Histological demonstration of increased vascular permeability in the dura mater of the rat

Neurogenic inflammation of the dura mater encephali has been suggested to play an important role in the pathophysiology of headaches. Although functional studies using extravasation techniques indicate an enhanced permeability of blood vessels after chemical or electrical stimulation of C-fibres supplying the dura mater, histological demonstration of leaky blood vessels is still a problem. We used the vascular labelling method combined with i.v. injection of colloidal silver solution to test the permeability increasing effect of intravenous administration of substance P, topical application of mustard oil or acidic phosphate buffer and local electrical stimulation of the exposed dura mater. Histological characteristics of increased vascular permeability were observed exclusively after mustard oil and acidic phosphate buffer. This observation may indicate different mechanisms of increased vascular permeability involving pinocytosis and formation of interendothelial gaps selectively visualized by the vascular labelling method.     

Interaction of rat tumor cells with blood vessels and lymphatics of the avian chorioallantoic membrane

It has generally been assumed that tumors do not induce lymphangiogenesis and only very recently animal models have been presented showing tumor-induced lymphangiogenesis. We have grown two types of rat tumor cells, 10AS pancreatic carcinoma and C6 glioma cells, on the chorioallantoic membrane (CAM) of chick and quail embryos. The suspended tumor cells rapidly formed solid tumors which invaded the CAM and were vascularized by CAM vessels. When grown on the CAM of quail embryos intratumoral endothelial cells could be specifically stained with the QH1 antibody. In C6 gliomas the vascular pattern was more regular than in 10AS carcinomas. The vessels often grew radially into the glioma and many of them were invested by smooth muscle alpha-actin-positive periendothelial cells. Lymphatics, which were identified by vascular endothelial growth factor receptor-3 (VEGFR-3) in situ hybridization were absent from C6 gliomas, although a weak expression of the lymphangiogenic growth factor, VEGF-C, could be detected in the C6 cells by Northern blot analysis. In contrast, 10AS cells, which expressed high levels of VEGF-C, induced ingrowth of lymphatics into the tumors, with BrdU-labeling rates of about 9% of lymphatic endothelial cells. Our studies demonstrate the heterogeneity of interactions of tumor cells with blood vessels and lymphatics and show that sufficient quantities and/or quality of lymphangiogenic growth factors are crucial for the induction of lymphatics in tumors.     

剪切力对动脉血管平滑肌细胞增殖分化的影响

目的：探讨剪切力作用下人脐动脉血管平滑肌细胞的增殖分化的变化。方法：利用改进的灌流及流动培养装置,通过蠕动泵提供稳定的剪切力,同时提供静态培养所需的其它条件,建立体外人脐动脉血管平滑肌流动培养模型。分别对体外培养的人脐动脉血管平滑肌细胞加载3,4,5,8,10dyn／cm^2的定长流剪切力24h,同时以静态培养的细胞为对照组。相差倒置显微镜观察玻片上细胞的数量并作细胞计数,α—actin免疫组化染色．结果：细胞记数显示,同对照组相比,七刀应力各组的增殖能力都有所下降,5dyn／cm^2切应力作用下,细胞增殖缓慢最为明显．α—actin免疫组化染色提示切应力各组的分化程度较对照组高。结论：初步研究表明,剪切力对人脐动脉血管平滑肌细胞的增殖有一定抑制作用,并且可能促进了细胞的分化。     

基于苦味受体表达的多组织细胞传感器及其应用

非口腔组织中的苦味受体(TAS2Rs)可能成为相关疾病治疗的新靶点.该研究将表达有TAS2Rs的细胞作为敏感元件,根据其生理特性与不同的传感器耦合,探究了味觉受体异位表达及其在个性化药物筛选中的应用,构建了针对不同疾病模型的个性化药物筛选平台.首先,基于细胞阻抗传感器以及内源性表达TAS2R38受体的结肠癌细胞,开发了...     

Morphologic and temporal analysis of vascular smooth muscle cell apoptosis induced by c-Myc and E1A

Apoptosis is a physiologic form of cell death present in many disease conditions. When the balance of mitosis versus apoptosis is altered, tumor-like growth or degeneration of tissues may ensue. This appears to occur in several diseases, including those of the cardiovascular system, where apoptosis plays a key role in atherosclerosis and restenosis following angioplasty. Since c-myc is upregulated in the pathogenesis of these diseases, we chose to study the sequential morphologic features of programmed cell death in vascular smooth muscle cells induced by c-myc and by the adenovirus early gene E1A. Morphology and timed events in apoptotic cell cultures were analyzed by scanning electron microscopy, transmission electron microscopy, and time-lapse videomicroscopy. We observed that both c-myc-and E1A-induced apoptosis (in serum-free medium) resulted in numerous, tightly packed clusters of apoptotic blebs, as well as in one or two asymmetrically larger blebs. Transmission electron microscopy analysis revealed the larger blebs contained mostly nuclear chromatin, whereas the many smaller fragments often had little or no chromatin. Time-lapse studies showed that apoptosis was induced at a slower rate in cells stably transfected with c-myc versus those stably transfected with E1A. The early changes of apoptosis, including cell shrinkage and intense blebbing, occurred in under 5 min in both cells. Slight alterations such as cell size and further rounding occurred up to 8 h following the initial changes of apoptosis. Rather than being a part of the apoptotic response, release from the culture floor almost entirely resulted from movement of the culture flask. These studies provide a framework of timed morphologic events for future mechanistic investigation into the key aspects of myc-and E1A-induced apoptosis in vascular smooth muscle.     

Lymphatic networks in the periodontal tissue and dental pulp as revealed by histochemical study.

The structural organization and fine distribution of the lymphatic networks in the periodontal tissues (gingiva, periodontal membrane, and alveolar process) and dental pulp of animals and humans were reviewed with special reference to histochemical examination by light and electron microscopy. The distinction between lymphatics and blood vessels was made on cryostat sections of undecalcified and calcified teeth treated with EDTA solution and whole mount preparations of periodontal membranes using 5'-nucleotidase (5'-Nase)-alkaline phosphatase (ALPase) double staining. This staining procedure allowed lymphatic vessels in the periodontal tissue and dental pulp to be differentiated from blood vessels. The specificity and localization of the enzyme reactions were confirmed by comparative histochemical studies of the same specimen with light microscopy and scanning or transmission electron microscopy. Well-developed 5'-Nase-positive lymphatic networks were observed on the tissue sections and whole mount preparations of the gingiva, periodontium, and dental pulp. More lymphatic vessels were seen in the root area of the periodontium than in the cervical area. In the dental pulp, lymphatic vessels were more numerous in the central part than in the peripheral odontoblastic layer. These distributions of the lymphatic capillary networks are discussed in relation to their ability to supply lymph to the teeth.     

Regulation of adrenomedullin expression and release

Adrenomedullin (AM) was originally identified in the extracts of human pheochromocytoma tissue, but this peptide is now known to be synthesized and secreted from many kinds of cells in the body, including vascular smooth muscle cells, endothelial cells, fibroblasts, cardiac myocytes, epithelial cells, and cancer cells. In this review, we summarize AM-secreting and AM gene-expressing cells in addition to the regulation of secretion and gene expression of AM. Although the data are still limited to deduce the general features of AM gene expression, synthesis, and secretion, AM is assumed to be classified into the new class of biologically active peptides, which is mainly expressed and secreted from non-endocrine type cells by the stimulation with inflammation-related substances. It is also interesting that serious physiological conditions such as inflammation or hypoxia potently stimulate AM expression and release, suggesting its unique physiological function distinct from other known biologically active peptides.     

Changes of inducible nitric oxide synthase in aortic cells during the development of hypertension: Effect of angiotensin II

Nitric oxide (NO) generation by inducible nitric oxide synthase (iNOS) in the vascular smooth muscle cells (VSMC), may play a role in blood vessel tone regulation. Lipopolysaccharide (LPS) induced iNOS activity and subsequent nitrite production by cultured aortic VSMC, from SHR with an established chronic blood pressure elevation (adult SHR) or during the period preceding the development of hypertension (young SHR) and from age-matched normotensive Wistar (W) rats were compared. Angiotensin II (Ang II) effect was also evaluated. Both basal LPS-induced iNOS activity and nitrite accumulation were significantly lower in young SHR VSMC compared to young W rat cells. In contrast, adult hypertensive and normotensive rat cells did not differ in NO generation. Besides, young SHR cells exhibited a significant smaller iNOS activity and nitrites than adult SHR cells. After 24h-incubation with Ang II, both variables were markedly reduced in all groups. The proportional reduction of iNOS activity and nitrites by Ang II was not different between hypertensive and normotensive rat cells, at any age. However, this Ang II inhibitory effect was greater in both adult SHR and W cells than in VSMC from young rats. In conclusion, a reduced LPS-induced iNOS activity and NO generation was observed in VSMC form spontaneously hypertensive rats before the raise of blood pressure, but not in adult hypertensive rat cells. Additionally, an inhibitory effect of angiotensin II on these variables is described. We can speculate that the impairment in vascular smooth muscle NO production precedes the development of hypertension in SHR and may play a pathophysiologic role in the early blood pressure elevation in genetically hypertensive rats.     

Freeze-substitution and isothermal freeze-fixation studies to elucidate the pattern of ice formation in smooth muscle at 252 K (-21°C)

Taenia coli muscle was cooled to 252 K in the presence of the cryoprotectant dimethyl-sulphoxide, at cooling rates known to reduce viability by significantly different amounts. The reduction in viability was known to be related to ice formation. Freeze-substitution and isothermal freeze-fixation studies were carried out to determine the distribution of ice within the muscle at this temperature. Freeze-substitution using ethylene glycol was unsuccessful but a new method, using high concentrations of the cryoprotectant as the substituting solvent, was able to maintain ice configurations at this relatively high substitution temperature. The results of freeze-substitution in dimethylsulphoxide were confirmed by isothermal freeze-fixation when both techniques were conducted under identical cooling conditions. The results indicated that the functional differences produced by cooling muscle at either 0·3 K min^?1 or 2 K min^?1 were related to the distribution of the ice phase within the tissue.     

Functional expression and localization of P-glycoprotein at the blood brain barrier

Until recently, the blood-brain barrier was viewed as a static lipid membrane barrier. Physical attributes of the cerebral endothelial cells such as the presence of tight junctions, paucity of vesicles or caveolae, and high electrical resistance were believed to be the primary components that provide the membrane selectivity of the blood-brain barrier to a variety of circulating compounds from the periphery. However, results from molecular biology, immunocytochemistry, biochemistry, and transport studies show that the cerebral endothelial cells possess an asymmetrical array of metabolic enzymes (i.e., alkaline phosphatase, cytochrome P450 enzymes, glutathione transferases) and energy-dependent efflux transport proteins (i.e., P-glycoprotein and Multidrug-resistance proteins) that are instrumental to the barrier function. P-glycoprotein, a membrane-associated, energy-dependent, efflux transporter, is expressed in brain parenchyma (i.e., astrocytes and microglia) as well as in blood-brain and blood-cerebrospinal fluid barriers. Its function along the blood-brain barrier is believed to prevent the accumulation of potentially harmful compounds in the brain by actively removing them from the brain into the peripheral circulation. This is a brief review on the expression and activity of P-glycoprotein at the blood-brain barrier, which reports on the localization of the protein in rat brain capillaries in situ as well as in a well-characterized in vitro model of the blood-brain barrier, an immortalized rat brain endothelial cell line, the RBE4. Immunocytochemical analysis employing various P-glycoprotein monoclonal antibodies, demonstrated the presence of the protein along the plasma membrane, in plasmalemmal vesicles and nuclear envelope of rat cerebral endothelial cells, both in situ and in vitro. Western blot analysis revealed a single band with a molecular weight of 170-180 kDa, a size previously reported for P-glycoprotein, in RBE4 cells. In addition, results from functional studies show that the accumulation of the P-glycoprotein substrate digoxin by RBE4 monolayer cells is significantly enhanced in the presence of standard P-glycoprotein inhibitors (verapamil, cyclosporin A, PSC 833), protease inhibitors (saquinavir, ritonavir, indinavir), and the metabolic inhibitor, sodium azide. These results demonstrate the functional expression of P-glycoprotein in the immortalized rat brain endothelial cell line, RBE4. Novel in situ and in vitro intracellular locations of P-glycoprotein in cerebral endothelial cells have been identified suggesting that this transporter may play a significant role in the subcellular distribution of substrates in the brain.     

Morphological characterization of the progenitor blood cells in canine and feline umbilical cord

The umbilical cord blood (UCB) is an important source of hematopoietic stem cells with great deal of interest in regenerative medicine. The UCB cells have been extensively studied as an alternative to the bone marrow transplants. The challenge is to define specific methods to purify and characterize these cells in different animal species. This study is aimed at morphological characterization of progenitor cells derived from UCB highlighting relevant differences with peripheral blood of adult in dog and cats. Therefore, blood was collected from 18 dogs and 5 cats' umbilical cords from fetus in various developmental stages. The mononuclear cells were separated using the gradient of density Histopaque-1077. Characterization of CD34+ cells was performed by flow cytometric analysis and transmission electron microscopy. Granulocytes (ancestry of the basophiles, eosinophiles, and neutrophiles) and agranulocytes (represented by immature lymphocytes) were identified. We showed for the first time the ultrastructural features of cat UCB cells.     

Role of dendritic cells and Th2 lymphocytes in asthma: lessons from eosinophilic airway inflammation in the mouse

Asthma is a chronic disorder of the airways characterized by variable airway narrowing, mucus hypersecretion, and infiltration of the airway wall with eosinophils. It is now believed that asthma is controlled by Th2 lymphocytes producing cytokines such as IL-4, IL-5, IL-9, and IL-13. Animal models of eosinophilic airway inflammation and airway hyperreactivity have been developed to study the contribution of cells or mediators in the pathogenesis of asthma. In this review, we discuss the role of antigen presenting cells, CD4(+) and CD8(+) T lymphocytes, B lymphocytes, NK cells, and mast cells in the induction and maintenance of eosinophilic airway inflammation, mucus hypersecretion, and airway hyperreactivity.     

Fine structure and formation of the eggshell in scorpionfly Panorpa liui Hua (Mecoptera: Panorpidae)

The morphology and formation of the eggshell in the scorpionfly Panorpa liui Hua were examined with light microscopy and scanning and transmission electron microscopy. During oogenesis of the scorpionfly, the follicle cells multiply by mitotic divisions and diversify into four morphologically distinct subpopulations, three of which are engaged in the eggshell formation and mold various parts of chorion. The eggshell consists of three layers: An inner vitelline membrane, an outer chorion, and a precursor extrachorion. The chorion constitutes a very compact endochorion, a rough fibrillar exochorion, and a polygonal meshwork of elevated ridges. At proximal end of ovarioles the chorion of matured oocyte is covered with a loose membrane, which might be the remnant of follicle cells. The jelly substance, which acts as lubricant to protect the oviducts and ovulated eggs during ovulation, might add to the top of polygonal ridges as the outermost extrachorion after oviposition. The eggshell formation process in Panorpa is tentatively proposed. Microsc. Res. Tech. 2009. © 2009 Wiley-Liss, Inc.     

Study of the structure of the air and blood capillaries of the gas exchange tissue of the avian lung by serial section three-dimensional reconstruction

We have previously reconstructed the gas exchange tissue of the adult muscovy duck, Cairina moschata using a method of manually aligning sections and tracing the contours of the components of the gas exchange tissue. This reconstruction method demonstrated that the air capillaries are comprised of an expanded globular part interconnected by narrow air channels. The blood capillaries completely surround the air capillaries forming an anastomosing meshwork of short segments. However, the resulting reconstruction was limited in scope because of the laborious process of tracing the profiles of each component through the sequence of micrographs. We have now reconstructed a larger proportion of the exchange tissue by using a cross-correlation based alignment strategy and have demonstrated that the staining intensity of each of the exchange tissue components is sufficiently different to allow them to be identified by simple filtering and thresholding. The resulting reconstructions sample a much larger proportion of the exchange tissue and demonstrate the heterogeneity of structures from different locations in the parabronchus. We have shown that a sheet-flow-type arrangement of blood capillaries surrounds the infundibulum; this represents an unexpected functional convergence with the arrangement of blood capillaries surrounding the mammalian alveoli. It is feasible, using this reconstruction strategy, to analyse the exchange tissue of a large number of avian species in order to determine structural correlates of function. The resulting reconstructions could be analysed in order to determine the basis of the functional efficiency and rigidity of the avian lung.     

Three-dimensional visualization and physiologic evaluation of bile canaliculi in the rat liver slice by confocal laser scanning microscopy.

We evaluated the morphology and physiologic function of the bile canaliculi (BC) in the rat liver slice (RLS) by confocal laser scanning microscopy (CLSM). Lucifer yellow (LY) dye was injected into the RLS, and the distribution of LY was serially evaluated. After the injection of LY, hepatocytes were initially visualized, followed by visualization of the BC. There was no significant difference in the distribution of LY between zones 1 and 3 in the hepatic lobule. In zone 1, the reticular distribution of the BC was observed, whereas the part of BC was linearly visualized in zone 3 along the course of sinusoids. When changes in the bile canalicular fluorescence (BCF) were serially evaluated, the BCF was decreased to the minimal level (88% of the value obtained immediately after the LY injection) 10 min after the LY injection, and it tended to increase thereafter. The intralobular hepatocyte fluorescence (ILHF) was decreased to 58.9% of the initial value during the first 40 min. However, the ILHF was transiently increased 30 min after the LY injection, suggesting the possibility of reabsorption of LY by hepatocytes. Three-dimensional (3-D) reconstruction images of the BC facilitated the evaluation of the stereoscopic structure of BC. Confocal laser scanning microscopy facilitated the evaluation of structures and physiologic function of the BC.     

The pars intercerebralis of the locust brain: a developmental and comparative study.

The anterior midline of the brain, also known as the pars intercerebralis, contains the largest collection of neurosecretory cells in the central nervous system of the grasshopper. In this study, we use immunocytochemical, intracellular staining, and histological methods to establish the ontogenies of the various cell types in the brain midline, and show how these cells contribute to the pars intercerebralis of the adult brain. We show that the adult pars intercerebralis develops from three distinct embryonic cell groups: (1) the median neurosecretory cells, which derive from a subset of neuroblasts in the protocerebral hemispheres, and which project axons to the corpora cardiaca; (2) the paired primary commissure pioneers, which derive directly from the mesectoderm of the dorsal median domain and whose axons project to the ventral nerve cord via the midline tract; and (3) the six progeny of the median precursor in the dorsal median domain, which share a common axonal projection with the primary commissure pioneers. Since the adult pars intercerebralis is a fusion product of these independent cellular components, it can only be understood in terms of its origins in the embryonic brain. When the expression pattern of the TERM-1 antigen is compared in subsets of median neurosecretory cells in a wide range of insect orders, the results suggests a common organizational Bauplan for the pars intercerebralis. This hypothesis is supported by the identification of putative homologs of the grasshopper primary commissure pioneers in all these insects.     

Goblet cell number in the ileum of rats denervated during suckling and weaning.

The enteric nervous system plays a role on the stimulation of secretory cells of intestinal epithelia. We have demonstrated that ablation of ENS stimulates epithelial cell proliferation. As goblet cells are important constituents of the epithelial sheet, it is mandatory to investigate separately this cell type. The myenteric plexus of the ileum of rats in postnatal development was partially removed by the serosal application of benzalkonium chloride (BAC). Three groups of animals were used: those where BAC application was at 13 days and sacrifice was at 15 (13/28-day-old) or 23 days after treatment (13/36-day-old), and those where BAC was applied at 21 days and rats were killed 15 days after treatment (21/36-day-old) . The number of goblet cells in the ileum was estimated in sections stained by periodic acid-Schiff (PAS) histochemistry. In the 13/28 and 21/36 groups, the number of goblet cells was significantly higher after BAC treatment. These results suggest that the myenteric denervation may have an acute effect on the number of goblet cell in suckling and weanling rats, probably through submucous plexus.     

On the method of revealing enamel structure by acid etching. Aspects of optimization and interpretation

The study aimed at finding an optimal combination of acid concentration and etching time when nitric acid is used as etchant for the study of the finer details of human dental enamel structure. Four hundred 2–3‐mm‐thick segments of facio‐lingually sectioned human third molar crowns were assigned to 20 groups with 20 specimens in each group, each group differing with respect to acid concentration (0.1, 1, 2.5, and 5%) and etching time (15, 30, 45, 90, and 180 s). After etching and preparation, specimens were observed in the scanning electron microscope (SEM). Surface roughness/topography increased with increasing acid concentration and increasing etching time, but not in a linear fashion; generally, prisms tended to go from flat‐surfaced to cone‐shaped and prism sheaths from fissure‐like to wedge‐shaped. Intragroup variations and intergroup similarities were considerable. The two major enamel factors determining the etch effect are crystal orientation and prism sheath properties. Other factors, such as distribution of porosities and crystal quality, also contribute probably. Slight to moderate topography is best for observing the finer enamel structure, for example, etching with concentrations in the range 0.1–1% and with etching times in the range 15–90 s, the stronger the acid, the shorter the time. The depth effect of nitric acid is judged to be relatively small. Considerable variations in expression of prism cross‐striations were observed. SEM observations of acid‐etched enamel in carefully selected planes are a powerful method for the study of enamel structure, bearing in mind the artifactual aspects of the observed surface.