The mesentery as the epicenter for intestinal regeneration.

The mesentery, a newly minted organ, plays various anatomical and physiological roles during animal development. In echinoderms, and particularly in members of the class Holothuroidea (sea cucumbers) the mesentery plays an additional unique role: it is crucial for the process of intestinal regeneration. In these organisms, a complete intestine can form from cells that originate in the mesentery. In this review, we focus on what is known about the changes that take place in the mesentery and what has been documented on the cellular and molecular mechanisms involved. We describe how the events that unfold in the mesentery result in the formation of a new intestine.

Differential regulation of blood flow-induced neovascularization and mural cell recruitment by vascular endothelial growth factor and angiopoietin signalling.

KEY POINTS: Combining nitric oxide (NO)-mediated increased blood flow with angiopoietin-1-Tie2 receptor signalling induces arteriolargenesis - the formation of arterioles from capillaries - in a model of physiological angiogenesis. This NO-Tie-mediated arteriolargenesis requires endogenous vascular endothelial growth factor (VEGF) signalling. Inhibition of VEGF signalling increases pericyte coverage in microvessels. Together these findings indicate that generation of functional neovasculature requires close titration of NO-Tie2 signalling and localized VEGF induction, suggesting that the use of exogenous VEGF expression as a therapeutic for neovascularization may not be successful. ABSTRACT: Signalling through vascular endothelial growth factor (VEGF) receptors and the tyrosine kinase with IgG and EGF domains-2 (Tie2) receptor by angiopoietins is required in combination with blood flow for the formation of a functional vascular network. We tested the hypothesis that VEGF and angiopoietin-1 (Ang1) contribute differentially to neovascularization induced by nitric oxide (NO)-mediated vasodilatation, by comparing the phenotype of new microvessels in the mesentery during induction of vascular remodelling by over-expression of endothelial nitric oxide synthase in the fat pad of the adult rat mesentery during inhibition of angiopoietin signalling with soluble Tie2 (sTie2) and VEGF signalling with soluble Fms-like tyrosine kinase receptor-1 (sFlt1). We found that NO-mediated angiogenesis was blocked by inhibition of VEGF with sFlt1 (from 881 ± 98% increase in functional vessel area to 279 ± 72%) and by inhibition of angiopoietin with sTie2 (to 337 ± 67%). Exogenous angiopoietin-1 was required to induce arteriolargenesis (8.6 ± 1.3% of vessels with recruitment of vascular smooth muscle cells; VSMCs) in the presence of enhanced flow. sTie2 and sFlt1 both inhibited VSMC recruitment (both 0%), and VEGF inhibition increased pericyte recruitment to newly formed vessels (from 27 ± 2 to 54 ± 3% pericyte ensheathment). We demonstrate that a fine balance of VEGF and angiopoietin signalling is required for the formation of a functional vascular network. Endogenous VEGF signalling prevents excess neovessel pericyte coverage, and is required for VSMC recruitment during increased nitric oxide-mediated vasodilatation and angiopoietin signalling (NO-Tie-mediated arteriogenesis). Therapeutic vascular remodelling paradigms may therefore require treatments that modulate blood flow to utilize endogenous VEGF, in combination with exogenous Ang1, for effective neovascularization.

The TRPV1 channel in rodents is a major target for antinociceptive effect of the probiotic Lactobacillus reuteri DSM 17938.

Certain probiotic bacteria have been shown to reduce distension-dependent gut pain, but the mechanisms involved remain obscure. Live luminal Lactobacillus reuteri (DSM 17938) and its conditioned medium dose dependently reduced jejunal spinal nerve firing evoked by distension or capsaicin, and 80% of this response was blocked by a specific TRPV1 channel antagonist or in TRPV1 knockout mice. The specificity of DSM action on TRPV1 was further confirmed by its inhibition of capsaicin-induced intracellular calcium increases in dorsal root ganglion neurons. Another lactobacillus with ability to reduce gut pain did not modify this response. Prior feeding of rats with DSM inhibited the bradycardia induced by painful gastric distension. These results offer a system for the screening of new and improved candidate bacteria that may be useful as novel therapeutic adjuncts in gut pain. Certain bacteria exert visceral antinociceptive activity, but the mechanisms involved are not determined. Lactobacillus reuteri DSM 17938 was examined since it may be antinociceptive in children. Since transient receptor potential vanilloid 1 (TRPV1) channel activity may mediate nociceptive signals, we hypothesized that TRPV1 current is inhibited by DSM. We tested this by examining the effect of DSM on the firing frequency of spinal nerve fibres in murine jejunal mesenteric nerve bundles following serosal application of capsaicin. We also measured the effects of DSM on capsaicin-evoked increase in intracellular Ca(2+) or ionic current in dorsal root ganglion (DRG) neurons. Furthermore, we tested the in vivo antinociceptive effects of oral DSM on gastric distension in rats. Live DSM reduced the response of capsaicin- and distension-evoked firing of spinal nerve action potentials (238 ± 27.5% vs. 129 ± 17%). DSM also reduced the capsaicin-evoked TRPV1 ionic current in DRG neuronal primary culture from 83 ± 11% to 41 ± 8% of the initial response to capsaicin only. Another lactobacillus (Lactobacillus rhamnosus JB-1) with known visceral anti-nociceptive activity did not have these effects. DSM also inhibited capsaicin-evoked Ca(2+) increase in DRG neurons; an increase in Ca(2+) fluorescence intensity ratio of 2.36 ± 0.31 evoked by capsaicin was reduced to 1.25 ± 0.04. DSM releasable products (conditioned medium) mimicked DSM inhibition of capsaicin-evoked excitability. The TRPV1 antagonist 6-iodonordihydrocapsaicin or the use of TRPV1 knock-out mice revealed that TRPV1 channels mediate about 80% of the inhibitory effect of DSM on mesenteric nerve response to high intensity gut distension. Finally, feeding with DSM inhibited perception in rats of painful gastric distension. Our results identify a specific target channel for a probiotic with potential therapeutic properties.

Characterization of Perivascular Nerve Distribution in Rat Mesenteric Small Arteries.

The distribution pattern of perivascular nerves in some branches of rat mesenteric arteries was studied. Mesenteric arteries isolated from 8-week-old Wistar rats were divided into the 1st-, 2nd-, and 3rd-order branches. The distribution of perivascular nerves in each branch was immunohistochemically evaluated using antibodies against neuropeptide Y (NPY), tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP), substance P (SP), and neuronal nitric oxide synthase (nNOS). The density of NPY-, TH-, CGRP-, and SP-like immunoreactive (LI) nerves in the 2nd and 3rd branches was significantly greater than that in the 1st branch, and a negative relationship was found between nerve density and arterial diameter, except for TH-LI nerves. The density of NPY- and TH-LI nerves in all branches, which was similar, was greater than that of CGRP- (except for NPY-LI nerves in the 1st branch), SP-, or nNOS-LI nerves. Double immunostaining revealed that TH-LI nerves made contact with nNOS-LI, CGRP-LI, and SP-LI nerves and that CGRP-LI nerves made contact with TH-, NPY-, or nNOS-LI nerves, while TH-LI and CGRP-LI nerves nearly merged with NPY-LI and SP-LI nerves, respectively. These results suggest that the each branch of mesenteric arteries is densely innervated by vasoconstrictor nerves containing NPY, TH, and vasodilator CGRP nerves. They also suggest that the intense density of perivascular nerves in the 2nd and 3rd branches may contribute to maintaining vascular tone.

InlA promotes dissemination of Listeria monocytogenes to the mesenteric lymph nodes during food borne infection of mice.

Intestinal Listeria monocytogenes infection is not efficient in mice and this has been attributed to a low affinity interaction between the bacterial surface protein InlA and E-cadherin on murine intestinal epithelial cells. Previous studies using either transgenic mice expressing human E-cadherin or mouse-adapted L. monocytogenes expressing a modified InlA protein (InlA(m)) with high affinity for murine E-cadherin showed increased efficiency of intragastric infection. However, the large inocula used in these studies disseminated to the spleen and liver rapidly, resulting in a lethal systemic infection that made it difficult to define the natural course of intestinal infection. We describe here a novel mouse model of oral listeriosis that closely mimics all phases of human disease: (1) ingestion of contaminated food, (2) a distinct period of time during which L. monocytogenes colonize only the intestines, (3) varying degrees of systemic spread in susceptible vs. resistant mice, and (4) late stage spread to the brain. Using this natural feeding model, we showed that the type of food, the time of day when feeding occurred, and mouse gender each affected susceptibility to L. monocytogenes infection. Co-infection studies using L. monocytogenes strains that expressed either a high affinity ligand for E-cadherin (InlA(m)), a low affinity ligand (wild type InlA from Lm EGDe), or no InlA (ΔinlA) showed that InlA was not required to establish intestinal infection in mice. However, expression of InlA(m) significantly increased bacterial persistence in the underlying lamina propria and greatly enhanced dissemination to the mesenteric lymph nodes. Thus, these studies revealed a previously uncharacterized role for InlA in facilitating systemic spread via the lymphatic system after invasion of the gut mucosa.

Functional adaptation of bovine mesenteric lymphatic vessels to mesenteric venous hypertension.

Lymph flow is the primary mechanism for returning interstitial fluid to the blood circulation. Currently, the adaptive response of lymphatic vessels to mesenteric venous hypertension is not known. This study sought to determine the functional responses of postnodal mesenteric lymphatic vessels. We surgically occluded bovine mesenteric veins to create mesenteric venous hypertension to elevate mesenteric lymph flow. Three days after surgery, postnodal mesenteric lymphatic vessels from mesenteric venous hypertension (MVH; n = 7) and sham surgery (Sham; n = 6) group animals were evaluated and compared. Contraction frequency (MVH: 2.98 ± 0.75 min(-1); Sham: 5.42 ± 0.81 min(-1)) and fractional pump flow (MVH: 1.14 ± 0.30 min(-1); Sham: 2.39 ± 0.32 min(-1)) were significantly lower in the venous occlusion group. These results indicate that postnodal mesenteric lymphatic vessels adapt to mesenteric venous hypertension by reducing intrinsic contractile activity.

Increased secretion of insulin and proliferation of islet ß-cells in rats with mesenteric lymph duct ligation.

BACKGROUND & AIMS: It has been suggested that intestinal lymph flow plays an important role in insulin secretion and glucose metabolism after meals. In this study, we investigated the influence of ligation of the mesenteric lymph duct on glucose metabolism and islet ß-cells in rats. METHODS: Male Sprague-Dawley rats (10 weeks old) were divided into two groups: one underwent ligation of the mesenteric lymph duct above the cistern (ligation group), and the other underwent a sham operation (sham group). After 1 and 2 weeks, fasting plasma concentrations of glucose, insulin, triglyceride, glucose-dependent insulinotropic polypeptide (GIP), and the active form of glucagon-like peptide-1 (GLP-1) were measured. At 2 weeks after the operation, the oral glucose tolerance test (OGTT) and intravenous glucose tolerance test (IVGTT) were performed. After the rats had been sacrificed, the insulin content of the pancreas was measured and the proliferation of ß-cells was assessed immunohistochemically using antibodies against insulin and Ki-67. RESULTS: During the OGTT, the ligation group showed a significant decrease in the plasma glucose concentration at 120 min (p<0.05) and a significant increase in the plasma insulin concentration by more than 2-fold at 15 min (p<0.01). On the other hand, the plasma GIP concentration was significantly decreased at 60 min (p<0.01) in the ligated group, while the active form of GLP-1 showed a significantly higher level at 90 min (1.7-fold; p<0.05) and 120 min (2.5-fold; p<0.01). During the IVGTT, the plasma insulin concentration in the ligation group was significantly higher at 2 min (more than 1.4-fold; p<0.05). Immunohistochemistry showed that the ratios of ß-cell area/acinar cell area and ß-cell area/islet area, and also ß-cell proliferation, were significantly higher in the ligation group than in the sham group (p<0.05, p<0.01 and p<0.01, respectively). The insulin content per unit wet weight of pancreas was also significantly increased in the ligation group (p<0.05). CONCLUSIONS: In rats with ligation of the mesenteric lymph duct, insulin secretion during the OGTT or IVGTT was higher, and the insulin content and ß-cell proliferation in the pancreas were also increased. Our data show that mesenteric lymph duct flow has a role in glucose metabolism.

Effect of histamine on contractile activity of smooth muscles in bovine mesenteric lymph nodes.

The effects of histamine and mechanisms of its action on the capsular smooth muscle cells of mesenteric lymph nodes were examined on isolated capsular strips under isometric conditions. Histamine (1×10(-8)-5×10(-7) M) decreased the tone of capsular smooth muscle cells and the frequency of phasic contractions. At high concentrations (more than 5×10(-6) M), histamine increased the amplitude and frequency of phasic contractions against the background of increased tonic stress. The effects of histamine were dose-dependent and were realized via direct stimulation of H(1)- and H(2)-receptors on the membrane of smooth muscle cells.

Update on the mesentery: structure, function, and role in disease.

Over the past 5 years, systematic investigation of the mesenteric organ has expanded and shown that the mesentery is the organ in and on which all abdominal digestive organs develop and remain connected to. In turn, this observation has clarified the anatomical foundation of the abdomen and the fundamental order at that level. Findings related to the shape and development of the mesentery have illuminated its function, advancing our understanding of the pathobiology, diagnosis, and treatment of several abdominal and systemic diseases. Inclusion of the mesentery in surgical resections alters the course of benign and malignant diseases. Mesenteric-based scoring systems can enhance the radiological interpretation of abdominal disease. Emerging findings reconcile observations across scientific and clinical fields and have been assimilated into reference curricula and practice guidelines. This Review summarises the developmental, anatomical, and clinical advances made since the mesentery was redesignated as an organ in 2016.

Cell dedifferentiation and epithelial to mesenchymal transitions during intestinal regeneration in H. glaberrima.

BACKGROUND: Determining the type and source of cells involved in regenerative processes has been one of the most important goals of researchers in the field of regeneration biology. We have previously used several cellular markers to characterize the cells involved in the regeneration of the intestine in the sea cucumber Holothuria glaberrima. RESULTS: We have now obtained a monoclonal antibody that labels the mesothelium; the outer layer of the gut wall composed of peritoneocytes and myocytes. Using this antibody we studied the role of this tissue layer in the early stages of intestinal regeneration. We have now shown that the mesothelial cells of the mesentery, specifically the muscle component, undergo dedifferentiation from very early on in the regeneration process. Cell proliferation, on the other hand, increases much later, and mainly takes place in the mesothelium or coelomic epithelium of the regenerating intestinal rudiment. Moreover, we have found that the formation of the intestinal rudiment involves a novel regenerative mechanism where epithelial cells ingress into the connective tissue and acquire mesenchymal phenotypes. CONCLUSIONS: Our results strongly suggest that the dedifferentiating mesothelium provides the initial source of cells for the formation of the intestinal rudiment. At later stages, cell proliferation supplies additional cells necessary for the increase in size of the regenerate. Our data also shows that the mechanism of epithelial to mesenchymal transition provides many of the connective tissue cells found in the regenerating intestine. These results present some new and important information as to the cellular basis of organ regeneration and in particular to the process of regeneration of visceral organs.

Determinants of valve gating in collecting lymphatic vessels from rat mesentery.

Secondary lymphatic valves are essential for minimizing backflow of lymph and are presumed to gate passively according to the instantaneous trans-valve pressure gradient. We hypothesized that valve gating is also modulated by vessel distention, which could alter leaflet stiffness and coaptation. To test this hypothesis, we devised protocols to measure the small pressure gradients required to open or close lymphatic valves and determine if the gradients varied as a function of vessel diameter. Lymphatic vessels were isolated from rat mesentery, cannulated, and pressurized using a servo-control system. Detection of valve leaflet position simultaneously with diameter and intraluminal pressure changes in two-valve segments revealed the detailed temporal relationships between these parameters during the lymphatic contraction cycle. The timing of valve movements was similar to that of cardiac valves, but only when lymphatic vessel afterload was elevated. The pressure gradients required to open or close a valve were determined in one-valve segments during slow, ramp-wise pressure elevation, either from the input or output side of the valve. Tests were conducted over a wide range of baseline pressures (and thus diameters) in passive vessels as well as in vessels with two levels of imposed tone. Surprisingly, the pressure gradient required for valve closure varied >20-fold (0.1-2.2 cmH(2)O) as a passive vessel progressively distended. Similarly, the pressure gradient required for valve opening varied sixfold with vessel distention. Finally, our functional evidence supports the concept that lymphatic muscle tone exerts an indirect effect on valve gating.

Oil palm vegetation liquor: a new source of phenolic bioactives.

Waste from agricultural products represents a disposal liability, which needs to be addressed. Palm oil is the most widely traded edible oil globally, and its production generates 85 million tons of aqueous by-products annually. This aqueous stream is rich in phenolic antioxidants, which were investigated for their composition and potential in vitro biological activity. We have identified three isomers of caffeoylshikimic acid as major components of oil palm phenolics (OPP). The 2,2-diphenyl-1-picrylhydrazyl assay confirmed potent free radical scavenging activity. To test for possible cardioprotective effects of OPP, we carried out in vitro LDL oxidation studies as well as ex vivo aortic ring and mesenteric vascular bed relaxation measurements. We found that OPP inhibited the Cu-mediated oxidation of human LDL. OPP also promoted vascular relaxation in both isolated aortic rings and perfused mesenteric vascular beds pre-contracted with noradrenaline. To rule out developmental toxicity, we performed teratological studies on rats up to the third generation and did not find any congenital anomalies. Thus, these initial studies suggest that OPP is safe and may have a protective role against free radical damage, LDL oxidation and its attendant negative effects, as well as vascular constriction in mitigating atherosclerosis. Oil palm vegetation liquor thus represents a new source of phenolic bioactives.

Mechanical properties of lymph node capsule.

We studied the relationship between the length and tension in isolated strips of the capsules of bovine mesenteric lymph nodes. The deformation-strain and radius-pressure relationships were established in the lymph node. The capsule possesses high distensibility, modulus of elasticity at optimal tension was 0.09×10(5) N/m2. Smooth muscle activation produces a 6-fold increase of modulus of elasticity. Maximum active stress in the capsule was recorded at a length of 1.1 L0 and maximal active pressure (4.5 cm H2O) at 0.9 L0.

Measurement of oxygen in the microcirculation using phosphorescence quenching microscopy.

According to the classical concept of Krogh, O(2) is delivered to the tissues solely by capillaries and intra-capillary resistance to O(2) diffusion is negligible. Over the past three decades longitudinal PO(2) and SO(2) gradients in arterioles have been observed with a transmural PO(2) gradient in small arterioles of only 1-2 mmHg. Application of phosphorescence quenching microscopy to measurements of PO(2) in arterioles of the rat mesentery by Tsai et al. (1998) found a large transmural PO(2) in these arterioles. That led to the provocative conclusion that the arteriolar wall is the major sink for O(2) in the microcirculation. Our studies indicate that many of these results can be explained by photo-activated O(2) consumption following phosphor excitation, combined with a large excitation area and high frequency of flash excitation. We have developed the basic principles for phosphorescence quenching microscopy including the need to use a small excitation area, a low excitation frequency and a scanning excitation for stationary samples.

Plasma appearance rate of intraperitoneal macromolecular tracer underestimates peritoneal lymph flow.

The magnitude of peritoneal lymph flow is an issue of great controversy in peritoneal dialysis (PD) research. Because no single lymphatic duct drains the entire peritoneal cavity, peritoneal lymph flow is indirectly measured as lymphatic removal of intraperitoneal macromolecular tracer. In rats, the peritoneal clearance (K) of such a tracer is 5 times the approximately 8 microL/min determined from the tracer appearance rate in blood (Cl). The fractional contribution of tissues bordering the peritoneal cavity to the overall Cl was determined to be diaphragm, 55%; viscera, 30%; and abdominal wall, 15%. The present study determines whether direct measurement of visceral peritoneal lymph flow matches the 30% (approximately 2.5 microL/min) contribution of the visceral peritoneal lymph flow as measured indirectly by the Cl method. The mesenteric lymph duct that exclusively drains lymph from the gut, liver, and mesentery was cannulated in 15 rats, and lymph flow from the duct was collected at hourly intervals up to 6 hours under near-normal physiologic conditions and under conditions of simulated PD. Changes in mesenteric lymph flow that resulted from a challenge with 3 mL intravenous saline were captured using real-time video. We observed no significant differences between the hourly lymph volumes collected over 6 hours in naïve animals (n = 5, p > 0.05). Under conditions of simulated PD with dialysis fluid in the peritoneal cavity, the mesenteric duct lymph flow averaged 8.67 +/- 1.41 microL/min (n = 10). That flow is similar to reported data on total peritoneal Cl in rats; and 4 times the 2.5 microL/min visceral peritoneal contribution to the total peritoneal Cl. The intravenous saline challenge significantly increased mesenteric lymph duct output to 30.9 +/- 1.6 microL/min (n = 5, p < 0.01) and reduced the lymph-to-plasma concentration ratio (L/P) by 43%. The reflection coefficient for total proteins (sigma(prot)) across the intestinal capillaries as calculated from the filtration rate-dependent L/P ratio when the transcapillary fluid escape rate and the mesenteric lymph flow were both high was more than 0.87. We concluded that (A) under near-normal physiologic conditions, the mesenteric lymph duct flow is steady, but quite low; (B) under conditions of simulated PD, the mesenteric lymph duct flow increases significantly from the physiologic norm; (C) mesenteric lymph duct flow is sensitive to the peritoneal fill volume; (D) during simulated PD, the fractional visceral peritoneal lymph flow measured indirectly from plasma appearance of intraperitoneal tracer underestimates the directly measured mesenteric duct lymph flow; and (E) the increased transcapillary fluid escape rate is rapidly buffered by augmentation of mesenteric lymph duct output.

[Effect of red blood cell transfusion on the oxygenation of mesenteric tissue in premature infants].

Objective: To investigate the effect of red blood cell transfusion on the oxygenation of mesenteric tissue in premature infants. Methods: In this prospective cohort study, preterm infants with gestational age <37 weeks who were treated with red blood cell transfusions were enrolled from June 2017 to March 2018 in Department of Neonatology, Children's Hospital of Fudan University. The infants were categorized into feeding intolerance group and feeding tolerance group according to the feeding intolerance standard. Near-infrared spectroscopy was applied to continuously monitor intestinal oxygen saturation from 2 h before red blood cell transfusion to 48 h after red blood cell transfusion. Intergroup differences of basic conditions were analyzed with t test, Mann-Whitney U test and χ(2) test. Mixed linear model was used to compare intragroup and intergroup differences in intestinal oxygen saturation over time. Results: A total of 73 cases with gestational age <37 weeks were enrolled, of whom 41 were males and 32 were females, with mean gestational age of (30±4)weeks and mean birth weight of (1 543±688)g; there were 33 cases in feeding intolerance group and 42 cases in feeding tolerance group. The average intestinal oxygen saturations at 2 h before blood transfusion, during blood transfusion, 2, 6, 12, 24, and 48 h after transfusion were 0.50±0.07, 0.52±0.07, 0.52±0.08, 0.51±0.08, 0.51±0.07, 0.51±0.08, and 0.51±0.07 respectively in feeding intolerance group and were 0.51±0.04, 0.55±0.04, 0.57±0.05, 0.57±0.04, 0.56±0.04, 0.56±0.04, and 0.56±0.05 respectively in feeding tolerance group. Compared with 2 h before transfusion, intestinal oxygen saturation were increased during transfusion in both group (feeding intolerance group t=4.992, P=0.000; feeding tolerance group t=9.615, P=0.000), however this effect lasted until 48 h after transfusion in feeding tolerance group (t=5.519, 12.409, 10.033, 9.133, 7.983, all P=0.000). Additionally, the increasement of intestinal oxygen saturation over time were lower in feeding intolerance group(F=8.876, P=0.000). Besides, the level of intestinal oxygen saturation was positively correlated with postmenstrual age (PMA)(F=4.863, P=0.031). In infants with PMA<30 weeks, particularly in feeding intolerance group, the level of intestinal oxygen saturation significantly decreased at 2 h after transfusion (t=23.063, P=0.002). Conclusions: Feeding status and PMA may play a role in development of transfusion-associated necrotizing enterocolitis. Red blood cell transfusion may increase the risk for mesenteric ischemia and is more likely to cause necrotizing enterocolitis in preterm infants with PMA <30 weeks as well as feeding intolerance. Clinical Trail: Children's Hospital of Fudan University, NCT02544100.

A computational modeling of blood flow in asymmetrically bifurcating microvessels and its experimental validation.

Microvascular transport is complex due to its heterogeneity. Many researchers have been developing mathematical and computational models in predicting microvascular geometries and blood transport. However, previous works were focused on developing simulation models, not on validating suggested models with microvascular geometry and blood flow in the real microvasculature. In this paper, we suggest a computational model for microvascular transport with experimental validation in its geometry and blood flow. The geometry is generated by controlling asymmetric conditions of microvascular network. Also, the blood flow in microvascular networks is predicted by considering in vivo viscosity, Poiseuille flow model, and hematocrit redistribution by plasma skimming. The suggested model is validated by the measured data in rat mesentery. Also, the microvascular transport in a case of mouse cortex is predicted and compared against experimental data to check applicability of the suggested model.

Biomonitoring the cardiorenal effects of Luehea divaricata Mart.: An ethnoguided approach.

ETHNOPHARMACOLOGICAL RELEVANCE: Luehea divaricata Mart. (Malvaceae) is an important medicinal species that is widely used as a diuretic in the Brazilian Pantanal region. An ethanolic supernatant that was obtained from an infusion of leaves of this species (ESLD) was recently shown to exert hypotensive and diuretic activity. Nevertheless, the secondary metabolites that are responsible for this activity and the molecular mechanisms of pharmacological action remain unknown. AIM: We performed a detailed study to identify possible active metabolites that are present in different ESLD fractions and investigated their effects on renal and peripheral arteriolar tone. We further evaluated their interrelations with sustained diuretic and hypotensive actions. MATERIALS AND METHODS: The ESLD was first obtained from L. divaricata leaves, and liquid-liquid fractionation was performed. The fractions were analyzed by liquid chromatography-mass spectrometry. An ethyl acetate fraction (AceFr), n-butanolic fraction (ButFr), and aqueous fraction (AqueFr) were then orally administered in male Wistar rats in a single dose or daily for 7 days. The doses were previously defined based on the yield that was obtained from each fraction. Hydrochlorothiazide was used as a positive control. Blood pressure, heart rate, urinary volume, pH, density, and urinary sodium, potassium, chloride, and calcium levels were measured. Serum levels of nitrite, thiobarbituric acid reactive species, nitrotyrosine, aldosterone, vasopressin, and plasma angiotensin converting enzyme activity were also measured. Finally, the direct effects of the ButFr on renal and mesenteric arteriolar tone and the role of nitric oxide and prostaglandins in the renal and hemodynamic effects were investigated. RESULTS: Of the fractions that were tested, only the ButFr exerted significant diuretic and saluretic effects. The AceFr and ButFr also had acute hypotensive effects, but only the ButFr maintained its response after 7 days of treatment. Prolonged treatment with the ButFr increased serum nitrite levels and significantly reduced oxidative and nitrosative markers of stress. Additionally, the ButFr caused a vasodilatory response in the renal and mesenteric arteriolar beds through the release of nitric oxide and prostaglandins. Finally, the diuretic and hypotensive effects of the ButFr were completely blocked by pretreatment with Nω-nitro-L-arginine methyl ester and indomethacin, thus demonstrating the direct involvement of nitric oxide and prostaglandins in these effects. CONCLUSION: The ButFr that was obtained from Luehea divaricata exerted sustained diuretic and hypotensive effects. These effects were apparently attributable to the release of nitric oxide and prostaglandins, which reduce renal and peripheral arteriolar tone and lead to an increase in the glomerular filtration rate and a reduction of global peripheral resistance. These findings suggest that the ButFr may be a potential complementary therapy for several conditions in which diuretic and hypotensive effects are required.

In vivo behavior of neutrophils from two patients with distinct inherited leukocyte adhesion deficiency syndromes.

The selectins and the beta 2-integrins (CD11/CD18) mediate distinct adhesive interactions between neutrophils and endothelial cells. Selectins are believed to initiate binding by mediating neutrophil rolling, whereas beta 2-integrins are required for subsequent activation-induced firm sticking and emigration. In vitro evidence suggests that two endothelial cell selectins, P- and E-selectin, can mediate rolling by binding to the carbohydrate ligand sialyl-Lewisx (sLex) on neutrophil surface glycoconjugates. To test the relative contribution of selectins and beta 2-integrins in vivo we used intravital microscopy to study the behavior of neutrophils from two patients with distinct inherited leukocyte adhesion deficiency syndromes. Neutrophils from a patient suffering from CD18 deficiency showed normal rolling behavior but were incapable of sticking or emigrating upon chemotactic stimulation. Neutrophils from a second patient with a newly described adhesion deficiency had normal CD18 but did not express sLex. These neutrophils rolled poorly and also failed to stick in venules under shear force. Under static conditions, however, chemoattractant-induced sticking and emigration could be observed. This demonstrates that both selectin-carbohydrate-mediated initiation of adhesion and subsequent activation-induced beta 2-integrin engagement are essential for the normal function of human neutrophils in vivo.

Increases in vanilloid TRPV1 receptor protein and CGRP content during endotoxemia in rats.

The aim of the present study was to determine whether the transient receptor potential vanilloid (TRPV1) receptor protein as well as the calcitonin gene-related peptide (CGRP) content could be enhanced after the i.p. administration of 5 mg/kg lipopolysaccharide (LPS) to Sprague-Dawley rats. In tongue tissue, used as a representative model of TRPV1 receptors expression, there was a significant increase in the abundance of TRPV1 receptor protein 6 h after LPS administration. In mesenteric arteries, the density of the CGRP-positive nerves as well as the release of CGRP induced by 10 microM anandamide was also significantly increased 6 h after LPS administration. The relaxant responses induced by anandamide in mesenteric beds isolated from either untreated or LPS-treated rats were abolished after a 2 h exposure to 10 microM capsaicin. Moreover, anandamide-induced relaxations of untreated mesenteries were potentiated by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 0.1 microM), but not by its inactive analogue 4alpha-phorbol (0.1 microM). The potentiation of anandamide effects caused by the PKC activator was accompanied by a significant increase in the overflow of CGRP induced by anandamide in the untreated rats. It is proposed that the overexpression of the TRPV1 receptors and the increased content of CGRP could contribute to the enhancement of anandamide effects during the endotoxemic shock. An eventual phosphorylation event linked to the overflow of CGRP could also participate in the enhanced relaxation caused by anandamide in endotoxemia.