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. Received: 4 April 1997 / Accepted: 19 June 1997     

Mapping of cytoskeletal components in the hippocampal formation of the tree shrew (Tupaia belangeri)

The distribution of the major cytoskeletal components in frontal cryosections of the hippocampal formation of adult male tree shrews (Tupaia belangeri) was immunohistochemically investigated by using commercially available antibodies. Actin-immunolabeling was evident in all layers of the dentate gyrus as well as in the regio superior (CA1) and the regio inferior (CA3). Neurofilament 160 was detected only in the molecular layer of the dentate gyrus and in the axons of the granule cells (mossy fibers). For beta-tubulin, the microtubule associated proteins (MAPs) MAP2AB, MAP2ABC and Tau, immunoreactivity was evident within the granule cells and within the somatodendritic compartment of pyramidal neurons. Granule cells and the somata of the pyramidal neurons were intensely labeled for kinesin. Our findings show the elaborate expression of cytoskeletal proteins in the hippocampal formation of the tree shrew, relatively similar to what is seen in other species but with also some important differences, such as the immunonegativity of the axonal compartment for Tau in the tree shrew, which is contrary to what we see in the mouse (unpublished data). These findings provide useful insights regarding the organization of the hippocampal formation of the tree shrew and are fundamental for further research in this field.     

Histamine-immunoreactive neurons in the brain of the teleost Gasterosteus aculeatus L. Correlation with hypothalamic tyrosine hydroxylase- and serotonin-immunoreactive neurons

The distribution of putative histaminergic neurons in the brain of a teleost, the three-spined stickleback, was investigated by means of immunocytochemistry using specific antibodies against histamine (HA), and conventional microscopy as well as confocal laser scanning microscopy. Histamine-immunoreactive (HAir) neurons form discrete populations ventral to the nucleus of the posterior recess (NRP) and in the nucleus saccus vasculosus (NSV), which belong to the periventricular hypothalamic nuclei. The neuronal somata are subependymally located, and do not possess apical neurites contacting the cerebrospinal fluid. They give rise to both long-range and local axonal projections. The local projections give rise to a field of dense punctate immunoreaction dorsal to the NRP and lateral to the NSV. Long-range projections are comprised of ascending projections to the thalamus, habenula, preoptic area and dorsal telencephalon; and descending projections via the posterior tuberal nucleus, ventrally to the nucleus interpeduncularis, and dorsally into the central gray. HAir neurons occur together with serotoninergic cerebrospinal fluid-contacting (CSFc) neurons in the NRP, and with tyrosine hydroxylase-immunoreactive (THir) neurons in the NSV. Although HAir elements occur together with THir ones in many brain areas, direct contacts between the two neurotransmitter systems are rare. The putative histaminergic neurons in the brain of the three-spined stickleback constitute a very discrete neuronal system, with a major projection area in the dorsal telencephalon in a region which is considered homologous with the dorsal pallium of land vertebrates.     

“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 diverticular 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 canaciluar 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.     

变异链球菌生物膜结构观察

目的　建立变异链球菌生物膜模型 ,用激光共聚焦扫描显微镜 (CLSM)观察变异链球菌生物膜结构。方法　在盖玻片上分别形成 6、12、18、2 4、4 8、72h变异链球菌生物膜 ,将得到的各时段生物膜荧光染色后 ,用CLSM观察生物膜的断层扫描图像、生物膜厚度、每层红光绿光的面积 ,计算生物膜中细菌密度和活菌百分比 ,用软件处理扫描数据 ,得到生物膜的三维重建图像。结果　变异链球菌生物膜具有空间立体结构 ,形态多样 ,其中细菌密集 ,由死细菌和活细菌组成 ,还有丰富的基质和管道系统。 2 4h生物膜平均厚度最大 ,生物膜内层、中间层的细菌密度相对较大 ,而外层较低 ,72h内各时间段生物膜中活菌百分比由内往外逐渐增加。结论　变异链球菌生物膜有一定的厚度 ,具有三维立体空间结构 ,结构形态具有多样、不均质、开放的特点。     

探索双重荧光染色并利用两种不同显微镜在Hep-2细胞上观察抗核抗体荧光模式的临床价值

目的 探索荧光显微镜,激光扫描共聚焦显微镜以及双重荧光染色法在Hep-2细胞上观察抗核抗体荧光模式的临床价值.方法 选取临床上抗核抗体呈阳性的血清样本结合到抗原片上,先后用异硫氰酸荧光素(FITC)和4 ',6-二脒基-2-苯基吲哚(DAPI)两种荧光染料孵育,之后在两种不同的显微镜下观察.结果 FITC荧光出现的部位是抗原抗体结合处,DAPI荧光出现的部位为细胞核,当通过DAPI荧光对细胞核定位后,可使得FITC的荧光模式更易于观察.本实验通过利用两种荧光标记的方法在两种显微镜上分别观察了斑点型、均质型、着丝点型、核点型、核膜型、胞浆颗粒型——抗高尔基体抗体.结论 综合各种因素,利用双荧光染色法染色,并在激光扫描共聚焦显微镜下观察,对于在Hep-2细胞上观察抗核抗体荧光模式最具临床价值.     

Deformation and failure of cartilage in the tensile mode

The aim of this study was to visualize, at the ultrastructural level, the deformation and failure mechanism of cartilage matrix in the tensile mode. Full-thickness dumbbell-shaped specimens were prepared from adult bovines. There were two specimen groups; in the 'parallel' group the specimen axis was parallel to the split lines defining the preferential orientation of the collagen in the articular surface, and in the 'perpendicular' group the specimen axis was perpendicular to the split lines. Specimens were placed with the articular surface uppermost and subjected to a graded series of strain within individual mini-tension devices, while observed with stereomicroscopy and confocal laser scanning microscopy. Thereafter, the changes in the ultrastructure were observed with both scanning and transmission electron microscopy. The mechanism of cartilage failure in the tensile mode comprised the following stages, whether the strain was applied parallel or perpendicular to the split line. (1) At 0% strain a fibrillar meshwork within the articular surface was predominantly orientated in the direction of the split line. (2) As strain increased, the fibrillar meshwork became more orientated in the parallel group and reorientated in the perpendicular group in the direction of the applied strain. (3) After complete reorientation of the fibrillar meshwork in the direction of the applied strain, the initial sign of failure was rupture of the fibrillar meshwork within the articular surface. (4) Subsequently, the rupture rapidly propagated into the deeper layers. Greater strains were required for fibrillar reorientation and complete rupture in the 'perpendicular group' than in the parallel group.     

Dynamic changes in the microvascular architecture of rat hair follicles during the hair cycle

We examined dynamic changes in the microvascular architecture of rat hair follicles during the hair cycle by scanning electron microscopy. Anagen hair follicles were surrounded by dense networks of capillaries at their lower parts. Below the tips of telogen hair follicles, there were collapsed networks of capillaries. Transmission electron microscopy showed that the capillaries around the bulbar region of anagen hair follicles possessed many fenestrations, while those of the collapsed networks located below the tips of telogen hair follices possessed virtually no fenestrations. Our results indicate that the dense network of capillaries around the lower part of the anagen hair follicle collapses with the involution of the follicle during tologen, and in turn develops with a new follicular downgrowth to form the basket-like capillary network of the anagen follicle. We also found that trans-endothelial transport occured much more frequently through the capillaries around anagen hair follicles than through those around telogen follicles.     

脑血管内皮形态与血液缘流保护性屏障

用共聚焦激光扫描显微镜,在开放颅窗的大耳白家兔模型上活体观测激光断层扫描光学切片,切片厚2μm或5μm,家兔血浆用荧光素标记、图像分析系统测量分析不同口径血管的轴流、缘流的荧光辉度,并描绘出辉度曲线。结果表明:在大脑皮质内动脉的口径在50μm以上者,辉度曲线呈"平台"形,在20μm以下者,辉度曲线呈"抛物线"形,这与在研究血管内皮形态时提出的"咀液缘流保护性屏障假说"相符合,证明了"血液缘流保护性屏障"的存在,随文附有血管内皮形态学扫描电镜研究资料。     

光动力疗法对牙菌斑生物膜活性及结构的影响

目的 探讨光动力疗法(PDT)对4种主要致龋菌形成的混合菌菌斑生物膜活性及结构的影响.方法以变形链球菌、血链球菌、嗜酸性乳杆菌和黏性放线菌为实验菌株,建立牙菌斑生物膜模型.实验分为3组:PDT组,洗必泰处理组,生理盐水处理组.平板菌落计数观察牙菌斑生物膜活性,并运用激光共聚焦显微镜(CLSM)对处理后的牙菌斑生物膜进行...     

Scanning and transmission electron microscopic study of visceral and parietal peritoneal regions in the rat

The visceral peritoneum of intraabdominal organs (spleen, stomach, liver, small intestine), omentum majus and the parietal peritoneum of the anterior abdominal wall and the diaphragm were studied in adult Wistar rats by combined scanning and transmission electron microscopy (SEM, TEM). In general, the peritoneal surface consisted of a mesothelium composed of cubic, flat or intermediate cell types delimited by a basal lamina. Cubic mesothelial cells predominated in parenchymal organs (spleen, liver) and were characterized by prominent and indentated nuclei, a cytoplasm richly supplied with organelles, a dense microvillous coat, basal invaginations and elaborate intercellular contacts. Flat mesothelial cells were observed in the intestinal, omental and parietal peritoneum (tendinous diaphragm, abdominal wall) and showed elongated nuclei, scant cytoplasm, a poorly developed organelle apparatus and sparsely distributed microvilli. An intermediate mesothelial cell type was described within the gastric peritoneum characterized by a central cytoplasmic protrusion at the nuclear region containing most of the cytoplasmic organelles and by thin finger-like cytoplasmic processes. The submesothelial connective tissue layer was composed of collagen fiber bundles, fibroblasts and free cells (macrophages, granulocytes, mast cells) and contained blood and lymphatic vessels. In the spleen, elastic fibers formed a membranous structure with intercalated smooth muscle cells. Mesothelial openings were observed as tunnel-like invaginations within the hepatic peritoneum and as clusters of peritoneal stomata within the parietal peritoneum of the anterior abdominal wall and the muscular diaphragm. The round or oval openings of the peritoneal stomata were frequently occluded by overlapping adjacent mesothelial cells and their microvillous coat or obstructed by cellular material. At the side of the peritoneal stomata the mesothelial cell layer was interrupted to allow a direct access to the underlying submesothelial lymphatic system. The mesothelium and lymphatic endothelium shared a common basal lamina. The endothelial cells were discontinuous and displayed valve-like plasmalemmatic interdigitations facilitating an intercellular transport of fluids and corpuscular elements from the peritoneal cavity to the submesothelial lymphatic lacunae. The findings underline the morphological heterogeneity of the peritoneum in visceral and parietal regions, suggesting different functional implications, and further support the presence of extra-diaphragmatic peritoneal stomata.     

Anti-biofilm efficacy of silver nanoparticles against MRSA and MRSE isolated from wounds in a tertiary care hospital

Purpose: Different approaches have been used for preventing biofilm-related infections in health care settings. Many of these methods have their own de-merits, which include chemical-based complications; emergent antibiotic resistant strains, etc. The formation of biofilm is the hallmark characteristic of Staphylococcus aureus and S. epidermidis infection, which consists of multiple layers of bacteria encased within an exopolysachharide glycocalyx. Nanotechnology may provide the answer to penetrate such biofilms and reduce biofilm formation. Therefore, the aim of present study was to demonstrate the biofilm formation by methicillin resistance S. aureus (MRSA) and methicillin resistance S. epidermidis (MRSE) isolated from wounds by direct visualisation applying tissue culture plate, tube and Congo Red Agar methods. Materials and Methods: The anti-biofilm activity of AgNPs was investigated by Congo Red, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) techniques. Results: The minimum inhibitory concentration (MIC) was found to be in the range of 11.25-45 μg/ml. The AgNPs coated surfaces effectively restricted biofilm formation of the tested bacteria. Double fluorescent staining (propidium iodide staining to detect bacterial cells and fluorescein isothiocyanate concanavalin A (Con A-FITC) staining to detect the exopolysachharides matrix) technique using CLSM provides the visual evidence that AgNPs arrested the bacterial growth and prevent the glycocalyx formation. In our study, we could demonstrate the complete anti-biofilm activity AgNPs at a concentration as low as 50 μg/ml. Conclusions: Our findings suggested that AgNPs can be exploited towards the development of potential anti-bacterial coatings for various biomedical and environmental applications. In the near future, the AgNPs may play major role in the coating of medical devices and treatment of infections caused due to highly antibiotic resistant biofilm.     

共聚焦激光扫描显微镜(MRC600)活体观测家兔大脑皮质内微循环的动物模型制作方法及其应用探讨

目的 :用共聚焦激光扫描显微镜活体观测家兔大脑皮质内微血管构筑及其微循环提供更近似于人类的动物模型。方法 :在开放颅窗的家兔动物模型上 ,荧光素标记血浆 ,罗丹明 6G标记WBC ,用共聚焦激光扫描显微镜活体观测正常状态下家兔大脑皮质内微循环 ,并经图像分析系统测量数据 ,用SAS软件包进行统计学分析。结果 :本研究探索用开放颅窗的家兔动物模型进行大脑皮质微循环的研究取得成功 ,作者认为采用开放颅窗的家兔动物模型使观察大脑皮质的深度达 2 5 0～ 30 0 μm ,对大脑皮质内微血管构筑及微循环进行活体观测 ,可进行连续断层扫描 ,或在同一层面进行连续定位扫描监测 ,所摄取图像经监视器及计算机摄入并存盘待分析 ,用图像分析系统进行观测血管口径 ,血流速度 ,血液流态 ,白细胞 ,红细胞运动等微循环指标 ,也可用标准Bio Radthruview软件三维重建 ,观察各种血管形态 ,研究大脑皮质微血管构筑。结论 :开放颅窗的家兔动物模型是更近似于人类的动物模型 ,适合用于脑血管疾病发病机理的研究     

Structural development of PGP9.5-immunopositive myenteric plexus in embryonic rats

To investigate relationships between changes in the 3-dimensional structure of the myenteric plexus and the time at which functional movement of intestine begins in mammalian embryos, whole mounts of embryonic rat intestine were examined under confocal laser scanning microscopy on spacer-equipped glass slides after immunostaining with antiprotein gene product 9.5 antibody. At embryonic day 12.5, very few nerve cells were scattered throughout the small intestine, but no immunostained structures were apparent on the anal side of the large intestine. At embryonic day 13.5, immunostained fibers appeared on the oral side of the large intestine. Nerve cells and associated fiber bundles formed neuronal networks with large meshes in both intestines. Marked increases in number of nerve fibers and decreases in mesh size were seen in the small intestine between embryonic days 13.5 and 15.5. Similar changes were found in the large intestine between embryonic days 13.5 and 16.5. After embryonic day 16.5, nerve cells were arranged parallel to circular muscle fibers, and networks formed by cell fibers elongated until the neonatal period in both intestines. Meconium passed through the large intestine from embryonic day 17.5. Thin fiber bundles extended from the ganglion to the inner side of the myenteric layer, parallel (and occasionally extending) to the circular muscle fibers. Formation of nerve fiber networks and arrangement of nerve cells parallel to circular muscle fibers probably relate to movement coordination for inner circular muscle fibers in the intestinal wall, and development of this neural network may be important for acquiring intestinal movements before birth.     

Image cross-correlation spectroscopy: A new experimental biophysical approach to measurement of slow diffusion of fluorescent molecules

The technique of image cross-correlation spectroscopy has been applied to measurements of diffusion of fluorescent beads in polymer solution. The technique is based on measurement and analysis of temporal fluctuations of the intensity observed in fluorescence confocal microscope images. As with other fluctuation techniques, fluorescence fluctuations arise from stochastic concentration fluctuations about the equilibrium concentration. The dynamics of the fluctuations depend on the number of fluorescent molecules in the observation volume. This work presents the theory and illustrates how the technique can be applied to measurement of diffusion of fluorescent beads in solutions of various viscosities. Further we extend the concept of cross-correlation to studies on the cell surface to detect the relative distribution of molecules (receptors) on the cell surface, in which intensity fluctuations arising from samples containing two distinct types of labelled molecules are cross-correlated using two detectors with different wavelength sensitivities.     

Three-dimensional organization of the elastic fibres in the rat lung

Background: The elastic framework of the distal lung has been studied by light microscopy (LM) and transmission electron microscopy (TEM). The preservation of the elastic fibres, for the three-dimensional observation in their relative positions, is difficult because they lack support when the normal methods of tissue processing are used. The goal of the present study was to understand the three-dimensional ultrastructure and organization of the elastic fibres of the lung preserved in their relative positions. Methods: A combination of intravascular resin injection and formic acid digestion was used. The resin cast of the microvasculature acted as a scaffold to preserve the in vivo arrangement of the elastic fibres that are, otherwise, easily collapsible. Scanning electron microscopy (SEM) samples were further processed for TEM in order to confirm that the fibres were indeed components of the elastic system. Results: SEM demonstrated a fine framework of elastic fibres, representing remnants of the alveolar walls, with the casted capillaries interwoven with the network of elastin. Each individual elastic fibre is composed of a small bundle of discrete fibrils. Some of these fibrils emerge from the fibre and join other fibres, producing an anastomosing appearance. Several elastic fibres link the walls of the intrapulmonary conducting airways, the vessels walls and the alveolar network, thus establishing an interrelated and interlaced framework. Conclusions: The method we have applied to visualize the elastic fibres of the lung is a unique approach to define the spatial organization of the pulmonary elastic fibres. We have demonstrated here the close relationship between the elastic fibres and the capillaries of the septal alveoli. The arrangement of the interwoven network of elastin and its relationship with the capillaries offers the structural setting for the distending capacity of the alveolar wall. © 1995 Wiley-Liss, Inc.     

Apoptosis and occurrence of Bcl-2, Bak, Bax, Fas and FasL in the developing and adult rat endocrine pancreas

Apoptotic cell death is thought to play a crucial role in the manifestation of insulin- and non-insulin dependent diabetes mellitus. Therefore, apoptosis and apoptotic markers were studied in the rat endocrine pancreas to get insight into the possible life cycle of Langerhans islets.The islets were investigated at 13 time points between day E19 and 18 months. At each time point, histologic sections were treated with the direct fluorescein-labelled TUNEL method and immunostained for pancreatic hormones (glucagon, insulin), apoptotic promoters (Bak, Bax, Fas, Fas Ligand) as well as for the anti-apoptotic peptide Bcl-2. All tissue sections were investigated using confocal laser scanning microscopy under identical settings for semiquantitative estimation of staining intensity. TUNEL-positive cells occurred in all pre- or postnatal stages.The findings indicated a biphasic apoptotic activity in the endocrine pancreas during the lifetime of rats. The first phase began at E19 and peaked at P5 accompanied by a considerable increase in Bak fluorescence staining intensity, while the second phase began at P30 and peaked at 18 months with increasing amounts of Fas and FasL staining intensities in the islet cells. The presented in situ data may be important for understanding the increased age-related vulnerability of islet cells and for studies of isolated and cultivated rat islets. Accepted: 9 May 2000