Three distinct types of Ca(2+) waves in Langendorff-perfused rat heart revealed by real-time confocal microscopy.

Although Ca(2+) waves in cardiac myocytes are regarded as arrhythmogenic substrates, their properties in the heart in situ are poorly understood. On the hypothesis that Ca(2+) waves in the heart behave diversely and some of them influence the cardiac function, we analyzed their incidence, propagation velocity, and intercellular propagation at the subepicardial myocardium of fluo 3-loaded rat whole hearts using real-time laser scanning confocal microscopy. We classified Ca(2+) waves into 3 types. In intact regions showing homogeneous Ca(2+) transients under sinus rhythm (2 mmol/L [Ca(2+)](o)), Ca(2+) waves did not occur. Under quiescence, the waves occurred sporadically (3.8 waves. min(-1) x cell(-1)), with a velocity of 84 microm/s, a decline half-time (t(1/2)) of 0.16 seconds, and rare intercellular propagation (propagation ratio <0.06) (sporadic wave). In contrast, in presumably Ca(2+)-overloaded regions showing higher fluorescent intensity (113% versus the intact regions), Ca(2+) waves occurred at 28 waves x min(-1) x cell(-1) under quiescence with a higher velocity (116 microm/s), longer decline time (t(1/2) = 0.41 second), and occasional intercellular propagation (propagation ratio = 0.23) (Ca(2+)-overloaded wave). In regions with much higher fluorescent intensity (124% versus the intact region), Ca(2+) waves occurred with a high incidence (133 waves x min(-1) x cell(-1)) and little intercellular propagation (agonal wave). We conclude that the spatiotemporal properties of Ca(2+) waves in the heart are diverse and modulated by the Ca(2+)-loading state. The sporadic waves would not affect cardiac function, but prevalent Ca(2+)-overloaded and agonal waves may induce contractile failure and arrhythmias.

Subjective response to neuroleptics: the effect of a questionnaire about neuroleptic side effects.

BACKGROUND: A patient's subjective response to neuroleptics is an important factor in pharmacotherapy of schizophrenia. In this study, we conducted an intervention to assess the effect of a questionnaire about neuroleptic side effects. We hypothesized that paying more attention to a patient's subjective distress associated with neuroleptic side effects would improve the patient's subjective response to neuroleptics. So we made a questionnaire about neuroleptic side effects, and used this questionnaire repeatedly in the usual clinical setting as an intervention. METHOD: We administered this study to 210 outpatients who met the following criteria: (1) diagnosis of schizophrenia or schizoaffective disorder as defined by DSM-IV and (2) no worsening of symptoms in the past 6 months. The patients were divided into the intervention and the control groups. Patients of the intervention group filled out the questionnaire four times during 6 months and were given routine clinical care. Patients of the control group had routine clinical care only. The 10-item Drug Attitude Inventory (DAI-10) was used to evaluate the patients' subjective responses to neuroleptics. RESULTS: After 6 months, the patients' subjective responses to neuroleptics assessed by the DAI-10 significantly improved in the intervention group (p<0.05 for within-group comparison). The most improved response was that the patients felt they were taking medications of their own free choice. CONCLUSION: Paying more attention to the patient's subjective distress associated with neuroleptic side effects may have encouraged patients to participate in pharmacotherapy on their own initiative. This study suggests that our 19-item questionnaire is a useful tool to improve a patient's subjective response to neuroleptics.

Highly sensitive fluorescence detection of metastatic lymph nodes of gastric cancer with photo-oxidation of protoporphyrin IX.

BACKGROUND: The establishment of a precise and rapid method to detect metastatic lymph nodes (LNs) is essential to perform less invasive surgery with reduced gastrectomy along with reduced lymph node dissection. We herein describe a novel imaging strategy to detect 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence in excised LNs specifically with reduced effects of tissue autofluorescence based on photo-oxidation of PpIX. We applied the method in a clinical setting, and evaluated its feasibility. METHODS: To reduce the unfavorable effect of autofluorescence, we focused on photo-oxidation of PpIX: Following light irradiation, PpIX changes into another substance, photo-protoporphyrin, via an oxidative process, which has a different spectral peak, at 675 nm, whereas PpIX has its spectral peak at 635 nm. Based on the unique spectral alteration, fluorescence spectral imaging before and after light irradiation and subsequent originally-developed image processing was performed. Following in vitro study, we applied this method to a total of 662 excised LNs obtained from 30 gastric cancer patients administered 5-ALA preoperatively. RESULTS: Specific visualization of PpIX was achieved in in vitro study. The method allowed highly sensitive detection of metastatic LNs, with sensitivity of 91.9% and specificity of 90.8% in the in vivo clinical trial. Receiver operating characteristic analysis indicated high diagnostic accuracy, with the area under the curve of 0.926. CONCLUSIONS: We established a highly sensitive and specific 5-ALA-induced fluorescence imaging method applicable in clinical settings. The novel method has a potential to become a useful tool for intraoperative rapid diagnosis of LN metastasis.

Raman spectra of some snake venom components.

The Raman spectra of aqueous solutions of cobrotoxin, erabutoxin b (short-chain neurotoxins), alpha-bungarotoxin (a long-chain neurotoxin), cardiotoxin, cytotoxin I (cardiotoxins), 6-carboxyl-modified cobrotoxin and cobrotoxin treated with 5 M guanidine-HCl (denatured neurotoxins) were investigated. The main-chain structure of the toxins was found to consist predominantly of beta-pleated sheets and random-coils. The relative amounts of the beta-conformations were estimated to be in order of: cardiotoxins approximately short-chain neurotoxins greater than long-chain neurotoxins greater than Laticauda semifasciata III (exceptionally weak neurotoxin). Most of the disulfide-bridges of every toxin take the gauche-gauche structure about the CC-SS-CC linkage. However, the number of gauche-gauche structures is not the same among the neurotoxins. Tyr-25 of cobrotoxin is found to form a strong hydrogen bond similar to that of other short-chain neurotoxins investigated earlier.

Effects of basal [Ca2+]i on calcium handling in Ca(2+)-overloaded rat cultured heart muscle cells.

To elucidate the relationship between intracellular free Ca2+ concentration ([Ca2+]i) and Ca(2+)-signalling by the sarcoplasmic reticulum (SR) in Ca(2+)-overloaded heart muscle cells, the direct effects of "basal" [Ca2+]i on calcium waves were investigated by altering the membrane potential. When basal inter-calcium wave (BCW) [Ca2+]i was maintained at a high level, (i) calcium waves showed more gradual and more rapidly suppressed increase in [Ca2+]-profile (P < 0.005), and (ii) calcium waves occurred at a significantly higher frequency and velocity (259% and 137%), than when low BCW [Ca2+]i was maintained. Similar investigations on inhibition of the Na(+)-Ca2+ exchanger, however, showed that membrane potential did not elicit direct effects on calcium waves. These results showed that the elevation of BCW [Ca2+]i per se directly influences Ca(2+)-signalling in heart muscle cells through non-equilibrated release-restoration Ca(2+)-handling by the SR.

Real time in situ confocal imaging of calcium wave in the perfused whole heart of the rat.

To understand the calcium handling in whole heart having automaticity of the sinus node, we have developed a system of in situ imaging the intracellular calcium ion concentration in the perfused whole heart of the rat. The system consists of a stage-fixed upright microscope equipped with a real-time confocal laser scanning device of a multipinhole type with a water-immersion objective lens for observation. This in situ imaging system rendered observations and analyses of the rapidly changing images of intracellular calcium dynamics possible in the whole rat heart loaded with fluo-3. The scanning was conducted at a video rate of 30 frames per second, and the confocal effects included both X and Y planes. Calcium waves were frequently interrupted by calcium transients from either external electro-stimulation pulses or spontaneous sinus rhythm. Our findings suggest that abnormal calcium waves in minute areas cannot disturb the excitation-contraction coupling in the whole heart if the myocardial cells have orderly end-on-end intercellular electric paths.

High temporal resolution video imaging of intracellular calcium.

We have developed a system for imaging intracellular free calcium ion concentration ([Ca2+]i) at the highest rate possible with conventional video equipment. The system is intended to facilitate quantitative study of rapid changes in [Ca2+]i in cells that move. It utilizes intensified video cameras with nearly ideal properties and digital image processing to produce two images that can be ratioed without artifacts. Two dichroic mirrors direct images of cellular Indo-1 fluorescence at two different wavelengths to two synchronized video cameras, each consisting of a fast micro-channel plate image intensifier optically coupled with a tapered fiber optic bundle to a CCD image sensor. The critical technical issues in this dual-image system are: (1) minimization and correction of the small geometric and other types of differences in the images provided by the two cameras; and (2) the signal-to-noise ratio that can be achieved in single frames. We have used this system to obtain images of [Ca2+]i at 16.7 ms intervals in voltage-clamped single cardiac cells perfused internally with Indo-1 (pentapotassium salt). The images indicate that, except for the nuclear regions, [Ca2+]i is uniform during normal excitation-contraction coupling. In contrast, changes in [Ca2+]i propagate in rapid 'waves' during the spontaneous release of Ca2+ that accompanies certain 'Ca2(+)-overload conditions.'

Secretagogue-induced calcium wave shows higher and prolonged transients of nuclear calcium concentration in mast cells.

To clarify the mechanism of secretagogue (compound 48/80)-induced calcium signaling in rat peritoneal mast cells, we analyzed serial confocal calcium images with high spatial and temporal resolution using different Ca(2+)-probes. The Ca(2+)-wave began at the periphery of the cytoplasm, and then spread to the center of the nucleus. Nuclear [Ca2+]i was clearly higher than cytoplasmic [Ca2+]i. The heterogeneity of [Ca2+]i continued until about 2 min after degranulation. These results suggest the existence of an intranuclear Ca(2+)-store which possesses a Ca(2+)-releasing mechanism similar to that in the cytoplasm.

Intranuclear topological distribution of HIV-1 trans-activators.

Subcellular localization of human immunodeficiency virus type I (HIV-1) Tat and Rev was examined using a confocal laser scanning microscope (CLSM). In transfected COS-7 cells, Tat resided exclusively in the perinocleolar region, while Rev infiltrated fully into the nucleoli. The chimeric Tat in which the nucleolar targeting signal was replaced by that of Rev, which retains trans-acting activity of Tat, remained still in the perinucleolar region as wild-type Tat. Perinucleolar distribution of Tat protein suggests the existence of a novel nucleolar architecture that affects transcription.

Different regulation of connexin26 and ZO-1 in cochleas of developing rats and of guinea pigs with endolymphatic hydrops.

Using confocal microscopy and morphometry, we analyzed the expression of connexin26 (Cx26) and ZO-1 in rat cochlea during the postnatal period to elucidate spatiotemporal changes in gap junctions and tight junctions during auditory development. We also studied changes in these junctions in experimental endolymphatic hydrops in the guinea pig. In the adult rat cochlear lateral wall, Cx26 was detected in fibrocytes in the spiral ligament and in the basal cell layer of the stria vascularis, whereas ZO-1 was detected in the apical surfaces of marginal cells and in the basal cell layer. During postnatal development, Cx26 expression increased mainly in the spiral ligament, whereas ZO-1 expression increased in the basal cell layer. The morphometry of Cx26 showed a sigmoid time course with a rapid increase on postnatal day (PND) 14, whereas that of ZO-1 showed a marked increase on PND 7. In experimental endolymphatic hydrops, the expression of Cx26 significantly decreased, whereas there were no obvious changes in the expression of ZO-1. These results indicate that gap junctions and tight junctions in the cochlea increase in a different spatiotemporal manner during the development of auditory function and that gap junctions and tight junctions in the cochlea are differentially regulated in experimental endolymphatic hydrops. (J Histochem Cytochem 49:573-586, 2001)

Differential expression of gap junction proteins connexin26, 32, and 43 in normal and crush-injured rat sciatic nerves. Close relationship between connexin43 and occludin in the perineurium.

We immunohistochemically and morphometrically examined the expression of gap junction protein connexin (Cx) in normal and crush-injured rat sciatic nerves using confocal laser scanning microscopy. Cx26 was localized in the perineurium and Cx43 was present in the perineurium and the epineurium, whereas Cx32 was confined to the paranodal regions of the nodes of Ranvier. Double labeling for connexins and laminin revealed that Cx43 was localized in multiple layers of the perineurium, whereas Cx26 was confined to the innermost layer. Double labeling for connexins and a tight junction protein, occludin, showed that occludin frequently coexisted with Cx43 but existed separately from Cx26 in the perineurium. After crush injury, the pattern of normal Cx32 expression was initially lost but recovered, whereas Cx43 rapidly appeared in the endoneurium and its expression was subsequently attenuated. Although crush injury produced no apparent alteration in Cx43 and occludin in the perineurium, a rapid increase and a subsequent decrease in the frequency of Cx26-positive spots during nerve regeneration were shown by morphometric analysis. These results indicate that Cx26, Cx32, and Cx43 are expressed differently in various types of cells in peripheral nerves and that their expressions are differentially regulated after injury. The expression of connexins and occludin in the perineurium suggests that perineurial cells are not uniform in type and that the regulation of gap junctions and tight junctions is closely related in the perineurium.

Abnormalities in gap junctions and Ca2+ dynamics in cardiomyocytes at the border zone of myocardial infarcts.

Abnormalities in gap junction function and Ca2+ dynamics are believed to be important factors in arrhythmogenesis after myocardial infarction. To elucidate the relationship between changes in Ca2+ dynamics and gap junctions, we analyzed by real-time in situ Ca2+ imaging of fluo-3 loaded whole hearts the spatiotemporal occurrence of Ca2+ waves and the localization of connexin43 (Cx43) at the border zone of myocardial infarcts induced in the rat by coronary ligation. At early time points (2-4 hours postligation), different regions of the left ventricle showed distinct changes in cytosolic free Ca2+ concentrations [Ca2+]i. While some cardiomyocytes of infarcted regions exhibited high levels of resting fluo-3 fluorescence, at border zones frequent Ca2+ waves were observed. Some of the waves were abolished by spontaneous Ca2+ transients and others were not. Intact myocardium apart from infarcted regions exhibited homogenous Ca2+ transients. Confocal imaging of Cx43 and actin filaments in the rat heart fixed 2 hours after coronary ligation revealed that Cx43 was markedly decreased in the area of myocyte necrosis with contraction bands and in the neighboring myocardium. These results suggest that abnormal expression and function of gap junctions could be associated with Ca2+ waves at the border zone of myocardial infarcts, possibly through Ca2+ overload.

Pulmonary hypertension in a patient with rheumatoid arthritis.

A 53-year-old woman who had suffered from severe rheumatoid arthritis developed pulmonary hypertension. Her small arteries in the lung showed plexogenic arteriopathy with fibrous intimal hyperplasia. There was also vasculitis of the small arteries in other organs and mural thrombosis in the pulmonary stem and abdominal aorta. The plexogenic arteriopathy which was responsible for pulmonary hypertension appears to be the result of vasculitis in association with rheumatoid arthritis.

Detection and evaluation of asymptomatic myocarditis in schoolchildren: report of four cases.

STUDY OBJECTIVE: Data on the prevalence of myocarditis in children are limited. Autopsy studies have shown that myocarditis is often undiagnosed. We attempted to investigate the clinical features of asymptomatic myocarditis in four schoolchildren detected during a mass ECG screening in schoolchildren. DESIGN AND SETTING: To evaluate asymptomatic myocarditis, we clinically examined 12 schoolchildren who were referred to Shiga University of Medical Science Hospital, Otsu, Japan, because of abnormal ST or T waves detected during ECG screening. None of the 12 children had experienced any episodes suggesting cardiac disease or Kawasaki disease. Cardiac function and myocardial viability were assessed by two-dimensional echocardiography (2-DE), thallium-201 (201Tl) myocardial scintigraphy, and cardiac catheterization. Endomyocardial biopsy specimens were examined histologically. PATIENTS: Endomyocardial biopsy specimens revealed histologic evidence of myocarditis in 4 of the 12 children with abnormal ST or T waves. RESULTS: Abnormal tracer perfusion was observed on 201Tl myocardial scintigrams in these four children, but the results of coronary arteriography were normal. 2-DE showed left ventricular hypokinesis in one child and left ventricular enlargement in one of the four children with histologic evidence of myocarditis. A second endomyocardial biopsy specimen was obtained in two of four children, showing persistent myocarditis in one child. CONCLUSIONS: This type of screening program and indepth evaluation using 2-DE and 201Tl myocardial scintigraphy appear to be helpful in identifying children with myocarditis. The present histologic investigations suggested that even asymptomatic myocarditis might result in persistent heart damage.

Ischaemia-induced temporal expression of connexin43 in rat heart.

To investigate the regulation of cell-to-cell coupling in myocardial ischaemia, the three-dimensional expression of connexin43 (Cx43) during experimental ischaemia was examined using a confocal laser scanning microscope. After induction of myocardial infarction in rats, serial optical sections were obtained from the left ventricular myocardium at various times (3 h to 60 days after ligation). The expression of Cx43 was detected immunohistochemically with FITC-labelled anti-rat Cx43 antibody. Fluorescent dots of Cx43 remained along the intercalated disc and decreased in number around the infarct up to 12 h after ligation. Cx43-expression disappeared completely within 48 h after ligation. After day 4, and especially on days 8 and 15 after ligation, the edges of the cardiomyocytes bordering the infarcted area manifested numerous sarcoplasmic tentacles that reacted positively to anti-desmin antibody. Distinct expression of Cx43 was observed extensively on the tentacles, although no cardiomyocytes remained viable around them. By day 60 after ligation, atypical expression of Cx43 had disappeared. These findings suggest that ischaemia induces temporally abnormal expression of Cx43, which might be responsible for abnormal conduction around the infarct.

"Varicoid change" of bile canaliculi in rat liver at an early phase of ischaemia-reperfusion injury.

To elucidate early changes and the mechanism of ischaemia-reperfusion liver injury, we investigated three-dimensional microstructural changes of cellular actin filaments in rat livers using confocal laser scanning microscopy. The liver tissues of a reperfusion group were examined 12 h after removal of a vascular clamp. Fixed tissues were stained with fluorescein-labelled phalloidin to obtain stereoscopic images of the actin filaments and these were compared with histological findings. The images of bile canaliculi showed that multiple abnormal minute diverticula arose from the canalicular membranes and fused with one another, resulting in irregular dilation of the bile canaliculi. These changes were observed after 15 min of ischaemia and reperfusion in which no significant necrosis was seen. The frequency and degree of these changes were strictly dependent on the periods of ischaemia (15-60 min). We called these bile canacilular lesions "varicoid changes". The liver of an ischaemia group taken after persistent clamping without reperfusion did not show these changes. Our findings suggest that the varicoid change in the bile canaliculi is probably due to alterations in the actin polymerization-depolymerization cycle and is a pathognomonic change of ischaemia-reperfusion liver injury.

Regulation of apoptotic cell death in the pyloric glands of the canine stomach.

In gastrointestinal epithelia, apoptosis has been thought to play a part in the shedding of postmitotic cells into the lumen. However, we have found that apoptosis more frequently in the generative cell (G) zone (the lower one third of the pit) than in the luminal zone (the upper one third of the pit) and the gland zone in the canine pyloric gland. To analyse the regulation of apoptotic cell death in each zone, we labelled S-phase cells by single and repeated injections of bromodeoxyuridine (BrdU) i.v. at intervals of 8 h. We found that 30% of apoptoses in the G zone were flash-labelled by BrdU and might derive from cells in or just after the S phase. The incidence of apoptosis and mitotic index did not change significantly after repeated injections of BrdU until the 49-h point, when the incidence of apoptosis increased and the mitotic index decreased significantly in the G zone, while the incidence of apoptosis decreased in the luminal zone. The BrdU-induced increase of apoptosis and cell-cycle arrest at the 49-h point may be caused by enhanced DNA mispairs that are elicited by incorporation of BrdU, in particular using the template of BrdU incorporated DNA. Apoptosis in the luminal zone may be down-regulated by reduced cell production in the G-zone.

Direct retinal projections to GRP neurons in the suprachiasmatic nucleus of the rat.

The retinal projections to gastrin-releasing peptide (GRP)-expressing neurons in the rat suprachiasmatic nucleus (SCN) were investigated by double immunofluorescence and immunoelectron microscopy. Optic nerve terminals labeled by cholera toxin B subunit (CTb) which was transported from the retinal ganglion cells were intermingled with GRP-immunoreactive cell bodies and processes in the ventrolateral portion of the SCN. Ultrastructural analysis revealed that CTb-immunoreactive retinal terminals made synaptic contacts with GRP-immunoreactive dendritic processes. These results demonstrated that photic information is directly input from the optic nerve to GRP neurons in the SCN and these GRP neurons may be involved in circadian entrainment by light.

Correlation of histological inflammation in needle biopsy specimens with serum prostate- specific antigen levels in men with negative biopsy for prostate cancer.

OBJECTIVES: To reveal the possible contribution of histological inflammation within the prostate to the abnormal elevation of serum prostate-specific antigen (PSA) levels in patients with needle biopsy negative for prostate cancer. METHODS: We reviewed negative needle biopsy specimens obtained in 93 patients. The degree of acute and chronic inflammation as evaluated histologically was compared with serum PSA levels in conjunction with age and prostate volume. RESULTS: Both age (P <0.01) and prostate volume (P <0.0001) correlated significantly with serum PSA levels and were significantly greater in patients with abnormal serum PSA levels (greater than 4.0 ng/mL) than in those with normal serum PSA levels (4.0 ng/mL or less) (P <0.01). The presence of histological inflammation within the prostate also correlated significantly with serum PSA levels. Multiple regression analysis demonstrated prostate volume to be the only independent determinant of serum PSA levels (P <0.01). In patients with a prostate volume larger than 25 mL, only prostate volume correlated significantly with serum PSA levels (P <0. 05). On the other hand, the degree of acute inflammation as represented by polymorphonuclear leukocyte infiltration was the only parameter correlating significantly with serum PSA levels (P <0.05) in patients with a prostate volume smaller than 25 mL. CONCLUSIONS: Histologically defined acute inflammation within the prostate is a significant contributor to elevated serum PSA levels, especially in patients with small prostates. In the assessment of needle biopsy results negative for prostate cancer, it might be helpful to evaluate the degree of histological inflammation, especially in terms of the necessity of subsequent repeated biopsies.

Regulation of cytosol-nucleus pH gradients by K+/H+ exchange mechanism in the nuclear envelope of neonatal rat astrocytes.

In order to study the subcellular heterogeneity of intracellular H+ concentration in reactive astrocytes, the pH in the nucleus and cytosol of cultured astrocytes was measured using a confocal laser scanning microscope (CLSM) and pH indicator dye, 5'(and 6')-carboxyseminaphthofluorescein (carboxy SNAFL-1). The change in intracellular pH was indexed by the fluorescence ratio (F535/F610) at an excitation wavelength of 514.5 nm. The in vitro fluorescence ratio increased as pH decreased. This ratio in the nucleus was significantly lower than that in the cytosol of astrocytes when perfused by HEPES-buffered Hanks' balanced salt solution (HHBSS) at pH 7.4. Acid stimulations of cells (pH 5.0) raised the fluorescence ratio in both nucleus and cytosol. However, the increase in the fluorescence ratio of the nucleus was less than that of cytosol. Treatment with a K+/H+ ionophore, nigericin (20 microM), reversibly nullified this cytosol-nucleus pH gradient. These findings suggest that a buffering mechanism(s) for maintaining of intracellular pH exists between the nucleus and cytosol, and a K+/H+ exchanger may act on the nuclear envelope to eventuate intranuclear pH maintenance in the living cells.