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Yasutoshi Murayama,
Daisuke Ichikawa,
Noriaki Koizumi,
Shuhei Komatsu,
Atsushi Shiozaki,
Yoshiaki Kuriu,
Hisashi Ikoma,
Takeshi Kubota,
Masayoshi Nakanishi,
Yoshinori Harada,
Hitoshi Fujiwara,
Kazuma Okamoto,
Toshiya Ochiai,
Yukihito Kokuba, Tetsuro Takamatsu,
Eigo Otsuji
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ABSTRACT: Precise staging of gastric cancer is essential when selecting for the appropriate treatment approach. 5-Aminolevulinic acid (5-ALA) is metabolized and accumulated as protoporphyrin IX (PpIX), which is a photosensitizer. In this study, we evaluated the diagnostic usefulness of 5-ALA for demonstrating peritoneal dissemination in patients with gastric cancer.
Fluorescence laparoscopy (FL) was performed in 13 patients with gastric cancer. All patients received preoperative oral administration of 5-ALA. Fluorescence-light laparoscopies were sequentially performed intraoperatively.
In four out of the 13 patients, primary tumors were detected by FL and demonstrated serosal invasion on histological examination. Five out of the 13 patients demonstrated peritoneal metastases, and one patient demonstrated superficial liver micrometastases, by FL. All of these lesions were diagnosed as metastatic lesions by hematoxylin and eosin staining.
These findings show that FL diagnosis with 5-ALA is accurate and suitable for the detection of peritoneal metastases and of superficial liver micrometastases in patients with gastric cancer.
Anticancer research 12/2012; 32(12):5421-7. · 1.73 Impact Factor
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ABSTRACT: Detection of peripheral nerve tissues during surgery is required to avoid neural disturbance following surgery as an aspect of realizing better functional outcome. We provide a proof-of-principle demonstration of a label-free detection technique of peripheral nerve tissues, including myelinated and unmyelinated nerves, against adjacent tissues that employ spontaneous Raman microspectroscopy. To investigate the Raman spectral features of peripheral nerves in detail, we used unfixed sectioned samples. Raman spectra of myelinated nerve, unmyelinated nerve, fibrous connective tissue, skeletal muscle, tunica media of blood vessel, and adipose tissue of Wistar rats were analyzed, and Raman images of the tissue distribution were constructed using the map of the ordinary least squares regression (OLSR) estimates. We found that nerve tissues exhibited a specific Raman spectrum arising from axon or myelin sheath, and that the nerve tissues can be selectively detected against the other tissues. Moreover, myelinated and unmyelinated nerves can be distinguished by the intensity differences of 2,855 cm(-1), and 2,945 cm(-1), which are mainly derived from lipid and protein contents of nerve fibers. We applied this method to unfixed section samples of human periprostatic tissues excised from prostatic cancer patients. Myelinated nerves, unmyelinated nerves, fibrous connective tissues, and adipose tissues of the periprostatic tissues were separately detected by OLSR analysis. These results suggest the potential of the Raman spectroscopic observation for noninvasive and label-free nerve detection, and we expect this method could be a key technique for nerve-sparing surgery.
Histochemie 08/2012; · 2.59 Impact Factor
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ABSTRACT: Raman spectroscopy has long been used as a powerful tool for chemical composition analyses. Raman scattering light measurement has the advantage of being able to examine biomolecules in a nondestructive and non-labeling manner. However, molecular imaging of cells and tissues by using Raman microscopy had been hampered due to weak Raman signals and required long acquisition time. Recent advances in imaging devices, such as development of slit-scanning Raman microscopy, have enabled us to acquire high-resolution Raman images of biomedical samples. Thus, Raman molecular imaging now has wide application potential for in-situ functional analysis of biomolecules in living bodies, such as studies of intracellular drug pharmacokinetics and oxygen saturation of blood capillaries. There is a possibility that Raman scattering light measurement can be applied for functional diagnostic imaging of human bodies, taking advantage of its noninvasive merits in the future. This review briefly highlights recent topics in spontaneous Raman molecular imaging of cells and tissues.
Current pharmaceutical biotechnology 02/2012; · 3.40 Impact Factor
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ABSTRACT: Purpose Tissue autofluorescence study is a promising means of endoscopic detection of colonic neoplasia, but the mechanism of autofluorescence eruption has still not been verified. The purpose of this study was to precisely analyze the autofluorescence characteristics of freshly prepared normal rat colon under UVA and violet light excitation. Methods Excised rat colons were studied by using multichannel spectrophotometry, spectroscopic imaging, confocal microscopy, combined two-photon excited fluorescence and second-harmonic generation (SHG) microscopy, and fluorescence lifetime imaging microscopy. Results Spectroscopic analysis of freshly prepared colon sections revealed that the mucosa and the submucosa showed strong autofluorescence under UVA and violet light excitation. The combined images of two-photon and SHG microscopy revealed that the mucosal epithelia are the important source of autofluorescence. Nicotinamide adenine dinucleotide seems to be one of the major substances involved in the autofluorescence of the mucosal layer on 365-nm light excitation. The autofluorescence spectra of the luminal surfaces were identical to those of the mucosa on cross-sectional examinations with 365-nm excitation. The main origin of autofluorescence of the luminal surface with 365-nm excitation is the epithelial cells in the mucosa without overlay of submucosal fluorescence. Conclusion: The mucosal layer is the important source of the autofluorescence observed under excitation with UVA/violet light in multilayered colonic structures. Illumination of 365-nm wavelength light is a suitable means of analyzing the autofluorescence of mucosal epithelia.
Current pharmaceutical biotechnology 02/2012; · 3.40 Impact Factor
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ABSTRACT: Optical imaging to visualize biomolecules holds promise for early and precise detection of various diseases. Rapid development of technologies for laser oscillators, spectroscopes, and detectors has made it possible to study biochemical changes associated with diseases which were difficult to visualize in situ without labeling. In this paper, we introduced our recent non-labeling imaging studies by using Raman scattering light and autofluorescence. Our results suggest the potential of Raman scattering light and autofluorescence measurements for the non-labeling detection of various diseased tissues based on their molecular compositions.
Rinsho byori. The Japanese journal of clinical pathology 02/2012; 60(2):119-24.
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ABSTRACT: The mucosal layer of the colon contains metabolism-related fluorophores, such as reduced nicotinamide adenine dinucleotide (NADH), which might have the potential to serve as biomarkers for detecting neoplasia.
To examine NADH fluorescence in human colonic adenoma while eliminating the effect of hemoglobin absorption and to develop a novel imaging technique for precise detection of adenomas.
Cross-sectional study.
A total of 66 endoscopically resected colonic polyps were investigated. After serial acquisition of autofluorescence images between 450 and 490 nm illuminated with dual-wavelength excitation at 365 nm (F(365ex)) and 405 nm (F(405ex)) on cross sections of the samples, ratio images were created by dividing F(365ex) by F(405ex). The excitation-emission wavelength combinations in F(365ex) and F(405ex) were optimized for NADH fluorescence and reference fluorescence.
The F(365ex)/F(405ex) ratio in the tumorous (T) and normal (N) mucosa.
F(365ex)/F(405ex) ratio images showed a 1.81- and 1.12-fold higher signal intensity in the adenomas and hyperplastic polyps, respectively, than in the adjacent normal mucosa. The ratio between signal intensities in tumorous mucosa and normal mucosa in F(365ex)/F(405ex) ratio images for tubular adenomas was significantly higher than that for hyperplastic polyps. The signal intensity in F(365ex)/F(405ex) ratio images was not correlated with the hemoglobin concentration index evaluated by reflection images at 550 nm and 610 nm. Diminutive adenomas (<5 mm) and large adenomas were well discriminated in F(365ex)/F(405ex) ratio images.
Ex vivo experiment.
These results suggest that the precise measurement of NADH fluorescence intensity together with eliminating the influence of blood hemoglobin concentration serves as a method for visualizing colonic adenomas and that the dual-wavelength excitation method is a promising technique applicable to endoscopic detection of early colonic adenomas.
Gastrointestinal endoscopy 01/2012; 75(1):110-7. · 6.71 Impact Factor
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Hideo Nakamura,
Satoaki Matoba,
Eri Iwai-Kanai,
Masaki Kimata,
Atsushi Hoshino,
Mikihiko Nakaoka,
Maki Katamura,
Yoshifumi Okawa,
Makoto Ariyoshi,
Yuichiro Mita,
Koji Ikeda,
Mitsuhiko Okigaki,
Souichi Adachi,
Hideo Tanaka, Tetsuro Takamatsu,
Hiroaki Matsubara
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ABSTRACT: Diabetic cardiomyopathy is characterized by energetic dysregulation caused by glucotoxicity, lipotoxicity, and mitochondrial alterations. p53 and its downstream mitochondrial assembly protein, synthesis of cytochrome c oxidase 2 (SCO2), are important regulators of mitochondrial respiration, whereas the involvement in diabetic cardiomyopathy remains to be determined.
The role of p53 and SCO2 in energy metabolism was examined in both type I (streptozotocin [STZ] administration) and type II diabetic (db/db) mice. Cardiac expressions of p53 and SCO2 in 4-week STZ diabetic mice were upregulated (185% and 152% versus controls, respectively, P<0.01), with a marked decrease in cardiac performance. Mitochondrial oxygen consumption was increased (136% versus control, P<0.01) in parallel with augmentation of mitochondrial cytochrome c oxidase (complex IV) activity. Reactive oxygen species (ROS)-damaged myocytes and lipid accumulation were increased in association with membrane-localization of fatty acid translocase protein FAT/CD36. Antioxidant tempol reduced the increased expressions of p53 and SCO2 in STZ-diabetic hearts and normalized alterations in mitochondrial oxygen consumption, lipid accumulation, and cardiac dysfunction. Similar results were observed in db/db mice, whereas in p53-deficient or SCO2-deficient diabetic mice, the cardiac and metabolic abnormalities were prevented. Overexpression of SCO2 in cardiac myocytes increased mitochondrial ROS and fatty acid accumulation, whereas knockdown of SCO2 ameliorated them.
Myocardial p53/SCO2 signal is activated by diabetes-mediated ROS generation to increase mitochondrial oxygen consumption, resulting in excessive generation of mitochondria-derived ROS and lipid accumulation in association with cardiac dysfunction.
Circulation Heart Failure 11/2011; 5(1):106-15. · 6.29 Impact Factor
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09/2011; , ISBN: 978-953-307-777-2
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ABSTRACT: Photoacoustic microscopy usually uses high-frequency photoacoustic waves, which provide not only high spatial resolution but also limitation of the penetration depth. In this study, we developed two-photon absorption-induced photoacoustic microscopy (TP-PAM) to improve the depth resolution without use of high-frequency photoacoustic waves. The spatial resolution in TP-PAM is determined by two-photon absorption. TP-PAM with a 20X objective lens (numerical aperture: 0.4) provides an optically-determined depth resolution of 44.9 ± 2.0 μm, which is estimated by the full width at half maximum of the photoacoustic signal from an infinitely small target, using low-frequency bandpass filtering of photoacoustic waves. The combination of TP-PAM and frequency filtering provides high spatial resolution.
Optics Express 07/2011; 19(14):13365-77. · 3.59 Impact Factor
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Ken Inoue,
Naoki Wakabayashi,
Yasutaka Morimoto,
Kiichirou Miyawaki,
Atsufumi Kashiwa,
Naohisa Yoshida,
Keimei Nakano,
Hisashi Takada,
Yoshinori Harada,
Nobuaki Yagi,
Yuji Naito, Tetsuro Takamatsu,
Toshikazu Yoshikawa
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ABSTRACT: Autofluorescence (AF) imaging, which can potentially differentiate tissue types based on differences in fluorescence emission, may be useful in the diagnosis and treatment of colorectal malignancies. This study was designed to assess the potential usefulness of AF colonoscopy for evaluating superficial colorectal neoplastic lesions.
A total of 49 colorectal lesions in 43 patients were investigated. All superficial colorectal neoplastic lesions were identified with white light (WL) colonoscopy. Each detected lesion was investigated by WL colonoscopy, AF colonoscopy, and chromoendoscopy using 0.2% indigo carmine dye. Three endoscopists, blinded to each patient's history, evaluated the still images (as obtained with these three methods) in random order and evaluated their influence on the assessment of lesion visualization. All the lesions were biopsied or resected endoscopically, with the pathological results used as the gold standard.
For visualization of the surface appearance and differences in color of the lesions compared with the surrounding mucosa, AF colonoscopy was superior to WL colonoscopy (p < 0.01) and comparable to chromoendoscopy (Mann-Whitney U test). For visualization of the circumferential margin, AF colonoscopy was superior to WL colonoscopy (p < 0.05) but inferior to chromoendoscopy (p < 0.01).
AF colonoscopy may be a valuable tool for detection and diagnosis of superficial colorectal neoplastic lesions.
International Journal of Colorectal Disease 07/2010; 25(7):811-6. · 2.38 Impact Factor
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ABSTRACT: Accurate diagnosis of metastatic lymph nodes (LNs) is essential in choosing appropriate treatment for gastrointestinal carcinoma. Our aim was to evaluate the diagnostic power of 5-aminolevulinic acid (5-ALA) for LN metastasis in mouse rectal cancer. Colorectal cancer cell lines, isolated cells from normal LNs, and orthotopic mouse model incorporating enhanced green fluorescent protein-tagged and untagged human rectal cancer cells were studied after 5-ALA administration by using confocal microscopy, fluorescence stereomicroscopy, fluorescence lifetime imaging microscopy (FLIM), multichannel spectrophotometry and macroconfocal imaging system to precisely detect LN metastases. In vitro confocal microscopic analyses showed that all colorectal cancer cell lines tested were positive for 5-ALA-induced fluorescence, whereas isolated normal LN cells were negative. 5-ALA-induced protoporphyrin IX (PPIX) fluorescence, verified by FLIM and multichannel spectrophotometry, revealed LN metastases in mice-bearing human rectal cancer cells. Occult LN metastases, unrecognized on white-light imaging and simplified hematoxylin-eosin analyses, were readily detectable on 5-ALA-induced PPIX fluorescence imaging. In vivo macroconfocal images clearly revealed PPIX-fluorescence-positive cancer cells in draining lymph vessels and nodes. Together with specific speckled patterns of PPIX-fluorescence in metastatic lesions, the PPIX-fluorescence intensity ratio of metastatic and nonmetastatic lesions discriminated metastasis with 100% sensitivity and 100% specificity in excised whole LN samples. These results show that fluorescence diagnosis with 5-ALA is very accurate in the detection of LN micrometastases of mouse rectal cancer, suggesting that this feasible diagnostic approach is applicable to target sectioning of metastases of resected fresh whole node samples in pathology laboratories. (c) 2009 UICC.
International Journal of Cancer 07/2009; 125(10):2256-63. · 5.44 Impact Factor
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ABSTRACT: Label-free imaging is desirable for elucidating morphological and biochemical changes of heart tissue in vivo. Spontaneous Raman microscopy (SRM) provides high chemical contrast without labeling, but presents disadvantage in acquiring images due to low sensitivity and consequent long imaging time. Here, we report a novel technique for label-free imaging of rat heart tissues with high-speed SRM combined with resonance Raman effect of heme proteins. We found that individual cardiomyocytes were identified with resonance Raman signal arising mainly from reduced b- and c-type cytochromes, and that cardiomyocytes and blood vessels were imaged by distinguishing cytochromes from oxy- and deoxy-hemoglobin in intact hearts, while cardiomyocytes and fibrotic tissue were imaged by distinguishing cytochromes from collagen type-I in infarct hearts with principal component analysis. These results suggest the potential of SRM as a label-free high-contrast imaging technique, providing a new approach for studying biochemical changes, based on the molecular composition, in the heart.
Biochemical and Biophysical Research Communications 06/2009; 382(2):370-4. · 2.48 Impact Factor
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ABSTRACT: Most molecular imaging technologies require exogenous probes and may have some influence on the intracellular dynamics of target molecules. In contrast, Raman scattering light measurement can identify biomolecules in their innate state without application of staining methods. Our aim was to analyze intracellular dynamics of topoisomerase I inhibitor, CPT-11, by using slit-scanning confocal Raman microscopy, which can take Raman images with high temporal and spatial resolution. We could acquire images of the intracellular distribution of CPT-11 and its metabolite SN-38 within several minutes without use of any exogenous tags. Change of subcellular drug localization after treatment could be assessed by Raman imaging. We also showed intracellular conversion from CPT-11 to SN-38 using Raman spectra. The study shows the feasibility of using slit-scanning confocal Raman microscopy for the non-labeling evaluation of the intracellular dynamics of CPT-11 with high temporal and spatial resolution. We conclude that Raman spectromicroscopic imaging is useful for pharmacokinetic studies of anticancer drugs in living cells.
Histochemie 05/2009; 132(1):39-46. · 2.59 Impact Factor
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ABSTRACT: Low-energy laser irradiation (low-level laser therapy) (LELI/LLLT/photobiomodulation) has been found to modulate various biological effects, especially those involved in promoting cell proliferation. Synovial fibroblasts are important in maintaining the homeostasis of articular joints and have strong chondrogenetic capacity. Here, we investigated the effect and molecular basis of LELI on synovial fibroblast proliferation.
HIG-82 rabbit synovial fibroblasts were cultured, and laser irradiation (660 nm) was applied at the power density of 40 mW/cm(2) for 2 minutes, corresponding to laser fluence of 4.8 J/cm(2). The effect of LELI on cell proliferation, cell cycle progression, and expression of cyclin-dependent kinase inhibitors (CKIs) were investigated. We also examined whether the effects of LELI on HIG-82 cell proliferation were affected by cAMP content, which is known to influence the cell cycle via inducing CKIs.
LELI promoted HIG-82 synovial fibroblast proliferation and induced cytoplasmic localization of cyclin-dependent kinase inhibitor p15 (INK4B/CDKN2B). Moreover, the proliferation of HIG-82 synovial fibroblasts was reduced by cAMP, while cAMP inhibitor, SQ22536, induced p15 cytoplasmic localization and as a result, elevated synovial fibroblast proliferation was observed. In addition, the promotive effect of LELI-induced HIG-82 synovial fibroblast proliferation was abolished by cAMP treatment. Our findings suggest that cAMP may be involved in the effect of LELI on synovial fibroblast proliferation.
We revealed the effect and molecular link involved in synovial fibroblast proliferation induced by 660-nm LELI. Our study provides new insights into the mechanisms by which LELI has biological effects on synovial fibroblast proliferation. These insights may contribute to further investigation on biological effects and application of LELI in regenerative medicine.
Lasers in Surgery and Medicine 04/2009; 41(3):232-9. · 2.75 Impact Factor
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Naofumi Takehara,
Yoshiaki Tsutsumi,
Kento Tateishi,
Takehiro Ogata,
Hideo Tanaka,
Tomomi Ueyama,
Tomosaburo Takahashi, Tetsuro Takamatsu,
Masanori Fukushima,
Masashi Komeda,
Masaaki Yamagishi,
Hitoshi Yaku,
Yasuhiko Tabata,
Hiroaki Matsubara,
Hidemasa Oh
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ABSTRACT: This study was designed to determine whether controlled release of basic fibroblast growth factor (bFGF) might improve human cardiosphere-derived cell (hCDC) therapy in a pig model of chronic myocardial infarction.
Current cell therapies for cardiac repair are limited by loss of the transplanted cells and poor differentiation.
We conducted 2 randomized, placebo-controlled studies in immunosuppressed pigs with anterior myocardial infarctions. Four weeks after coronary reperfusion, 14 pigs were randomly assigned to receive an intramyocardial injection of placebo medium with or without bFGF-incorporating hydrogel implantation. As a second study, 26 pigs were randomized to receive controlled release of bFGF combined with or without hCDCs or bone marrow-derived mesenchymal stem cell transplantation 4 weeks after reperfusion.
Controlled release of bFGF in ischemic myocardium significantly augmented the formation of microvascular networks to enhance myocardial perfusion and contractile function. When combined with cell transplantation, the additive effects of bFGF were confined to hCDC-injected animals, but were not observed in animals receiving human bone marrow-derived mesenchymal stem cell transplantation. This was shown by increased donor-cell engraftment and enhanced cardiomyocyte differentiation in the transplanted hearts, resulting in synergistically improved ventricular function and regional wall motion and reduced infarct size.
Controlled delivery of bFGF modulates the post-ischemic microenvironment to enhance hCDC engraftment and differentiation. This novel strategy demonstrates significant functional improvements after myocardial infarction and may potentially represent a therapeutic approach to be studied in a clinical trial in human heart failure.
Journal of the American College of Cardiology 01/2009; 52(23):1858-65. · 14.16 Impact Factor
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ABSTRACT: The primary cilium is an antenna-like structure extending from the surface of most vertebrate cells. Loss or mutation of ciliary proteins can lead to polycystic kidney disease and other developmental abnormalities. inv mutant mice develop multiple renal cysts and are a model for human nephronophthisis type 2. The mouse Inv gene encodes a 1062-amino-acid protein that is localized in primary cilia. In this study, we show that the Inv protein (also known as inversin) is localized at a distinctive proximal segment of the primary cilium, using GFP-tagged Inv constructs and anti-Inv antibody. We named this segment the Inv compartment of the cilium. Further investigation of the Inv protein showed that 60 amino acids at its C-terminal, which contains ninein homologous sequences, are crucial for its localization to the Inv compartment. Fluorescence recovery after photobleaching analysis revealed that the Inv protein was dynamic within this compartment. These results suggest that localization of the Inv protein to the Inv compartment is actively regulated. The present study revealed that the primary cilium has a distinct molecular compartment in the body of the primary cilium with a specific confining and trafficking machinery that has not been detected previously by morphological examination.
Journal of Cell Science 01/2009; 122(Pt 1):44-54. · 6.11 Impact Factor
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ABSTRACT: Despite the importance of glucose metabolism for auditory function, the mechanisms of glucose transport in the cochlea are not completely understood. We hypothesized that gap junctions mediate intercellular glucose transport in the cochlea in cooperation with facilitative glucose transporter 1 (GLUT1). Immunohistochemistry showed that GLUT1 and the tight junction protein occludin were expressed in blood vessels, and GLUT1, the gap junction proteins connexin26 and connexin30, and occludin were also present in strial basal cells in the lateral wall of the rat cochlea. Gap junctions were found among not only these GLUT1-positive strial basal cells but also GLUT1-negative fibrocytes in the spiral ligaments and strial intermediate cells. Glucose imaging using 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (6-NBDG, MW 342) together with Evans Blue Albumin (EBA, MW 68,000) showed that 6-NBDG was rapidly distributed throughout the stria vascularis and spiral ligament, whereas EBA was localized only in the vessels. The gap junctional uncouplers heptanol and carbenoxolone inhibited the distribution of 6-NBDG in the spiral ligament without decreasing the fluorescence of EBA in the blood vessels. These findings suggest that gap junctions mediate glucose transport from GLUT1-positive cells (strial basal cells) to GLUT1-negative cells (fibrocytes in the spiral ligament and strial intermediate cells) in the cochlea.
Histochemie 10/2008; 131(1):89-102. · 2.59 Impact Factor
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Tetsuro Takamatsu
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ABSTRACT: Life-threatening ventricular tachyarrhythmias are common clinical complications in ischemic heart diseases, especially infarcted heart. Although electrophysiological mechanisms have been extensively clarified for the genesis of arrhythmias in myocardial infarct, arrhythmogenic substrates in the infarct that eventually lead to electrical derangements are not fully understood. This review focuses on the intracellular calcium ion (Ca2+) dynamics and connexin43 (Cx43) gap junctions that play pivotal roles in excitation/contraction processes and intercellular communication, respectively, in heart muscle cells. Recent development of Ca2+-sensitive fluorescent dyes as well as microscopy imaging techniques has contributed substantially to a more precise understanding of spatiotemporal aspects in the intra- and inter-cellular dynamics of Ca2+ in cardiomyocytes. Ca2+ waves, heterogeneous wave-like elevations of the intracellular Ca2+ concentrations ([Ca2+](i)) that develop under [Ca2+](i)-overloaded conditions of the injured myocardium, play an essential role in arrhythmias, especially in triggered arrhythmias. Alteration of Cx43-mediated electrical coupling, that is, gap junction remodeling that arises at myocyte-myocyte and myocyte-myofibroblast interfaces, would also be an important substrate for arrhythmias, especially re-entrant tachyarrhythmias. Clarification of these substrates would provide not only deeper insights into the upstream events of life-threatening tachyarrhythmias in the infarcted heart but also bases for new therapeutic strategies for cardiovascular diseases.
Pathology International 10/2008; 58(9):533-43. · 1.62 Impact Factor
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ABSTRACT: Intracellular Ca(2+) waves (CaWs) of cardiomyocytes are spontaneous events of Ca(2+) release from the sarcoplasmic reticulum that are regarded as an important substrate for triggered arrhythmias and delayed afterdepolarizations. However, little is known regarding whether or how CaWs within the heart actually produce arrhythmogenic membrane oscillation because of the lack of data confirming direct correlation between CaWs and membrane potentials (V(m)) in the heart. On the hypothesis that CaWs evoke arrhythmogenic oscillatory depolarization when they emerge synchronously and intensively in the heart, we conducted simultaneous fluorescence recording of intracellular Ca(2+) ([Ca(2+)](i)) dynamics and V(m) of ventricular myocytes on subepicardial surfaces of Langendorff-perfused rat hearts using in situ dual-view, rapid-scanning confocal microscopy. In intact hearts loaded with fluo4/acetoxymethyl ester and RH237 under perfusion with cytochalasin D at room temperature, individual myocytes exhibited Ca(2+) transients and action potentials uniformly on ventricular excitation, whereas low-K(+)-perfused (2.4 mmol/L) hearts exhibited CaWs sporadically between Ca(2+) transients without discernible membrane depolarization. Further [Ca(2+)](i) loading of the heart, produced by rapid pacing and addition of isoproterenol, evoked triggered activity and subsequent oscillatory V(m), which are caused by burst emergence of CaWs in individual myocytes. Such arrhythmogenic membrane oscillation was abolished by ryanodine or the Na(+)-Ca(2+) exchanger inhibitor SEA0400, indicating an essential role of CaWs and resultant Na(+)-Ca(2+) exchanger-mediated depolarization in triggered activity. In summary, we demonstrate a mechanistic link between intracellular CaWs and arrhythmogenic oscillatory depolarizations in the heart. Our findings provide a cellular perspective on abnormal [Ca(2+)](i) handling in the genesis of triggered arrhythmias in the heart.
Circulation Research 08/2008; 103(5):509-18. · 9.49 Impact Factor
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ABSTRACT: Alteration of connexin43 (Cx43)-mediated intercellular communication is known to promote susceptibility to ventricular tachyarrhythmias. However, the precise mechanism of the altered Cx43 responsible for arrhythmogenesis remains unclear. We sought to understand changes in impulse propagation of ventricular myocytes under dominant-negative (DN) inhibition of Cx43 in the development of arrhythmias.
Intercellular communication was inhibited in confluent monolayers of neonatal rat cultured myocytes by an adenoviral vector-mediated gene transfer for DNCx43-fused red fluorescence protein (RFP). A high-resolution, macro-zoom fluorescence imaging system was used to visualize both the fluo4- and RFP-fluorescence intensities as measures of Ca2+ transient propagation and distribution of DNCx43 inhibition, respectively, in the myocyte monolayers. DNCx43 inhibition of the monolayers resulted in not only a significant slowing of Ca2+ transient propagation velocity, but also a preferential emergence of spiral-wave reentrant arrhythmias elicited by rapid pacing. Detailed observations on the development of spiral waves revealed that the gene-transferred myocyte monolayers exhibited regional slowing of propagation and subsequent generation of wave break, resulting in reentrant arrhythmias. Furthermore, DNCx43-RFP-transferred monolayers showed higher fluorescence intensity of RFP at the break point than at the surrounding myocardium, indicating a culprit role of DNCx43 inhibition in the genesis of spiral reentry.
The present results indicate that regional heterogeneity in gap-junctional communication promotes, in addition to slowing of conduction velocity, susceptibility to reentrant tachyarrhythmias.
Cardiovascular Research 08/2008; 79(1):70-9. · 6.06 Impact Factor
