Contrasting effects of pseudoephedrine and papaverine in dextran sodium sulfate-induced colitis.

BACKGROUND: Dextran sodium sulfate (DSS) induces submucosal arteriolar constriction that reduces blood flow to the intestine, and the relevance of this decrease in flow needs further investigation. In the present study we examined the effects of a vasoconstrictor (pseudoephedrine) and a vasodilator (papaverine) on the outcome of DSS-induced colitis. METHODS: Mice were given DSS in drinking water for 6 days, with enemas on days 0, 1, 3, and 5 containing pseudoephedrine, papaverine, or no drug. At the conclusion of the 6-day protocol a disease activity index comprising weight loss, stool consistency, and rectal bleeding was evaluated, along with intravital microscopy observations of submucosal venular leukocyte and platelet adherence in the proximal colon and terminal ileum. RESULTS: Pseudoephedrine and papaverine had several contrasting effects on the outcome of DSS ingestion: pseudoephedrine induced the highest levels of weight loss, loose stools, venular platelet adherence, and overall disease activity index, while papaverine induced the highest levels of venular leukocyte adherence, but the lowest levels of rectal bleeding, loose stools, and overall disease activity index. CONCLUSIONS: The results suggest that vasoconstriction worsens the pathological consequences of DSS in the mouse model of colitis.

Roles of platelet and endothelial cell COX-1 in hypercholesterolemia-induced microvascular dysfunction.

Aspirin is a common preventative therapy in patients at risk for cardiovascular diseases, yet little is known about how aspirin protects the vasculature in hypercholesterolemia. The present study determines whether aspirin, nitric oxide-releasing aspirin (NCX-4016), a selective cyclooxygenase (COX)-1 inhibitor (SC560), or genetic deficiency of COX-1 prevents the inflammatory and prothrombogenic phenotype assumed by hypercholesterolemic (HC) venules. Aspirin or NCX-4016 (60 mg/kg) was administered orally for the last week of a 2-wk HC diet. COX-1-deficient (COX-1(-/-)) and wild-type (WT) mice were transplanted with WT (WT/COX-1(-/-)) or COX-1(-/-) (COX-1(-/-)/WT) bone marrow, respectively. HC-induced adhesion of platelets and leukocytes in murine intestinal venules, observed with intravital fluorescence microscopy, was greatly attenuated in aspirin-treated mice. Adhesion of aspirin-treated platelets in HC venules was comparable to untreated platelets, whereas adhesion of SC560-treated platelets was significantly attenuated. HC-induced leukocyte and platelet adhesion in COX-1(-/-)/WT chimeras was comparable to that in SC560-treated mice, whereas the largest reductions in blood cell adhesion were in WT/COX-1(-/-) chimeras. NCX-4016 treatment of platelet recipients or donors attenuated leukocyte and platelet adhesion independent of platelet COX-1 inhibition. Platelet- and endothelial cell-associated COX-1 promote microvascular inflammation and thrombogenesis during hypercholesterolemia, yet nitric oxide-releasing aspirin directly inhibits platelets independent of COX-1.

Platelet recruitment in the murine hepatic microvasculature during experimental sepsis: role of neutrophils.

OBJECTIVES: Sepsis is a major clinical problem that often results in the dysfunction or failure of multiple organs, including the liver. While inflammatory cell activation has been implicated as an early critical event in sepsis-induced liver dysfunction, there is growing evidence for the involvement of activated platelets in this pathologic process. METHODS: Intravital microscopy was used in this study to assess the magnitude and time course of platelet adhesion in the liver microcirculation during experimental sepsis and to determine whether the platelet accumulation is linked to leukocyte infiltration. The adhesion of platelets and leukocytes in terminal hepatic venules (THV) and sinusoids was quantified at 2, 4, and 6 h after abdominal sepsis induced by cecal ligation and puncture (CLP). RESULTS: While the rolling and firm adhesion of platelets and leukocytes in THV were not altered in the first 2 h after CLP, platelet recruitment was observed at 4 h and further elevated at 6 h after CLP. Leukocyte adhesion in THV exhibited a similar time course. A similar accumulation of blood cells in sinusoids was noted after CLP. This was accompanied by an increased number of nonperfused sinusoids. CLP-induced leukocyte and platelet recruitment in THV and sinusoids was attenuated in mice rendered neutropenic with anti-neutrophil serum. CONCLUSION: These findings indicate that sepsis is associated with a neutrophil-dependent recruitment of platelets in the liver microcirculation that impairs sinusoidal perfusion and may contribute to the liver dysfunction associated with sepsis.

Venular constriction of submucosal arterioles induced by dextran sodium sulfate.

BACKGROUND: Leukocyte and platelet adherence in postcapillary venules has been speculated to induce constriction of closely paired arterioles. This mechanism was investigated in the current study, in a model of intestinal inflammation induced by dextran sodium sulfate (DSS; 5,000 molecular weight). METHODS: Closely paired, parallel arterioles and venules in the submucosa of the mouse intestine were observed using fluorescent intravital microscopy. Arterioles in control mice were compared to arterioles in mice given 5% DSS in drinking water for 11 days. RESULTS: DSS induced an inflammatory response in which fluorescently labeled leukocytes and platelets were observed adhering to venules. Arterioles constricted and flow decreased significantly when the arterioles were paired with venules having adherent leukocytes and platelets, although the decreases in flow and diameter appeared to be more dependent on platelet than leukocyte adherence. No significant constriction was observed in arterioles paired with venules having minimal platelet adherence. Inhibition of thromboxane with 100 mg/kg ozagrel induced a significant dilation of arterioles in DSS mice that was absent in control mice. CONCLUSION: The results are consistent with a proposed mechanism in which thromboxane constricts submucosal arterioles when the arterioles are closely paired with platelet-bearing venules in DSS-induced inflammation.

Acute gastric changes after intracerebral hemorrhage in rats.

Severe intracerebral hemorrhage (ICH) produces gastric pathology in about 30% of the patient population, even after the standard treatment of H2 receptor blockers or proton pump inhibitors. This study was undertaken to establish a rat model of ICH-induced gastric ulcer. Adult male Sprague-Dawley rats (300-350 g) were divided into two hemorrhage groups and a sham control group. ICH was produced either by injection of 100 microl of autologous arterial blood or by injection of 4 microl saline containing 0.6 unit of bacterial collagenase VII into the right basal ganglia. Rats were sacrificed at 24, 48, 72 h, and 7 days after ICH to harvest brains and stomachs. Greater degrees of hemorrhage and brain edema were observed in collagenase-induced ICH. Motor behavior decreased significantly after 24 h in both models. The incidence of acute ulceration with destruction of the forestomach epithelium was extremely low at 8.7% in the collagenase injection model and 4.8% in the blood injection rats. Small, pinpoint hemorrhages (petechiae) were noticed in 38% of rats after blood injection and 22% after collagenase injection, in the glandular portion of the gastric mucosa with penetration of red blood cells and inflammatory cells into the gastric mucosa. Enhanced tumor necrosis factor alpha (TNFalpha) and cyclooxygenase 2 (COX-2) expressions were observed in gastric tissues after ICH with more intense staining occurring at 24 and 48 h. Due to the low incidence of ulceration, ICH-induced gastric ulceration in rodents may not appropriate for evaluating the potential human risk of gastric ulceration after ICH.

Intercellular adhesion molecule 1 is important for the development of severe experimental malaria but is not required for leukocyte adhesion in the brain.

Plasmodium berghei-infected mice, a well-recognized model of experimental cerebral malaria (ECM), exhibit a systemic inflammatory response. Most investigators hypothesize that leukocytes bind to endothelial cells via intercellular adhesion molecule 1 (ICAM-1), which causes endothelial damage, increased microvascular permeability, and, ultimately, death. ICAM-1-deficient mice on an ECM-susceptible C57BL/6 background were significantly (p = .04) protected from P. berghei mortality compared with ICAM-1 intact controls. ICAM-1 expression assessed by the dual radiolabeled monoclonal antibody technique was increased in the brain and lung in C57BL/6 mice on day 6 of P. berghei infection compared with uninfected controls (5.3-fold, p = .0003 for brain and 1.8-fold, p = .04 for lung). The increase in ICAM-1 expression coincided with significant (p < .05) increases in microvascular permeability in the brain and lung. In contrast to the hypothesized role for ICAM-1, in vivo analysis by intravital microscopy of leukocyte rolling and adhesion in brain microvasculature of mice revealed markedly increased levels of leukocyte rolling and adhesion in ICAM-1-deficient mice on day 6 of P. berghei infection compared with uninfected controls. In addition, ICAM-1 expression and microvascular permeability were increased in infected ECM-resistant BALB/c mice compared with uninfected BALB/c controls. These results collectively indicate that although ICAM-1 contributes to the mortality of experimental malaria, it is not sufficient for the development of severe experimental malaria. In addition, ICAM-1 expressed on the endothelium or on leukocytes is not required for leukocyte rolling or adhesion to the brain microvasculature of mice during P. berghei malaria. Leukocyte rolling and adhesion in the brain vasculature during P. berghei malaria use different ligands than observed during inflammation in other vascular beds.

P-selectin contributes to severe experimental malaria but is not required for leukocyte adhesion to brain microvasculature.

Plasmodium berghei-infected mice, a well-recognized model of experimental cerebral malaria (ECM), exhibit many of the hallmarks of a systemic inflammatory response, with organ damage in brain, lung, and kidneys. Identification of the molecules mediating pathogenesis of the inflammatory response, such as leukocyte adhesion, may lead to new therapies. Indeed, mice lacking the cell adhesion molecule P-selectin were significantly (P = 0.005) protected from death due to P. berghei malaria compared with C57BL/6 controls despite similar parasitemia (P = 0.6) being found in both groups of mice. P-selectin levels assessed by the quantitative dual radiolabeled monoclonal antibody technique increased significantly (P < 0.05) in several organs in C57BL/6 mice infected with P. berghei, supporting the concept of a systemic inflammatory response mediating malarial pathogenesis. Intravital microscopic analysis of the brain microvasculature demonstrated significant (P < 0.001) leukocyte rolling and adhesion in brain venules of P. berghei-infected mice compared with those found in uninfected controls. The maximum leukocyte adhesion occurred on day 6 of P. berghei infection, when the mice become moribund and exhibit marked vascular leakage into the brain, lung, and heart. However, P-selectin levels were significantly (P < 0.005) increased in brain, lung, and kidneys during P. berghei malaria in ECM-resistant BALB/c mice compared with those found in uninfected BALB/c controls, indicating that increased P-selectin alone is not sufficient to mediate malarial pathogenesis. Leukocyte adhesion to brain microvessels of P-selectin-deficient mice with P. berghei malaria was similar to that observed in control mice. Collectively, these results indicate that P-selectin is important for the development of malarial pathogenesis but is not required for leukocyte adhesion in brain.

Regulation of endothelial cell adhesion molecule expression in an experimental model of cerebral malaria.

OBJECTIVE: Plasmodium falciparum malaria in humans and animal models of this disease have revealed changes in the infected host that are consistent with a systemic inflammatory response. Although it has been proposed that endothelial cell adhesion molecules (CAM) contribute to the adhesive interactions of Plasmodium-infected erythrocytes and immune cells with vascular endothelial cells, ECAM expression has not been systematically studied in Plasmodium-infected animals. METHODS: In this study, the dual radiolabeled monoclonal antibody method was used to quantify the expression of different ECAMs (ICAM-1, VCAM-1, P-selectin, E-selectin) in different regional vascular beds of Plasmodium berghei ANKA-inffected mice (PbA), a well-recognized model of human cerebral malaria. The roles of T lymphocytes and certain cytokines (TNF-alpha, IL-12, IFN-gamma) in mediating the infection-induced expression of ICAM-1 and P-selectin were assessed by using relevant mutant mice. RESULTS: Wild-type (WT) mice exhibited highly significant increases in the expression of ICAM-1, VCAM-1, and P-selectin (but not E-selectin) in all vascular beds on the 6th day of PbA infection. The PbA-induced upregulation of ICAM-1 was significantly blunted in mice that were either deficient in IFN-alpha, IL-12 (but not TNF1b) or T lymphocytes (Rag-1 deficiency); however, these responses were tissue specific. CONCLUSIONS: These findings indicate that vascular endothelial cells in most regional circulations assume an inflammatory phenotype and that cytokines and immune cells mediate this response in a tissue-specific manner.

The transcytosis of divalent metal transporter 1 and apo-transferrin during iron uptake in intestinal epithelium.

Caco-2 cells grown in bicameral chambers are a model system to study intestinal iron absorption. Caco-2 cells exhibit constitutive transport of iron from the apical (luminal) chamber to the basal (serosal) chamber that is enhanced by apo-transferrin in the basal chamber, with the apo-transferrin undergoing endocytosis to the apical portion of the cell. With the addition of iron to the apical surface, divalent metal transporter 1 (DMT1) on the brush-border membrane (BBM) undergoes endocytosis. These findings suggest that in Caco-2 cells DMT1 and apo-transferrin may cooperate in iron transport through transcytosis. To prove this hypothesis, we determined by confocal microscopy that, after addition of iron to the apical chamber, DMT1 from the BBM and Texas red apo-transferrin from the basal chamber colocalized in a perinuclear compartment. Colocalization was also observed by isolating endosomes from Caco-2 cells after ingestion of ultra-small paramagnetic particles from either the basal or apical chamber. The isolated endosomes contained both transferrin and DMT1 independent of the chamber from which the paramagnetic particles were endocytosed. These findings suggest that iron transport across intestinal epithelia may be mediated by transcytosis.