Imaging pheromone sensing in a mouse vomeronasal acute tissue slice preparation.

Peter Karlson and Martin Lüscher used the term pheromone for the first time in 1959 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules secreted and/or contained in biological fluids, such as urine, a liquid known to be a main source of pheromones. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions, hierarchical organisations and sexual interactions and are consequently directly correlated with the survival of a given species. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO), a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs). Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize. Two main vomeronasal receptor families, V1Rs and V2Rs, are composed respectively by 240 and 120 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs) and formyl peptide receptors (FPRs) are also expressed in VSNs. Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons TRPC2 independent transduction channels have been suggested. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex. Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

Effectiveness of heparin versus 0.9% saline solution in maintaining the permeability of central venous catheters: a systematic review

Abstract OBJECTIVE Determining which is the most effective solution (heparin flush compared to 0.9% saline flush) for reducing the risk of occlusions in central venous catheters (CVC) in adults. METHOD The systematic review followed the principles proposed by the Cochrane Handbook; critical analysis, extraction and synthesis of data were performed by two independent researchers; statistical analysis was performed using the RevMan program 5.2.8. RESULTS Eight randomized controlled trials and one cohort study were included and the results of the meta-analysis showed no difference (RR=0.68, 95% CI=0.41-1.10; p=0.12). Analysis by subgroups showed that there was no difference in fully deployed CVC (RR=1.09, CI 95%=0.53-2.22;p=0.82); Multi-Lumen CVC showed beneficial effects in the heparin group (RR=0.53, CI 95%=0.29-0.95; p=0.03); in Double-Lumen CVC for hemodialysis (RR=1.18, CI 95%=0.08-17.82;p=0.90) and Peripherally inserted CVC (RR=0.14, CI 95%=0.01-2.60; p=0.19) also showed no difference. CONCLUSION Saline solution is sufficient for maintaining patency of the central venous catheter, preventing the risks associated with heparin administration.

Occurrence, morphology and ultrastructure of the Dufour gland in Melipona bicolor Lepeletier (Hymenoptera, Meliponini)

The occurrence, morphology and ultrastructure of the Dufour gland in Melipona bicolor Lepeletier, 1836 are presented. The Dufour gland is not present in workers. In virgin queens the gland cells show characteristics of low activity, which are described in the text. In physogastric queens the gland epithelium is higher and the cells more active than in virgin queens, showing numerous basal plasmic membrane invaginations impregnated by an electrondense material, increased apical invaginations and accumulation of substances that will be released to the gland lumen in the subcuticular space. Therefore, the data show that the Dufour gland is more developed in physogastric than in virgin queens, indicating a possible involvement of the Dufour gland in the reproduction of this species.

Off-resonance angiography: a new method to depict vessels--phantom and rabbit studies.

PURPOSE: To evaluate the utility of inversion recovery with on-resonant water suppression (IRON) in combination with injection of the long-circulating monocrystalline iron oxide nanoparticle (MION)-47 for contrast material-enhanced magnetic resonance (MR) angiography. MATERIALS AND METhods: Experiments were approved by the institutional animal care committee. Eleven rabbits were imaged at baseline before injection of a contrast agent and then serially 5-30 minutes, 2 hours, 1 day, and 3 days after a single intravenous bolus injection of 80 micromol of MION-47 per kilogram of body weight (n = 6) or 250 micromol/kg MION-47 (n = 5). Conventional T1-weighted MR angiography and IRON MR angiography were performed on a clinical 3.0-T imager. Signal-to-noise and contrast-to-noise ratios were measured in the aorta of rabbits in vivo. Venous blood was obtained from the rabbits before and after MION-47 injection for use in phantom studies. RESULTS: In vitro blood that contained MION-47 appeared signal attenuated on T1-weighted angiograms, while characteristic signal-enhanced dipolar fields were observed on IRON angiograms. In vivo, the vessel lumen was signal attenuated on T1-weighted MR angiograms after MION-47 injection, while IRON supported high intravascular contrast by simultaneously providing positive signal within the vessels and suppressing background tissue (mean contrast-to-noise ratio, 61.9 +/- 12.4 [standard deviation] after injection vs 1.1 +/- 0.4 at baseline, P < .001). Contrast-to-noise ratio was higher on IRON MR angiograms than on conventional T1-weighted MR angiograms (9.0 +/- 2.5, P < .001 vs IRON MR angiography) and persisted up to 24 hours after MION-47 injection (76.2 +/- 15.9, P < .001 vs baseline). CONCLUSION: IRON MR angiography in conjunction with superparamagnetic nanoparticle administration provides high intravascular contrast over a long time and without the need for image subtraction.

Coronary MR angiography clinical applications and potential for imaging coronary artery disease.

Over the past decade, CMRA has emerged as a unique clinical imaging tool with applications in selected populations. Patients with suspected coronary artery anomalies and patients with Kawasaki disease and coronary aneurysms are among those for whom CMRA has demonstrated clinical usefulness. For assessment of patients with atherosclerotic CAD, CMRA is useful for detection of patency of bypass grafts. At centers with appropriate expertise and resources, CMRA also appears to be of value for exclusion of severe proximal multivessel CAD in selected patients. Data from multicenter trials will continue to define the clinical role of CMRA, particularly as it relates to assessment of CAD. Future developments and enhancements of CMRA promise better lumen and coronary artery wall imaging. This may become the new target in noninvasive evaluation of CAD.

Multi-phase postmortem CT angiography: recognizing technique-related artefacts and pitfalls.

BACKGROUND AND PURPOSE: Multi-phase postmortem CT angiography (MPMCTA) is increasingly being recognized as a valuable adjunct medicolegal tool to explore the vascular system. Adequate interpretation, however, requires knowledge about the most common technique-related artefacts. The purpose of this study was to identify and index the possible artefacts related to MPMCTA. MATERIAL AND METHODS: An experienced radiologist blinded to all clinical and forensic data retrospectively reviewed 49 MPMCTAs. Each angiographic phase, i.e. arterial, venous and dynamic, was analysed separately to identify phase-specific artefacts based on location and aspect. RESULTS: Incomplete contrast filling of the cerebral venous system was the most commonly encountered artefact, followed by contrast agent layering in the lumen of the thoracic aorta. Enhancement or so-called oedematization of the digestive system mucosa was also frequently observed. CONCLUSION: All MPMCTA artefacts observed and described here are reproducible and easily identifiable. Knowledge about these artefacts is important to avoid misinterpreting them as pathological findings.

cAMP-dependent chloride secretion mediates tubule enlargement and cyst formation by cultured mammalian collecting duct cells.

Polycystic kidney diseases result from disruption of the genetically defined program that controls the size and geometry of renal tubules. Cysts which frequently arise from the collecting duct (CD) result from cell proliferation and fluid secretion. From mCCD(cl1) cells, a differentiated mouse CD cell line, we isolated a clonal subpopulation (mCCD-N21) that retains morphogenetic capacity. When grown in three-dimensional gels, mCCD-N21 cells formed highly organized tubular structures consisting of a palisade of polarized epithelial cells surrounding a cylindrical lumen. Subsequent addition of cAMP-elevating agents (forskolin or cholera toxin) or of membrane-permeable cAMP analogs (CPT-cAMP) resulted in rapid and progressive dilatation of existing tubules, leading to the formation of cystlike structures. When grown on filters, mCCD-N21 cells exhibited a high transepithelial resistance as well as aldosterone- and/or vasopressin-induced amiloride-sensitive and -insensitive current. The latter was in part inhibited by Na(+)-K(+)-2Cl(-) cotransporter (bumetanide) and chloride channel (NPPB) inhibitors. Real-time PCR analysis confirmed the expression of NKCC1, the ubiquitous Na(+)-K(+)-2Cl(-) cotransporter and cystic fibrosis transmembrane regulator (CFTR) in mCCD-N21 cells. Tubule enlargement and cyst formation were prevented by inhibitors of Na(+)-K(+)-2Cl(-) cotransporters (bumetanide or ethacrynic acid) or CFTR (NPPB or CFTR inhibitor-172). These results further support the notion that cAMP signaling plays a key role in renal cyst formation, at least in part by promoting chloride-driven fluid secretion. This new in vitro model of tubule-to-cyst conversion affords a unique opportunity for investigating the molecular mechanisms that govern the architecture of epithelial tubes, as well as for dissecting the pathophysiological processes underlying cystic kidney diseases.

Artifact-free coronary magnetic resonance angiography and coronary vessel wall imaging in the presence of a new, metallic, coronary magnetic resonance imaging stent.

BACKGROUND: Coronary in-stent restenosis cannot be directly assessed by magnetic resonance angiography (MRA) because of the local signal void of currently used stainless steel stents. The aim of this study was to investigate the potential of a new, dedicated, coronary MR imaging (MRI) stent for artifact-free, coronary MRA and in-stent lumen and vessel wall visualization. METHODS AND RESULTS: Fifteen prototype stents were deployed in coronary arteries of 15 healthy swine and investigated with a double-oblique, navigator-gated, free-breathing, T2-prepared, 3D cartesian gradient-echo sequence; a T2-prepared, 3D spiral gradient-echo sequence; and a T2-prepared, 3D steady-state, free-precession coronary MRA sequence. Furthermore, black-blood vessel wall imaging by a dual-inversion-recovery, turbo spin-echo sequence was performed. Artifacts of the stented vessel segment and signal intensities of the coronary vessel lumen inside and outside the stent were assessed. With all investigated sequences, the vessel lumen and wall could be visualized without artifacts, including the stented vessel segment. No signal intensity alterations inside the stent when compared with the vessel lumen outside the stent were found. CONCLUSIONS: The new, coronary MRI stent allows for completely artifact-free coronary MRA and vessel wall imaging.

Gene deletion of dopamine beta-hydroxylase and alpha1-adrenoceptors demonstrates involvement of catecholamines in vascular remodeling.

In vitro studies have shown that stimulation of alpha1-adrenoceptors (ARs) directly induces proliferation, hypertrophy, and migration of arterial smooth muscle cells and adventitial fibroblasts. In vivo studies confirmed these findings and showed that catecholamine trophic activity becomes excessive after experimental balloon injury and contributes to neointimal growth, adventitial thickening, and lumen loss. However, past studies have been limited by selectivity of pharmacological agents. The aim of this study, in which mice devoid of norepinephrine and epinephrine synthesis [dopamine beta-hydroxylase (DBH-/-)] or deficient in alpha1-AR subtypes expressed in murine carotid (alpha1B-AR-/- and alpha1D-AR-/-) were used, was to test the hypothesis that catecholamines contribute to wall hypertrophy after injury. At 3 wk after injury of wild-type mice, lumen area and carotid circumference increased significantly, and hypertrophy of media and adventitia was in excess of that needed to restore circumferential wall stress to normal. In DBH-/- and alpha1B-AR-/- mice, increases in lumen area, circumference, and hypertrophy of the media and adventitia were reduced by 50-91%, resulting in restoration of wall tension to nearly normal (DBH-/-) or normal (alpha1B-AR-/-). In contrast, in alpha1D-AR-/- mice, increases in lumen area, circumference, and wall hypertrophy were unaffected and wall thickening remained in excess of that required to return tension to normal. When examined 5 days after injury, proliferation and leukocyte infiltration were inhibited in DBH-/- mice. These studies suggest that the trophic effects of catecholamines are mediated primarily by alpha1B-ARs in mouse carotid and contribute to hypertrophic growth after vascular injury.

Role of lipase in the regulation of upper gastrointestinal function in humans.

The role of lipase in the regulation of upper gastrointestinal function is poorly understood. We studied the effect of orlistat, a new, potent, and highly specific lipase inhibitor, on gastric emptying, cholecystokinin (CCK) release, and pancreaticobiliary secretion. Three groups of studies were performed in nine healthy volunteers, using the double-indicator technique with a triple-lumen duodenal tube, polyethylene glycol 4000 as a duodenal perfusion marker, and 99mTc-diethylenetriamine pentaacetic acid as a meal marker. Gastric emptying, pancreaticobiliary output, and postprandial plasma CCK levels were measured after ingestion of the following isocaloric 500-ml liquid meals with or without 200 mg orlistat: 1) a pure fat meal (10% Intralipid), 2) a meal containing free fatty acids, or 3) an albumin-glucose meal. All experiments were performed in a randomized, placebo-controlled, crossover design. Orlistat markedly inhibited lipase activity in all three experiments. Orlistat given with the fat meal reduced CCK release and output of lipase, trypsin, and bilirubin and accelerated the rate of gastric emptying (P < 0.05). After ingestion of the free fatty acid or albumin-glucose meal, orlistat had no significant effect on any of these parameters. We conclude that lipase plays an important, nutrient-specific role in the regulation of gastric emptying and pancreaticobiliary secretion after ingestion of fatty meals in humans.

ENaC inhibition stimulates Cl- secretion in the mouse cortical collecting duct through an NKCC1-dependent mechanism.

In cortical collecting ducts (CCDs) perfused in vitro, inhibiting the epithelial Na(+) channel (ENaC) reduces Cl(-) absorption. Since ENaC does not transport Cl(-), the purpose of this study was to determine how ENaC modulates Cl(-) absorption. Thus, Cl(-) absorption was measured in CCDs perfused in vitro that were taken from mice given aldosterone for 7 days. In wild-type mice, we observed no effect of luminal hydrochlorothiazide on either Cl(-) absorption or transepithelial voltage (V(T)). However, application of an ENaC inhibitor [benzamil (3 μM)] to the luminal fluid or application of a Na(+)-K(+)-ATPase inhibitor to the bath reduced Cl(-) absorption by ∼66-75% and nearly obliterated lumen-negative V(T). In contrast, ENaC inhibition had no effect in CCDs from collecting duct-specific ENaC-null mice (Hoxb7:CRE, Scnn1a(loxlox)). Whereas benzamil-sensitive Cl(-) absorption did not depend on CFTR, application of a Na(+)-K(+)-2Cl(-) cotransport inhibitor (bumetanide) to the bath or ablation of the gene encoding Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) blunted benzamil-sensitive Cl(-) absorption, although the benzamil-sensitive component of V(T) was unaffected. In conclusion, first, in CCDs from aldosterone-treated mice, most Cl(-) absorption is benzamil sensitive, whereas thiazide-sensitive Cl(-) absorption is undetectable. Second, benzamil-sensitive Cl(-) absorption occurs by inhibition of ENaC, possibly due to elimination of lumen-negative V(T). Finally, benzamil-sensitive Cl(-) flux occurs, at least in part, through transcellular transport through a pathway that depends on NKCC1.

CD47 knockout mice exhibit improved recovery from spinal cord injury.

Recent data have implicated thrombospondin-1 (TSP-1) signaling in the acute neuropathological events that occur in microvascular endothelial cells (ECs) following spinal cord injury (SCI) (Benton et al., 2008b). We hypothesized that deletion of TSP-1 or its receptor CD47 would reduce these pathological events following SCI. CD47 is expressed in a variety of tissues, including vascular ECs and neutrophils. CD47 binds to TSP-1 and inhibits angiogenesis. CD47 also binds to the signal regulatory protein (SIRP)α and facilitates neutrophil diapedesis across ECs to sites of injury. After contusive SCI, TSP-1(-/-) mice did not show functional improvement compared to wildtype (WT) mice. CD47(-/-) mice, however, exhibited functional locomotor improvements and greater white matter sparing. Whereas targeted deletion of either CD47 or TSP-1 improved acute epicenter vascularity in contused mice, only CD47 deletion reduced neutrophil diapedesis and increased microvascular perfusion. An ex vivo model of the CNS microvasculature revealed that CD47(-/-)-derived microvessels (MVs) prominently exhibit adherent WT or CD47(-/-) neutrophils on the endothelial lumen, whereas WT-derived MVs do not. This implicates a defect in diapedesis mediated by the loss of CD47 expression on ECs. In vitro transmigration assays confirmed the role of SIRPα in neutrophil diapedesis through EC monolayers. We conclude that CD47 deletion modestly, but significantly, improves functional recovery from SCI via an increase in vascular patency and a reduction of SIRPα-mediated neutrophil diapedesis, rather than the abrogation of TSP-1-mediated anti-angiogenic signaling.

Splenic arterial interventions: anatomy, indications, technical considerations, and potential complications.

Splenic arterial interventions are increasingly performed to treat various clinical conditions, including abdominal trauma, hypersplenism, splenic arterial aneurysm, portal hypertension, and splenic neoplasm. When clinically appropriate, these procedures may provide an alternative to open surgery. They may help to salvage splenic function in patients with posttraumatic injuries or hypersplenism and to improve hematologic parameters in those who otherwise would be unable to undergo high-dose chemotherapy or immunosuppressive therapy. Splenic arterial interventions also may be performed to exclude splenic artery aneurysms from the parent vessel lumen and prevent aneurysm rupture; to reduce portal pressure and prevent sequelae in patients with portal hypertension; to treat splenic artery steal syndrome and improve liver perfusion in liver transplant recipients; and to administer targeted treatment to areas of neoplastic disease in the splenic parenchyma. As the use of splenic arterial interventions increases in interventional radiology practice, clinicians must be familiar with the splenic vascular anatomy, the indications and contraindications for performing interventional procedures, the technical considerations involved, and the potential use of other interventional procedures, such as radiofrequency ablation, in combination with splenic arterial interventions. Familiarity with the complications that can result from these interventional procedures, including abscess formation and pancreatitis, also is important.

Temporal changes in the expression and distribution of adhesion molecules during liver development and regeneration

We have compared by immunocytochemistry and immunoblotting the expression and distribution of adhesion molecules participating in cell-matrix and cell-cell interactions during embryonic development and regeneration of rat liver. Fibronectin and the fibronectin receptor, integrin alpha 5 beta 1, were distributed pericellularly and expressed at a steady level during development from the 16th day of gestation and in neonate and adult liver. AGp110, a nonintegrin fibronectin receptor was first detected on the 17th day of gestation in a similar, nonpolarized distribution on parenchymal cell surfaces. At that stage of development haemopoiesis is at a peak in rat liver and fibronectin and receptors alpha 5 beta 1 and AGp110 were prominent on the surface of blood cell precursors. During the last 2 d of gestation (20th and 21st day) hepatocytes assembled around lumina. AGp110 was initially depolarized on the surface of these acinar cells but then confined to the lumen and to newly-formed bile canaliculi. At birth, a marked increase occurred in the canalicular expression of AGp110 and in the branching of the canalicular network. Simultaneously, there was enhanced expression of ZO-1, a protein component of tight junctions. On the second day postpartum, presence of AGp110 and of protein constituents of desmosomes and intermediate junctions, DGI and E-cadherin, respectively, was notably enhanced in cellular fractions insoluble in nonionic detergents, presumably signifying linkage of AGp110 with the cytoskeleton and assembly of desmosomal and intermediate junctions. During liver regeneration after partial hepatectomy, AGp110 remained confined to apical surfaces, indicating a preservation of basic polarity in parenchymal cells. A decrease in the extent and continuity of the canalicular network occurred in proliferating parenchyma, starting 24 h after resection in areas close to the terminal afferent blood supply of portal veins and spreading to the rest of the liver within the next 24 h. Distinct acinar structures, similar to the ones in prenatal liver, appeared at 72 h after hepatectomy. Restoration of the normal branching of the biliary tree commenced at 72 h. At 7 d postoperatively acinar formation declined and one-cell-thick hepatic plates, as in normal liver, were observed.

Molecular imaging of matrix metalloproteinases in atherosclerotic plaques.

Ischaemic stroke and myocardial infarction often result from the sudden rupture of an atherosclerotic plaque. The subsequent arterial thrombosis occluding the vessel lumen has been widely indicated as the crucial acute event causing peripheral tissue ischaemia. A complex cross-talk between systemic and intraplaque inflammatory mediators has been shown to regulate maturation, remodeling and final rupture of an atherosclerotic plaque. Matrix metalloproteinases (MMPs) are proteolytic enzymes (released by several cell subsets within atherosclerotic plaques), which favour atherogenesis and increase plaque vulnerability. Thus, the assessment of intraplaque levels and activity of MMP might be of pivotal relevance in the evaluation of the risk of rupture. New imaging approaches, focused on the visualisation of inflammation in the vessel wall and plaque, may emerge as tools for individualised risk assessment and prevention of events. In this review, we summarize experimental findings of the currently available invasive and noninvasive imaging techniques, used to detect the presence and activity of MMPs in atherosclerotic plaques.