistry, industry, biology, pharmaceutics, and medicine. Previous studies have investigated optical imaging and Raman spectroscopy for osteoarthritis (OA) diagnosis in weight-bearing joints such as hip and knee joints. However, to realize early diagnosis or a curable treatment, it is still challenging to understand the correlations with intrinsic factors or patients’ background.To elucidate the correlation between the Raman spectral features and pathological variations of human shoulder joint cartilage.Osteoarthritic cartilage specimens excised from the humeral heads of 14 patients who underwent shoulder arthroplasty were assessed by a confocal Raman microscope and histological staining. The Raman spectroscopic dataset of degenerative cartilage was further analyzed by principal component analysis and hierarchical cluster analysis.Multivariate association of the Raman spectral data generated three major clusters. The first cluster of patients shows a relatively high Raman intensity of collagen. The second cluster displays relatively low Raman intensities of proteoglycans (PGs) and glycosaminoglycans (GAGs), whereas the third cluster shows relatively high Raman intensities of PGs and GAGs. The reduced PGs and GAGs are typical changes in OA cartilage, which have been confirmed by safranin–O staining. In contrast, the increased Raman intensities of collagen, PGs, and GAGs may reflect the instability of the cartilage matrix structure in OA patients.The results obtained confirm the correlation between the Raman spectral features and pathological variations of human shoulder joint cartilage. Unsupervised machine learning methods successfully yielded a clinically meaningful classification between the shoulder OA patients. This approach not only has potential to confirm severity of cartilage defects but also to determine the origin of an individual’s OA by evaluating the cartilage quality.

Nanoinks composed of quantum dots (QDs) are applied in light-receiving devices and light-emitting devices such as solar cells and displays. However, since the most widely used QDs, PbS and CdS, are toxic and environmentally concerning, alternative materials need to be developed. We synthesized and analyzed Ag chalcogenide nanoparticles, including AgBiS2 and Ag2S nanoparticles, which are eco-friendly materials. AgBiS2 and Ag2S QD films were prepared by spin-coating nanoparticle solutions and subsequent heat treatment. The effects of the heat treatment on residual ligands and photoluminescence were determined by surface analysis. The photocurrent response of the AgBiS2 and Ag2S QD films was measured in the near-infrared region, and the effect of the heat treatment temperature was investigated. The results indicate that AgBiS2 and Ag2S are prospective materials for near-infrared photodetectors.

Chemotherapy-induced alopecia is one of the most difficult adverse events of cancer treatment for patients. However, it is still unknown why anticancer drugs cause hair loss. We aimed to clarify the mechanism of chemotherapy-induced alopecia in mice using an in vivo imaging technique with a two-photon microscope, which enables observation of the deep reaction in the living body in real time. In this study, ICR mice were injected intraperitoneally with cyclophosphamide (120 µg/g). Changes in the hair bulb morphology, subcutaneous vessel permeability, and vessel density were evaluated by two-photon microscopy and conventional methods. In order to determine whether there is a causal relationship between vascular permeability and hair loss, we combined cyclophosphamide (50 µg/g) with subcutaneous histamine. Using two‐photon microscopy and conventional examination, we confirmed that the hair bulbs became smaller, blood vessels around the hair follicle decreased, and vascular permeability increased at 24 hours after cyclophosphamide injection [corrected]. Apoptosis occurred in vascular endothelial cells around the hair follicle. Additionally, hair loss was exacerbated by temporarily enhancing vascular permeability with histamine. In conclusion, cyclophosphamide caused a decrease in vascular density and an increase in vascular permeability, therefore increased vascular permeability might be one of the causes of chemotherapy-induced alopecia.

In an RF ablation procedure for treating hepatocellular carcinoma and metastatic liver cancer, plucking out an ablation antenna from incompletely cauterized tumor tissues can cause neoplastic seeding; hence, it is demanded to properly evaluate the thermal denaturation of tumor tissue before plucking out the antenna. In the present ex vivo study, we show that the degree of thermal denaturation inside porcine liver tissue induced with RF current heating can be evaluated by binarized two-dimensional Nakagami parametric image indicating the weighted average of the Nakagami shape parameter m. Moreover, we present that the pre-Rayleigh distribution is dominant in ultrasonic backscattered envelopes obtained from incompletely cauterized liver tissue, whereas the Rayleigh distribution is dominant in those obtained from completely cauterized liver tissue which has coagulative necrosis region. The result indicates that the change in scatterer distribution would be occurred inside liver tissue by coagulative necrosis caused by RF current heating in this study.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer. It is an aggressive malignancy associated with poor prognosis because of recurrence, metastasis, and treatment resistance. Aberrant glycosylation of cancer cells triggers their migration and invasion and is considered one of the most important prognostic cancer biomarkers. The current study aimed to identify glycan alterations and their relationship with the malignant potential of PDAC. Using a lectin microarray, we evaluated glycan expression in 62 PDAC samples. Expression of fucosyltransferase 8 (FUT8), the only enzyme catalyzing core fucosylation, was investigated by immunohistochemistry. The role of FUT8 in PDAC invasion and metastasis was confirmed using an in vitro assay and a xenograft peritoneal metastasis mouse model. The microarray data demonstrated that core fucose-binding lectins were significantly higher in carcinoma than in normal pancreatic duct tissues. Similarly, FUT8 protein expression was significantly higher in carcinoma than in normal pancreatic duct tissues. High FUT8 protein expression was significantly associated with lymph-node metastases and relapse-free survival. FUT8 knockdown significantly reduced the invasion in PDAC cell lines and impaired peritoneal metastasis in the xenograft model. The findings of this study provide evidence that FUT8 plays a pivotal role in PDAC invasion and metastasis and might be a therapeutic target for this disease.

Chemotherapy after hepatectomy for colorectal liver metastasis has not been established, due to the toxic side effects, which are likely related to impaired drug clearance during liver regeneration. We investigated the pharmacokinetic and toxicodynamic evaluation of 5-fluorouracil (5-FU) during liver regeneration after major hepatectomy in a rat model. Thirty-six male Wistar rats were divided into control (C), control with chemotherapy (CC), hepatectomy (H), and hepatectomy with chemotherapy (HC) groups. The CC and HC groups were administered 5-FU for 4 days. Plasma 5-FU, liver weight, and liver dihydropyrimidine dehydrogenase (DPD) were measured. The ileal villous height was measured to determine adverse effects. The area under the curve and maximum plasma concentration of 5-FU increased by up to 51% and 32%, respectively, in the HC group compared to the CC group. The liver regeneration rate was significantly lower in the HC group than in the H group (67.3 ± 7.4 vs 33.0 ± 5.7%, p

In this study, we analysed the intracellular fatty acid profiles of Komagataella phaffii during methylotrophic growth. K. phaffii grown on methanol had significantly lower total fatty acid contents in the cells compared with glucose-grown cells. C18 and C16 fatty acids were the predominant fatty acids in K. phaffii, although the contents of odd-chain fatty acids such as C17 fatty acids were also relatively high. Moreover, the intracellular fatty acid composition of K. phaffii changed in response to not only carbon sources but also methanol concentrations: C17 fatty acids and C18:2 content increased significantly as methanol concentration increased, whereas C18:1 and C18:3 contents were significantly lower in methanol-grown cells. The intracellular content of unidentified compounds (Cn H2n O4 ), on the other hand, was significantly greater in cells grown on methanol. As the intracellular contents of these Cn H2n O4 compounds were significantly higher in a gene-disrupted strain for glutathione peroxidase (gpx1Δ) than in the wild-type strain, we presume that the Cn H2n O4 compounds are fatty acid peroxides. These results indicate that K. phaffii can coordinate intracellular fatty acid composition during methylotrophic growth in order to adapt to high-methanol conditions and that certain fatty acid species such as C17:0, C17:1, C17:2 and C18:2 may be related to the physiological functions by which K. phaffii adapts to high-methanol conditions.

Background Hirschsprung disease (HSCR) is characterized by the absence of an enteric nerve system (ENS). To remove aganglionosis, bowel reconstruction is only a curative treatment. It is mandatory to identify the extent of aganglionosis during surgery. Raman spectroscopy is a nondestructive chemical analysis technique that provides detailed information regarding molecular vibrations. The purpose of this study is to detect the ENS using Raman spectroscopy in the human intestine for diagnosis of HSCR. Methods The Raman spectra of each layer of the gastrointestinal wall were collected from surgical specimens of the human rectum. Based on collected spectral data, principal component analysis was performed to determine the ENS. Subsequently, the Raman spectra of HSCR sections were analyzed. Results Molecular structures of the gastrointestinal wall were characterized by Raman spectroscopy. Raman spectroscopy could discriminate between ganglion and muscle layers, and the spectra of the border between muscle layers in the aganglionosis were collagen-associated peaks. Either absence on presence of ENS was also confirmed in HSCR material. Conclusions Label-free detection of the ENS was successfully demonstrated using Raman spectroscopy. Since this is a preliminary study, the strategy which may contribute to differentiate between ganglionic and aganglionic segments using noninvasive techniques in HSCR should be evaluated by prospective studies in near future.

There has been significant changes and improvement in the wide-angle viewing systems and endoillumination in the last few years. Wide-angle viewing systems provide a panoramic view of the retina. Wide-angle illumination system has increased the safety profile while chandelier endoillumination has made bimanual surgery possible.

We examined 27-gauge vitrectomy in a consecutive series of cases to evaluate the outcome of a sutureless 27-gauge vitrectomy system. The findings of this study demonstrate that for the treatment of a variety of ocular diseases, the 27-gauge vitrectomy system is as safe and effective as larger-gauge vitrectomy systems., Purpose: To evaluate the safety and efficacy of 27-gauge vitrectomy for various vitreoretinal disorders. Methods: In this retrospective comparative study, 163 consecutive eyes with various diseases that underwent 27-gauge pars plana vitrectomy with or without ultraspeed transformer by a single surgeon from June 2012 through December 2014 were analyzed in regard to best-corrected visual acuity, intraocular pressure, intraoperative and postoperative complications, and surgery time. Results: In 2 eyes (1.2%), peripheral retina breaks were encountered intraoperatively, yet no other complications were found in those eyes. No cases required larger-gauge vitrectomy. Mean best-corrected visual acuity improved from 20/58 (logarithm of the minimum angle of resolution, 0.46 ± 0.64) preoperatively to 20/32 (logarithm of the minimum angle of resolution, 0.20 ± 0.40) postoperatively (P < 0.001). Mean follow-up was 16.7 months (range, 6–33 months). Intraocular pressure remained stable throughout the postoperative course. Hypotony was seen in 15 eyes (9.2%) at 1-day postoperative, yet that spontaneously improved within 1 week. No case of retinal detachment or endophthalmitis was recorded. In macular surgeries, such as idiopathic epiretinal membrane and macular hole combined with cataract surgery, the mean surgery time was 32.1 ± 6.9 minutes with ultraspeed transformer (n = 38) and 37.1 ± 7.7 minutes without ultraspeed transformer (n = 40) (P = 0.004). Conclusion: The 27-gauge pars plana vitrectomy was found to be safe and effective for treating various vitreoretinal disorders.

Aim The aim of this study was to examine the effect of bisphenol A (BPA) on the proliferation and motility potential of murine LM8 osteosarcoma cells. Materials and methods LM8 cells were treated for 3 days with or without 80 μM BPA. The effect of BPA on cell proliferation was determined by DNA measurement in the cultures and 5-bromo-2'-deoxyuridine (BrdU) incorporation study. Ethanol-fixed cells were stained with hematoxylin-eosin (H&E) to visualize cell morphology. Cell motility was assayed using inserts with uncoated membranes in invasion chambers. Expression of cell division cycle 42 (CDC42) was determined by immunofluorescence staining and western blotting. Results BPA reduced the DNA content of cultures and the number of BrdU-positive cells. BPA induced a change in morphology from cuboidal with multiple filopodia on the cell surface to spindle-shaped with a smooth cell surface. BPA-treated cells expressed less CDC42 and were less motile than untreated cells. Conclusion BPA inhibited DNA replication and cell proliferation. BPA inhibited filopodia formation and motile potential by inhibiting CDC42 expression in LM8 cells.

The use of WAVs in conjunction with wide-angle illumination, like a chandelier lighting, allows easier bimanual maneuvers because they can provide a view of all inside the ocular globe without eye rotation, eliminating concerns regarding fragility of small-gauge instruments.

To evaluate the surgical outcomes of the 27-gauge (G) vitrectomy system for the treatment of primary rhegmatogenous retinal detachment (RRD). This retrospective consecutive series multicenter study involved a total of 410 eyes of 406 patients who underwent 3-port transconjunctival 27G pars plana vitrectomy (PPV) for RRD between November 2014 and December 2016 and who were followed for a minimum of 3 months postoperative. The main outcome measure was primary reattachment, with the secondary outcome measures being final reattachment, improvement of visual acuity (VA), intraocular pressure (IOP), intraoperative and postoperative complications, and surgery time. Of the 410 treated eyes, primary reattachment was achieved in 392 (95.6%) and final reattachment was achieved in 410 (100%). In 226 eyes (55.1%) with macula-on RRD, the mean logarithm of the minimum angle of resolution (logMAR) VA improved from 0.16 ± 0.51 pre-surgery to 0.02 ± 0.14 post-surgery (P = 0.11). In 184 eyes (44.9%) with macula-off RRD, logMAR VA improved from 1.06 ± 0.77 pre-surgery to 0.26 ± 0.35 post-surgery (P

Chemotherapy after hepatectomy for colorectal liver metastasis has not been established, due to the toxic side effects, which are likely related to impaired drug clearance during liver regeneration. We investigated the pharmacokinetic and toxicodynamic evaluation of 5-fluorouracil (5-FU) during liver regeneration after major hepatectomy in a rat model.Thirty-six male Wistar rats were divided into control (C), control with chemotherapy (CC), hepatectomy (H), and hepatectomy with chemotherapy (HC) groups. The CC and HC groups were administered 5-FU for 4 days. Plasma 5-FU, liver weight, and liver dihydropyrimidine dehydrogenase (DPD) were measured. The ileal villous height was measured to determine adverse effects.The area under the curve and maximum plasma concentration of 5-FU increased by up to 51% and 32%, respectively, in the HC group compared to the CC group. The liver regeneration rate was significantly lower in the HC group than in the H group (67.3 ± 7.4 vs 33.0 ± 5.7%, p 0.001). The HC group had a significantly lower liver DPD than the CC group (4.4 ± 1.1 mg vs 6.9 ± 1.1 mg, p 0.01). The HC group had a significantly lower ileal villous height than the CC group (253 ± 40 μm vs. 318 ± 36 μm, p 0.05).Reduction of the total liver DPD following major hepatectomy caused increased plasma 5-FU levels and 5-FU-associated toxicity.

Background The molecular mechanisms underlying aortic valve calcification are poorly understood. Here, we aimed to identify the master regulators of calcification by comparison of genes in valve interstitial cells (VICs) with calcified and noncalcified aortic valves. Methods Calcified aortic valves were surgically excised from patients with aortic valve stenosis who required aortic valve replacements. Noncalcified and calcified sections were obtained from aortic valve leaflets. Collagenase-digested tissues were seeded into dishes, and VICs adhering to the dishes were cultured for 3 weeks, followed by comprehensive gene expression analysis. Functional analyses of identified proteins were performed by in vitro calcification assays. Tissue localization was determined by immunohistochemical staining for normal (n = 11) and stenotic valves (n = 30). Results We found 87 genes showing greater than a twofold change in calcified tissues. Among these genes, 68 were downregulated and 19 were upregulated. Cyclooxygenase-1 (COX1) messenger RNA and protein levels were upregulated in VICs from calcified tissues. The COX1 messenger RNA and protein levels in VICs were also strongly increased by stimulation with osteoblast differentiation medium. These were VIC-specific phenotypes and were not observed in other cell types. Immunohistochemical staining revealed that COX1-positive VICs were specifically localized in the calcified area of aortic valve tissues. Conclusions The VIC-specific COX1 overexpression played a crucial role in calcification by promoting osteoblast differentiation in aortic valve tissues.

Non-alcoholic steatohepatitis (NASH) is a common liver disorder caused by fatty liver. Because NASH is associated with fibrotic and morphological changes in liver tissue, a direct imaging technique is required for accurate staging of liver tissue. For this purpose, in this study we took advantage of two label-free optical imaging techniques, second harmonic generation (SHG) and auto-fluorescence (AF), using two-photon excitation microscopy (TPEM). Three-dimensional ex vivo imaging of tissues from NASH model mice, followed by image processing, revealed that SHG and AF are sufficient to quantitatively characterize the hepatic capsule at an early stage and parenchymal morphologies associated with liver disease progression, respectively., Highlights • Combination of two label-free optical imaging techniques for the evaluation of liver disease is proposed. • SHG is sufficient to quantitatively characterize the hepatic capsule at an early stage of NASH. • Auto-fluorescence is useful to evaluate the parenchymal morphologies associated with liver disease progression.

Bone mineral density (BMD) is a commonly used diagnostic indicator for bone fracture risk in osteoporosis. Along with low BMD, bone fragility accounts for reduced bone quality in addition to low BMD, but there is no diagnostic method to directly assess the bone quality. In this study, we investigated changes in bone quality using the Raman spectroscopic technique. Sciatic neurectomy (NX) was performed in male C57/BL6J mice (NX group) as a model of disuse osteoporosis, and sham surgery was used as an experimental control (Sham group). Eight months after surgery, we acquired Raman spectral data from the anterior cortical surface of the proximal tibia. We also performed a BMD measurement and micro-CT measurement to investigate the pathogenesis of osteoporosis. Quantitative analysis based on the Raman peak intensities showed that the carbonate/phosphate ratio and the mineral/matrix ratio were significantly higher in the NX group than in the Sham group. There was direct evidence of alterations in the mineral content associated with mechanical properties of bone. To fully understand the spectral changes, we performed principal component analysis of the spectral dataset, focusing on the matrix content. In conclusion, Raman spectroscopy provides reliable information on chemical changes in both mineral and matrix contents, and it also identifies possible mechanisms of disuse osteoporosis.

A high-performance liquid chromatography method with methyl acetoacetate derivatization via the Hantzsch reaction was developed for the analysis of formaldehyde (HCHO) in several water samples. Under optimized conditions, HCHO was detected within 4 min and was not affected by excessive derivatization reagents. The calibration curve constructed from the peak height of HCHO was linear, with a correlation coefficient of 0.9998. The relative standard deviation of the peak height from ten replicates was 0.29%. The detection and quantitative limits were 0.96 µg/L and 3.16 µg/L, respectively. A recovery test of HCHO was performed to compare the developed method with the official analysis method (DNPH method). The developed method was used to determine the HCHO levels in several water samples (tap water, river water, and waste water).

Raman spectroscopy provides a wealth of diagnostic information to the surgeon with in situ cancer detection and labelfree histopathology in clinical practice. However, to identify cancer diagnostic biomarkers in vivo is still challenging, because malignancy can be characterized not only by the cancer cells but also by the environmental factors. Here we investigate molecular dynamics in both cancer cells and their environment in xenograft models and spontaneous metastasis models using Raman spectroscopy and nonlinear optical imaging. We are constructing a custom-designed Raman spectral imaging system to reveal the metastasis process and to evaluate therapeutics toward the clinical application of the technique.

Raman spectroscopy provides a wealth of diagnostic information to the surgeon with in situ cancer detection and label-free histopathology in intraoperative conditions. Raman spectroscopy is a promising optical technique which can analyze biological tissues with light scattering. The difference in frequencies between the incident light and the scattering light are called Raman shifts, which correspond to the vibrational energy of the molecular bonds. Raman spectrum gives information about the molecular structure and composition in biological specimens. We had been previously reported that Raman spectroscopy could distinguish various histological types of human lung cancer cells from normal cells in vitro , and also confirmed that Raman spectra obtained from cancer cells and their environment including other cells and extracellular matrix in xenograft models and spontaneous metastasis models were distinguishable using Raman spectroscopy combined with fluorescence microscopy and photoluminescence imaging. Malignancy can be characterized not only by the cancer cells but also by the environmental factors including immune cells, stroma cells, secretion vesicles and extracellular matrix, but to identify and detect cancer diagnostic biomarkers in vivo on Raman spectroscopy is still challenging. Here we investigate morphological and molecular dynamics in advanced cancer specimens obtained from patients. We are also constructing a customdesigned Raman spectral imaging system for both in vitro and in vivo assay of tumor tissues to reveal the metastasis process and to evaluate therapeutic effects of anti-cancer drugs and their drug delivery toward the clinical application of the technique.

Osteoarthritis (OA) is a very common joint disease in the aging population. Main symptom of OA is accompanied by degenerative changes of articular cartilage. Cartilage contains mostly type II collagen and proteoglycans, so it is difficult to access the quality and morphology of cartilage tissue in situ by conventional diagnostic tools (X-ray, MRI and echography) directly or indirectly. Raman spectroscopy is a label-free technique which enables to analyze molecular composition in degenerative cartilage. In this proposal, we aim to develop Raman spectroscopic system for the quality assessment of articular cartilage during arthroscopic surgery. Toward this goal, we are focusing on the proteoglycan content and collagen fiber alignment in cartilage matrix which may be associated with degenerative changes in OA, and we designed an original Raman device for remote sensing during arthroscopic surgery. In this project, we define the grading system for cartilage defect based on Raman spectroscopy, and we complete the evaluation of the Raman probing system which makes it possible to detect early stage of degenerative cartilage as a novel tool for OA diagnosis using human subject.

To detect the bone quality loss in osteoporosis, we performed Raman spectroscopic analysis of sciatic nerve resection (NX) mice. Eight months after surgery, lower limbs were collected from the mice and fixed with 70% ethanol. Raman spectra of anterior cortical surface of the proximal tibia at 5 points in each bone were measured by RENISHAW inVia Raman Microscope. Excitation wave length was 785 nm. We also performed DXA and micro CT measurement to confirm the bone mineral density and bone microstructure in the osteoporotic model induced by sciatic nerve resection. In the result of Raman spectroscopy, we detected changes of Raman peak intensity ratio in carbonate/phosphate, mineral/combined proline and hydroxyproline and mineral/phenylalanine. In addition, in the result of micro CT, we found significant changes in VOX BV/TV, Trabecular number, thickness, cancellous bone mineral density, cortical thickness and cortical bone mineral density. The results suggest that not only the bone mineral density but also bone quality reduced in the NX mice. We conclude that Raman spectroscopy is a useful for bone quality assessment as a complementary technique for conventional diagnostics.

Bone modeling and remodeling are cellular events during which osteoblast lineage cells and osteoclasts interact. During these events, cells undergo drastic changes with time as they become differentiated. Their morphology, topology, and activity are affected by other cells and the extracellular matrices. Since the mechanisms underlying the cellular events of bone metabolism have not been elucidated, there is a need for systems to analyze these cellular networks and their microenvironments spatiotemporally at the cellular level. Here we report a novel in vitro system for reconstituting the bone cell network of osteoclasts, osteoblasts, and osteocytes in the mineralized nodule, allowing for observation of bone modeling and remodeling phenomena by 2-photon microscopy. Using this system, the change in morphology of osteoblasts from cuboidal to flat cells was observed and measured during the formation of mineralized nodules. Furthermore, the recruitment of osteoblasts to resorption pits and their replenishment by newly formed matrices were successfully observed, providing strong evidence for the coupling of bone resorption and bone formation at cellular level. During such remodeling cycle, flat osteoblasts that survived more than 7 weeks were recruited to resorption pits, where they became cuboidal osteoblasts that express osteocalcin. This novel system permitted the elucidation of cellular behavior during bone modeling and remodeling, and can be used to analyze cellular events involved in bone metabolism.

Despite the availability of several modalities of treatment, including surgery, pharmacological agents, and nerve blocks, neuropathic pain is often unresponsive and sometimes progresses to intractable chronic pain. Although exercise therapy is a candidate for treatment of neuropathic pain, the mechanism underlying its efficacy has not been elucidated. To clarify the molecular mechanism for pain relief induced by exercise, we measured Rnf34 and Pacap mRNA levels in the spinal cord dorsal horn of SNL rats, a model of neuropathic pain. SNL model rats exhibited stable mechanical hyperalgesia for at least 6 weeks. When the rats were forced to exercise on a treadmill, mechanical and thermal hyperalgesia were significantly ameliorated compared with the non-exercise group. Accordingly, gene expression level of Rnf34 and Pacap were also significantly altered in the time course analysis after surgery. These results suggest that exercise therapy possibly involves pain relief in SNL rats by suppressing Rnf34 and Pacap expression in the spinal cord.

To evaluate the bone quality in the osteoporosis, we generated sciatic nerve resection (NX) mice as an osteoporosis model and analyzed by Raman spectroscopy. Raman spectra were measured in anterior cortical surface of the proximal tibia at 5 points in each bone. After that, the samples were fixed with 70% ethanol. We then performed DXA and μCT measurement. Raman peak intensity ratios were significantly different between NX and Control. Those changes in the Raman peak intensity ratios may reflect loss of bone quality in the osteoporosis model. Raman spectroscopy is a promising technique for measuring the bone quality and bone strength.

Osteoarthritis (OA) is very common joint disease in the aging population. Main symptom of OA is accompanied by degenerative changes of articular cartilage. Raman spectroscopy is a label-free technique which enables to analyze molecular composition in degenerative cartilage. We generated an animal OA model surgically induced by knee joint instability and performed Raman spectroscopic analysis for the articular cartilage. In the result, Raman spectral data of the articular cartilage showed drastic changes in comparison between OA and control side. The relative intensity of phosphate band increases in the degenerative cartilage.

Raman spectroscopy provides a wealth of diagnostic information to the surgeon with in situ cancer detection and label-free histopathology in clinical practice. Raman spectroscopy is a developing optical technique which can analyze biological tissues with light scattering. The difference in frequencies between the incident light and the scattering light are called Raman shifts, which correspond to the vibrational energy of the molecular bonds. Raman spectrum gives information about the molecular structure and composition in biological specimens. We had been previously reported that Raman spectroscopy could distinguish various histological types of human lung cancer cells from normal cells in vitro. However, to identify and detect cancer diagnostic biomarkers in vivo on Raman spectroscopy is still challenging, because malignancy can be characterized not only by the cancer cells but also by the environmental factors including immune cells, stroma cells, secretion vesicles and extracellular matrix. Here we investigate morphological and molecular dynamics in both cancer cells and their environment in xenograft models and spontaneous metastasis models using Raman spectroscopy combined with fluorescence microscopy and photoluminescence imaging. We are also constructing a custom-designed Raman spectral imaging system for both in vitro and in vivo assay of tumor tissues to reveal the metastasis process and to evaluate therapeutic effects of anti-cancer drugs and their drug delivery toward the clinical application of the technique.

Osteoarthritis (OA) is very common joint disease in the aging population. Main symptom of OA is accompanied by degenerative changes of articular cartilage. Cartilage contains mostly type II collagen and proteoglycans, so it is difficult to access the quality and morphology of cartilage tissue in situ by conventional diagnostic tools (X-ray, MRI and echography) directly or indirectly. Raman spectroscopy is a label-free technique which enables to analyze molecular composition in degenerative cartilage. In this study, we generated an animal OA model surgically induced by knee joint instability, and the femurs were harvested at two weeks after the surgery. We performed Raman spectroscopic analysis for the articular cartilage of distal femurs in OA side and unaffected side in each mouse. In the result, there is no gross findings in the surface of the articular cartilage in OA. On the other hand, Raman spectral data of the articular cartilage showed drastic changes in comparison between OA and control side. The major finding of this study is that the relative intensity of phosphate band (960 cm -1 ) increases in the degenerative cartilage. This may be the result of exposure of subchondral bone due to thinning of the cartilage layer. In conclusion, Raman spectroscopic technique is sufficient to characterize articular cartilage in OA as a pilot study for Raman application in cartilage degeneration and regeneration using animal models and human subjects.

Yellow Cameleons are genetically encoded Ca2+ indicators in which cyan and yellow fluorescent proteins and calmodulin work together as a fluorescence (Förster) resonance energy transfer Ca2+-sensor probe. To achieve ultrasensitive Ca2+ imaging for low resting Ca2+ or small Ca2+ transients in various organs, we generated a transgenic mouse line expressing the highest-sensitive genetically encoded Ca2+ indicator (Yellow Cameleon-Nano 15) in the whole body. We then focused on the mechanism of exocytotic events mediated by intracellular Ca2+ signaling in acinar cells of the mice with an agonist and observed them by two-photon excitation microscopy. In the results, two-photon excitation imaging of Yellow Cameleon-Nano 15 successfully visualized intracellular Ca2+ concentration under stimulation with the agonist at nanomolar levels. This is the first demonstration for application of genetically encoded Ca2+ indicators to pancreatic acinar cells. We also simultaneously observed exocytotic events and an intracellular Ca2+ concentration under in vivo condition. Yellow Cameleon-Nano 15 mice are healthy and no significant deteriorative effect was observed on physiological response regarding the pancreatic acinar cells. The dynamic range of 165% was calculated from Rmax and Rmin values under in vivo condition. The mice will be useful for ultrasensitive Ca2+ imaging in vivo.

Recently, there has been growing interest in applying fluorescence imaging techniques to the study of various disease processes and complex biological phenomena in vivo. To apply these methods to clinical settings, several groups have developed protocols for fluorescence imaging using antibodies against tumor markers conjugated to fluorescent substances. Although these probes have been useful in macroscopic imaging, the specificity and sensitivity of these methods must be improved to enable them to detect micro-lesions in the early phases of cancer, resulting in better treatment outcomes. To establish a sensitive and highly specific imaging method, we used a fluorophore-conjugated anti-carcinoembryonic antigen (CEA) antibody to perform macroscopic and microscopic in vivo imaging of inoculated cancer cells expressing GFP with or without CEA. Macroscopic imaging by fluorescence zoom microscopy revealed that bio-conjugation of Alexa Fluor 594 to the anti-CEA antibody allowed visualization of tumor mass consisting of CEA-expressing human cancer cells, but the background levels were unacceptably high. In contrast, microscopic imaging using a two-photon excitation microscope and the same fluorescent antibody resulted in subcellular-resolution imaging that was more specific and sensitive than conventional imaging using a fluorescence zoom microscope. These results suggest that two-photon excitation microscopy in conjunction with fluorophore-conjugated antibodies could be widely adapted to detection of cancer-specific cell-surface molecules, both in cancer research and in clinical applications.