Increased expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and lymphocyte recruitment in murine gastritis induced by Helicobacter pylori.

Although T cell involvement in Helicobactor pylori-induced gastritis is known, mechanism about T cell recruitment is not understood. In this study we examined how mucosal addressin cell adhesion -molecule-1 (MAdCAM-1) is involved in lymphocyte recruitment in murine chronic gastritis induced by H. pylori. C57 BL/6 mice were infected with Sydney strain (SS1). Six months after infection, the stomach was removed. The expression of adhesion molecules, MAdCAM-1, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and the cell surface antigens CD4, CD8, CD45R/B220 or beta7-integrin were determined by immunohistochemistry. A significant increase in CD4 lymphocytes was observed in the body portion of stomach in SS1-infected mice and most of these CD4 cells express beta7-integrin, a known counter ligand for MAdCAM-1 molecule. Strong MAdCAM-1 expression was observed adjacent to these cells in the lamina propria as well as in the submucosa of SS1-infected stomach. Quantitative analysis showed that the area of MAdCAM-1 expression well correlated with the infiltration of beta7-integrin positive lymphocytes. On the other hand, expression of ICAM-1 or VCAM-1 in the lamina propria was few even in the SS1-infected stomach. Increased expression of MAdCAM-1 was well correlated to the location of lymphocytes, which express CD4 and beta7-integrin. These results suggest the possibility that MAdCAM-1 may be largely involved in the lymphocyte recruitment in the gastritis mucosa with H. pylori.

Intercellular cell adhesion molecule-1 regulates lymphocyte movement into intestinal microlymphatics of rat Peyer's patches.

The objective of this study was to determine whether specific adhesion molecules modulate lymphocyte movement from Peyer's patches into intestinal microlymphatics. The fluorochrome acridine orange was injected via a micropipette into Peyer's patches to fill lymphatics. The flux of labeled lymphocytes into intestinal microlymphatics was monitored with intravital fluorescence microscopy. The lymphatic microvessels in the perifollicular area of Peyer's patches were filled with lymphocytes, most of which remained within the lymphatics. Some lymphocytes became detached and were drained into intestinal lymph. Administration of antibodies directed against ICAM-1 significantly increased lymphocyte flux into interfollicular lymphatics. The immunohistochemical study showed intense ICAM-1 expression on the lymphocytes densely packed in the lymphatics surrounding follicles in Peyer's patches. A large number of lymphocytes are normally sequestered in the lymphatic network of Peyer's patches. This sequestration of lymphocytes is largely mediated by ICAM-1-dependent cell-cell interactions.

Involvement of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the pathogenesis of granulomatous colitis in rats.

Although increased expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) has been demonstrated in inflammatory sites of various diseases, its role in colitis remains unknown. In this study, we examined whether MAdCAM-1 is involved in the pathogenesis of granulomatous colitis induced by peptidoglycan-polysaccharide (PG-PS). Experimental colitis was induced by intramural injection of PG-PS to rat colon. After 3 weeks the colon was removed and the mucosal inflammation was assessed. The area of MAdCAM-1-positive venules and the subsets of infiltrating cells were determined in colonic mucosa by immunohistochemistry. In another experiment, monoclonal antibody against MAdCAM-1 was administered intraperitoneally to examine its attenuating effect on colitis. The intramural injection of PG-PS induced significant colonic inflammation with granuloma formation. The submucosa was drastically thickened with the infiltration of CD4 positive lymphocytes and ED-1 positive macrophages. Intense MAdCAM-1 expression was observed on endothelium of the submucosal venules in inflamed mucosa. Administration of anti-MAdCAM-1 antibody significantly attenuated the PG-PS-induced colonic damage and cell infiltration. Enhanced expression of MAdCAM-1 was demonstrated in venular endothelium of the inflamed colon in PG-PS-induced colitis. The attenuating effect of anti-MAdCAM-1 suggests the importance of the MAdCAM-1-dependent process in the formation of chronic granulomatous colitis.

Reduced sensitivity of inducible nitric oxide synthase-deficient mice to chronic colitis.

BACKGROUND: Overproduction of nitric oxide by the inducible form of nitric oxide synthase (iNOS) has been implicated in colitis. Different authors have postulated both toxic and protective effects of nitric oxide (NO) in the pathophysiology of active inflammation. The objective of this study was to examine the role of iNOS in experimental chronic colitis using iNOS-deficient mice. METHODS: For induction of colitis, mice received three cycles of 2% of dextran sodium sulfate (DSS) (M.W. 40,000) treatment in drinking water. The degree of colonic inflammation, leukocyte infiltration, and the expression of cell adhesion molecules were determined. INOS expression and nitrotyrosine were also determined by immunohistochemistry. RESULTS: After DSS treatment, a moderate colitis with marked cell infiltration was observed. Intense expression of iNOS was observed on infiltrating cells as well as on the colonic mucosal epithelium in these animals. In the iNOS-deficient mice, tissue damage was significantly diminished. No iNOS or nitrotyrosine staining was found in iNOS-deficient mice. The number of infiltrating cells and the expression of mucosal adressin cell adhesion molecule-1 were significantly attenuated in the DSS-treated colon of iNOS-deficient mice. CONCLUSION: Induction of iNOS seems to act as a critical toxic effector molecule in the pathogenesis of chronic colonic inflammation.

Direct measurement of erythrocyte deformability in diabetes mellitus with a transparent microchannel capillary model and high-speed video camera system.

To measure erythrocyte deformability in vitro, we made transparent microchannels on a crystal substrate as a capillary model. We observed axisymmetrically deformed erythrocytes and defined a deformation index directly from individual flowing erythrocytes. By appropriate choice of channel width and erythrocyte velocity, we could observe erythrocytes deforming to a parachute-like shape similar to that occurring in capillaries. The flowing erythrocytes magnified 200-fold through microscopy were recorded with an image-intensified high-speed video camera system. The sensitivity of deformability measurement was confirmed by comparing the deformation index in healthy controls with erythrocytes whose membranes were hardened by glutaraldehyde. We confirmed that the crystal microchannel system is a valuable tool for erythrocyte deformability measurement. Microangiopathy is a characteristic complication of diabetes mellitus. A decrease in erythrocyte deformability may be part of the cause of this complication. In order to identify the difference in erythrocyte deformability between control and diabetic erythrocytes, we measured erythrocyte deformability using transparent crystal microchannels and a high-speed video camera system. The deformability of diabetic erythrocytes was indeed measurably lower than that of erythrocytes in healthy controls. This result suggests that impaired deformability in diabetic erythrocytes can cause altered viscosity and increase the shear stress on the microvessel wall.

Image correlation method for measuring blood flow velocity in microcirculation: correlation 'window' simulation and in vivo image analysis.

To elucidate the function of the microcirculation system, it is very important to know the blood flow velocity and its distribution in the microvessels. We have developed an automated system for measuring blood flow velocity in microcirculation by image correlation. The 'window' in the image correlation method is equivalent to the sensors in various other measurement methods. We performed simulations with virtual blood flow images consisting of random dots before measuring actual ones, and examined the optimum window shape and size. We found that by reducing the size of a circular window to the size of erythrocytes we could measure in vivo blood flow images with high accuracy. We recorded them with a high-speed video camera system at high temporal resolution, and measured the velocity in microvessels of normal Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). SHR had higher blood velocity than WKY even though the vessel diameters were the same. Using this method to measure the blood flow velocity profile at the bent corner of SHR's arteriole at the heart systole, we found that erythrocytes flow faster at the inner side of the bend, so the vessel wall was exposed locally to higher shear stress in the hypertensive condition.

In vivo identification of parasinus macrophages in the mesenteric lymph node.

Macrophages beneath the marginal sinus in the lymph nodes may play a role in defense against microorganism. The purpose of this study was to directly visualize the parasinus macrophages in the mesenteric lymph node. Fluorescent latex particles were injected into the appendix submucosa of rats. The mesenteric lymph node was epi-illuminated and observed with a fluorescent microscope. Fluorescent particles entered the marginal sinus of the mesenteric lymph node through the afferent lymphatic vessels, and distributed diffusely all over the marginal sinus. The particles became aggregated and interspersed 3 hr after injection, suggesting that particles were incorporated by phagocytes. The number of these particle-laden phagocytes increased up to 12 hr after injection, and then declined. Some phagocytes migrated rapidly within the marginal sinus. Morphology of these phagocytes in cell suspension was consistent with macrophages. In conclusion, we successfully visualized parasinus macrophages in vivo, which incorporated foreign bodies and migrated within the marginal sinus.

Glycerol attenuates the adherence of leukocytes in rat pial venules after transient middle cerebral artery occlusion.

Intravenous infusion of glycerol has been used in patients with a cerebral infarction, expecting improvement in brain edema and cerebral blood flow (CBF). However, the mechanism of the improvement of CBF has not been clearly demonstrated. The aim of this study in the rat pial microvasculature after transient middle cerebral artery occlusion (MCAO) is to examine the effects of glycerol on leukocyte-endothelium interaction, which plays a critical role in the pathogenesis of brain injury by ischemia/reperfusion and concerns induction of secondary brain damage. Rhodamine 6G-labeled leukocytes at the brain surface were visualized with intra-vital fluorescence videomicroscopy through a closed cranial window and an analysis was made of the number of adherent leukocytes and the centerline leukocyte velocity in the venule before MCAO, after reperfusion of MCAO and after infusion of glycerol (Group 1) or saline (Group 2). The number of adherent leukocytes decreased and the centerline leukocyte velocity increased statistically significantly immediately after the infusion of glycerol in Group 1, but there was no significant change in Group 2. The infusion of glycerol washes away the adherent leukocytes and prevents them from interfering with the blood cell and plasma flow. Furthermore, secondary brain damage may be relieved by decreasing the adherence of leukocytes. In conclusion, modulating the adherence of leukocytes is one of the important factors in the neuroprotective effect of glycerol.

Is endoscopic paravascular injection of sclerosing agents reasonable in the control of GI bleeding?

BACKGROUND: The pharmacologic response and microvascular effects associated with the endoscopic injection of sclerosing agents around vessels (paravascular injection) to stop bleeding from the digestive tract remain to be clarified. METHODS: Using in vivo microscopy, we directly visualized submucosal microvessels of the rat stomach and intestine. We studied differences among sclerosing agents in thrombus formation and vascular diameter change that occur through a pharmacologic response and/or local compression after topical application or paravascular injection of the agents. RESULTS: Except for absolute ethanol, topical application of the agents did not cause constriction or thrombi in either arterioles or venules. Polidocanol topical application and paravascular injection significantly dilated arterioles. Injecting ethanolamine oleate near venules constricted them the longest and most effectively, but vasoconstriction in arterioles was transient. Injecting absolute ethanol formed long-lasting thrombi and caused vasoconstriction in venules, but arteriole thrombi persisted no more than 3 minutes. The vascular response to thrombin did not significantly differ from that to physiologic saline. CONCLUSION: The paravascular injection of ethanolamine oleate, because of its long-lasting vasoconstriction, or of absolute ethanol, because of its thrombogenic effect, is a valid therapeutic approach to treating venous bleeding. The efficacy of paravascular injection of sclerosing agents for treating acute arterial bleeding, however, is not supported in this experimental model.

Effects of moderate hypothermia on leukocyte- endothelium interaction in the rat pial microvasculature after transient middle cerebral artery occlusion.

Background and Purpose--It has been demonstrated that moderate hypothermia attenuates brain damage, but the mechanism whereby this is achieved has not been clearly shown. Recently, the role of leukocytes as mediators of secondary brain damage after brain ischemia has been discussed. The aim of this study is to examine the effects of moderate hypothermia on leukocyte-endothelium interaction in the rat pial microvasculature after transient middle cerebral artery occlusion (MCAO). Methods--Rhodamine 6G-labeled leukocytes in brain surface were visualized with intravital fluorescence videomicroscopy through a closed cranial window. We analyzed the number of leukocytes adhering to the venular and arteriolar endothelium before ischemic insult and up to 3 hours after reperfusion. Rats were divided into 4 experimental groups. Group I (n=6) consisted of sham-operated animals. Groups II (n=6) and III (n=6) received left MCAO for 1 hour under normothermia (36 degrees C to 37 degrees C, group II) and under moderate hypothermia (30 degrees C to 32 degrees C, group III). Group IV (n=4) received left common carotid artery occlusion for 1 hour under normothermia. Results--The number of adhering leukocytes in venules in groups II and IV increased significantly (P<0.001) after reperfusion compared with the group I, but that in group III did not increase significantly (P>0.05). The number of adhering leukocytes in arterioles in group II increased significantly (P<0.01) compared with the other groups, although the adhering leukocytes were not as numerous as those seen in venules. Conclusions--It is demonstrated that hypothermia attenuates adhering leukocytes in venules and arterioles after reperfusion of MCAO. The inhibition of the leukocyte function may be an important factor in the neuroprotective effect of hypothermia.

In vivo rat closed spinal window for spinal microcirculation: observation of pial vessels, leukocyte adhesion, and red blood cell velocity.

OBJECTIVES: An in vivo closed spinal window technique in rats was designed for observing the spinal microcirculation, such as the change of vessel diameter, leukocyte adhesion, and red blood cell (RBC) velocity, which has been very rarely examined in vivo in the spinal cord. METHODS: We made a very precise closed spinal window with a laminectomy at the C5 level using a dental acrylic resin and a cover glass (7 mm in diameter). Through this closed window, the dorsal surface of the rat cervical cord was observed with a video microscope, and the fluorescent images of rhodamine 6G-labeled leukocytes and fluorescein isothiocyanate-labeled RBCs were recorded and analyzed with a silicon-intensified target tube camera (30 frames/s) and an image-intensified high-speed video camera system (1000 frames/s). RESULTS: During CO2 inhalation, the pial arterioles responded with vasodilation of 12.4+/-10.4% (P<0.01) in 11 arterioles of seven rats. The adhering leukocytes significantly increased in 41 venular segments of seven rats after superfusion of the neutrophil chemoattractant, N-formyl-methionine-leucine-phenylalanine solution for 15 minutes (P<0.001) but not after superfusion of only artificial cerebrospinal fluid for 15 minutes. During these experiments, no adhering leukocyte was seen in the pial arterioles. Fluorescein isothiocyanate-labeled RBCs look like shooting stars in arterioles with silicon-intensified target tube camera processing at 30 frames per second, but individual fluorescein isothiocyanate-labeled RBCs could be recognized frame by frame with the image-intensified high-speed video camera system. In 13 arterioles of four rats, the RBC velocity was 5.3+/-2.0 mm per second. CONCLUSION: This closed spinal window technique in rats is available and applicable for the study of the spinal microcirculation, such as the pathophysiology of a secondary injury.

Measurement of RBC velocities in the rat pial arteries with an image-intensified high-speed video camera system.

The mean centerline red blood cell (RBC) velocity of the rat pial artery was measured using an image-intensified high-speed (1000 frames/s) video camera system and RBCs labeled with fluorescein isothiocyanate (FITC). Some investigations measuring RBC velocity have been made in most organs, but the RBC velocity of the pial artery has not yet been measured with this system using FITC labeled RBC. After recording the emission of the FITC labeled RBC through a closed cranial window using this system, the authors analyzed the videotape. The movement of each individual RBC for several milliseconds over a distance of 50 microm could be pursued. The mean centerline RBC velocity in normal rats varied between 1.0 and 9.0 mm/s (most of the measurements we taken in vessels ranging between 20 and 80 microm in diameter). As the diameter of the pial artery becomes smaller, the blood flow rate (pi x (diameter/2)2 x (mean centerline velocity/1.6)) tends to become smaller. During CO2 inhalation, the pial artery diameter, mean centerline RBC velocity, and blood flow rate increased with statistical significance. Mean centerline RBC velocities in the cerebral microcirculation could not be measured directly with accuracy using the older methods (30 frames/s). However, this method is useful for investigation of the cerebral microcirculation and is considered to be applicable for studying the behavior of leukocytes or platelets, which will be examined in a subsequent study.

Adenosine A2-receptor mediates ethanol-induced arteriolar dilation in rat stomach.

Topical application of ethanol to the gastrointestinal mucosa induces vasodilation. Using an in vivo microscopy technique, we studied the effect of topical ethanol on the submucosal microvessels that control mucosal blood flow in the rat stomach and identified vasoactive substances and receptors that mediate the ethanol vasoaction. Topical ethanol (1-20%) dilated submucosal arterioles dose dependently, but did not change venular diameters. An inhibitor of alcohol dehydrogenase, 1 mM 4-methylpyrazole, did not alter the ethanol vasoaction. Ethanol-induced arteriolar dilation was eliminated by adenosine deaminase, but other vasodilator inhibitors such as atropine, pyrilamine, indomethacin, human calcitonin gene-related peptide-(8-37), and N omega-nitro-L-arginine methyl ester did not prevent it. Ethanol-induced arteriolar dilation was inhibited by an adenosine A2-receptor antagonist, but not by an A1-receptor antagonist, whereas an A2-agonist, but not an A1-agonist, dose dependently dilated arterioles. Exogenous adenosine (10(-5)-10(-3) M) dilated arterioles to a similar extent as ethanol. This response was inhibited by an A2-antagonist. We conclude that nonmetabolized ethanol increases gastric mucosal blood flow via A2-receptors in submucosal arterioles.

Role of endothelin-1 in repeated electrical stimulation-induced microcirculatory disturbance and mucosal damage in rat stomach.

The aim of the present study was to clarify the involvement of endogenous endothelin in the pathogenesis of gastric mucosal damage. The rat stomach was exposed and repeated electrical stimulation (RES) was applied to the small arterial wall close to the lesser curvature. Significant mucosal haemorrhagic lesions (ulcer and erosion) were noted within 30 min after RES. Intravital microscopic observations revealed that an arteriolar constriction occurred in the submucosal layer of the rat stomach approximately 5 min after the completion of RES. Following the arteriolar constriction, the mucosal blood flow of the rat stomach, which was monitored by using a laser Doppler velocimeter, decreased to approximately 30% of the control value. The plasma immunoreactive endothelin-1 level in the regional blood of the stomach was significantly increased immediately after RES preceding the decrease in mucosal blood flow. Immunohistochemical studies revealed that endothelin-1 and big-endothelin-1 were detectable in the arteriolar endothelium around the muscularis mucosa, supporting the involvement of endothelin-1 in RES-induced mucosal ischaemia. In addition, BQ-123, a specific antagonist of the endothelin A (ETA) receptor, attenuated the reduction of blood flow and the development of haemorrhagic lesions observed in gastric mucosa subjected to RES. The results of the present study suggest that an excessive production of endothelin-1 in the arteriolar endothelium leads to microvascular derangements accompanied by haemorrhagic alterations of the gastric mucosa.

Differences in hemostasis among sclerosing agents in endoscopic injection sclerotherapy.

Endoscopic injection sclerotherapy is useful in stopping bleeding from esophageal varices. We compared the in vivo effects of sclerosants on thrombogenesis, hemostasis, and endothelial injury. We injected aethoxysclerol (AS) of ethanolamine oleate (EO) into the small veins of the rat intestine. The maximum thrombogenic index with AS was 30.7 and with EO was 9.2. The venous flow stopped sooner with EO than with AS. The thrombi caused by EO were mixed with red blood cells. Heparin pretreatment decreased the thrombogenic index with AS by 96.7%, but not that with EO. The area of the fluorescein-albumin conjugate that permeated from veins with AS was larger than that with EO. The fluorescent intensity with AS was lower than that with EO. We thus conclude that: (1) the size of thrombi is not necessarily proportional to the hemostatic efficacy; (2) change in a patient's coagulation may affect the potential of sclerosants; (3) the excellent hemostatic efficacy of EO is based on localized injury to the endothelium and the involvement of red blood cells aggregation; and (4) in vivo microscopy is useful in determining the rational selection of sclerosants.

In vivo visualization of lymphatic microvessels and lymphocyte migration through rat Peyer's patches.

BACKGROUND/AIMS: In the small intestine, lymphocytes migrate through Peyer's patches. The distribution of lymphatic microvessels in rat Peyer's patches and lymphocyte traffic through them were studied. METHODS: Vital dyes were injected via a micropipette into the Peyer's patches tissue to fill lymphatic microvessels and to stain lymphocytes in lymphatic microvessels. RESULTS: Direct microscopic observation revealed a dense plexus of lymphatic microvessels in the perifollicular and interfollicular areas. Injection of the dyes into the germinal center failed to delineate lymphatic microvessels. The lymphatic microvessels in the perifollicular area were filled with lymphocytes. Most lymphocytes in the perifollicular lymphatics stayed in the lymphatic microvessels. Some lymphocytes became detached and drained into lymphatic microvessels in the interfollicular areas. Lymphocytes then moved toward the submucosal lymphatics beneath the villi around the Peyer's patches. The interfollicular lymphatics did not display contractile activity but had valves. Opening and closing of valves was synchronized with the respiration and the back and forth flow of lymphocytes. CONCLUSIONS: There are numerous lymphocytes in a dense lymphatic network in the perifollicular and interfollicular areas of Peyer's patches. This well-developed lymphatic network has the potential capacity for storage of lymphocytes and modulation of lymphocyte migration.

Attenuating effect of antithrombin III on the fibrinolytic activation and microvascular derangement in rat gastric mucosa.

The roles for the fibrinolytic activation and disorder of coagulation in formation of gastric ulcer induced by microvascular derangement were investigated. The rat stomach was exposed and repeated electrical stimuli (RES) were applied on the small arterial wall close to the lesser curvature to induce mucosal microcirculatory disturbances. The level of tissue-type plasminogen activator (t-PA), a key enzyme for fibrinolytic activity, in the regional blood of the stomach was significantly elevated immediately after RES. At 5 min after RES, the leakage of FITC-labeled albumin and thrombus formation in the mucosal microvasculature were visually demonstrated by using an intravital microscopic system. At 30 min, hemorrhagic erosions and linear ulcers were observed in the gastric mucosa. Pretreatment with human antithrombin-III (AT-III) in the range of 0.1-10 U/kg dose-dependently attenuated both the fibrinolytic activation and microvascular alteration promoted by RES. Human AT-III also prevented RES-induced gastric mucosal injury. Thrombin inhibitory activity in the gastric vein decreased (69.0 +/- 2.1%) just after RES, and further reduced at 30 min (47.7 +/- 5.3%). The present study suggests a hypothesis that human AT-III has a preventive effect on the gastric mucosal hemorrhagic changes via attenuating the fibrinolytic activation and subsequent microcirculatory disturbances.

Regional differences in microvascular response in rat intestine during acute elevation of portal pressure.

The effect of acute elevation of portal pressure on the blood flow of rat intestinal microvessels was studied using a laser Doppler velocimeter and in vivo microscopy. The total intestinal blood flow decreased when portal pressure increased more than +15 cmH2O above the basal value. Blood flow in villus capillaries did not change at portal pressures of +5 to +15 cmH2O, but did decrease at +20 cmH2O. Blood flow in muscle capillaries decreased at all steps of portal hypertension. Red blood cell velocity was decreased by portal hypertension in large venules, but not in small venules of the submucosa. Large venules, but not small venules, dilated in acute portal hypertension. Large arterioles in the submucosa constricted, while small arterioles dilated at portal pressures of +10 to +15 cmH2O. In conclusion, the intestinal microvascular flow response differs according to the degree of portal hypertension and the location on the microvascular tree. Blood flow in villus capillaries and in small submucosal venules is maintained at a small degree of portal hypertension.