Neena Kalia

Studies on the gastric mucosal microcirculation. 2. Helicobacter pylori water soluble extracts induce platelet aggregation in the gastric mucosal microcirculation in vivo

Background—The exact mechanisms by whichHelicobacter pylori infection results in gastric mucosal injury are unclear. Aims—To assess in vivo whether H pylori extracts could initiate an inflammatory response in the rat gastric mucosal microcirculation. Methods—Extracts of H pylori, Escherichia coli, or distilled water were administered topically to the gastric mucosa of anaesthetised animals. Fluorescence in vivo microscopy assessed macromolecular leakage of labelled albumin from mucosal vessels, leucocyte adherence/rolling, and platelet activity for 90 minutes. Results—H pylori induced increases (p<0.001) in adherent platelet thrombi and circulating platelet emboli after five and 15 minutes respectively. Adherent platelet thrombi (mean of four per field of view) remained significantly increased throughout the experiment, but circulating emboli (maximum of five at 30 minutes) decreased with time. Leucocyte adherence did not occur although early transient rolling was observed. An 11% increase (p<0.02) in albumin leakage occurred after five minutes only. The induction of platelet aggregation was only observed following H pyloriadministration. Conclusion—This in vivo study demonstrated the ability of H pylori extracts to promote platelet aggregation within gastric mucosal microvessels. Recruitment of leucocytes was not observed. The results suggest that the early events associated with H pylori infection are platelet aggregation with perhaps subsequent leucocyte recruitment by activated platelets.

FK409 inhibits both local and remote organ damage after intestinal ischaemia

In addition to localized tissue injury, intestinal ischaemia–reperfusion (I/R) leads to remote organ damage, in particular to the lungs. Given that nitric oxide (NO) can attenuate I/R‐induced tissue injury in many situations, this study evaluated the effects of the NO donor, FK409, on leukocyte adhesion in the microcirculation of the intestinal villus and also assessed pulmonary tissue damage after intestinal I/R injury. PVG rats were subjected to 30 min intestinal ischaemia and a sub‐group of animals received the NO donor FK409 (10 mg/kg; i.v.) both 30 min prior to ischaemia and 30 min post‐reperfusion. The intestinal mucosal surface was visualized via an incision made in an exteriorized ileal segment and leukocyte adhesion in the villous microcirculation was determined by in vivo microscopy. Total and differential leukocyte counts from peripheral blood were evaluated. Lungs were removed at the end for histological assessment. Six out of ten untreated I/R animals failed to survive the 2 h reperfusion period, whereas all ten FK409‐treated animals survived. I/R induced a significant increase in villous leukocyte adhesion of untreated I/R animals (p<0.001) and this was significantly decreased by FK409 treatment (p<0.001). The total leukocyte count was significantly decreased in untreated I/R animals (p<0.001) and this primarily resulted from a reduction in circulating neutrophil numbers. This effect was not observed in FK409‐treated animals. Collapsed alveoli, thickened interstitial walls, and a dense neutrophilic infiltrate were apparent in the lungs of untreated I/R animals, whereas lung histology was normal in FK409‐treated animals. In conclusion, FK409 prevented mortality, significantly reduced villous leukocyte adhesion, maintained circulating leukocyte numbers, and prevented pulmonary tissue injury following intestinal I/R. FK409 may therefore be of value in reducing both local and remote tissue damage and improving outcome in situations where intestinal I/R injury is obligatory, such as small bowel transplantation. Copyright

Ketotifen abrogates local and systemic consequences of rat intestinal ischemia-reperfusion injury

Background:  Mast cell‐derived vasoactive and pro‐inflammatory mediators, particularly histamine, might contribute to local tissue damage and multiorgan dysfunction induced by intestinal ischemia/reperfusion (I/R). The purpose of the present study was to evaluate the effects of the mast cell stabilizer, ketotifen, on leukocyte adhesion within, and tissue leakage from the mucosal villous microcirculation after intestinal I/R.

Effects of Intestinal Ischemia–Reperfusion Injury on Rat Peripheral Blood Neutrophil Activation

Peripheral blood neutrophil activation status is indicative of remote organ damage after intestinal ischemia secondary to aortic aneurysm repair. However, the effects of direct intestinal ischemia–reperfusion (I/R) injury on neutrophil activation and its reflection of remote organ injury have not been evaluated. DA rats were subjected to 30 min of intestinal ischemia or sham surgery. Blood samples were taken before ischemia and 30, 60 120 min after reperfusion. Neutrophil counts were quantified and CD11b, CD62L NKR-P1 expression was assessed using flow cytometry. The sham procedure induced increases in neutrophil numbers (P < 0.001), which was transiently attenuated in animals subjected to intestinal I/R injury. CD11b expression increased in both groups, whereas CD62L and NKR-P1 (P < 0.01) expression decreased in both groups. These findings suggest that even mild surgical procedures induce demargination of neutrophils. Monitoring the peripheral blood for activated neutrophils is of no value in assessing the severity of direct intestinal I/R injury or predicting remote organ damage after intestinal ischemia.

Effects of Hypothermia and Rewarming on the Mucosal Villus Microcirculation and Survival After Rat Intestinal Ischemia–Reperfusion Injury

ObjectiveTo determine the effects of hypothermia and rewarming on changes in the villus microcirculation induced by intestinal ischemia–reperfusion (I/R). Summary Background DataThe small intestine is extremely sensitive to I/R injury, and although hypothermia can reduce cellular injury, its capacity to influence the villous microcirculation after intestinal I/R is unclear, especially after the return to normothermic conditions. MethodsCore body temperature of PVG rats was maintained at either 36° to 38°C (n = 12) or 30° to 32°C (n = 24) and then subjected to 30 minutes of intestinal ischemia. A subgroup of hypothermic animals (n = 12) were returned to normothermic conditions 120 minutes after clamp removal. The mucosal surface was visualized in an exteriorized ileal segment and macromolecular leak (MML) and leukocyte adhesion were monitored using in vivo microscopy (n = 6 in each group). MML from individual villi and numbers of adherent leukocytes within villi were determined for 2 to 4 hours after clamp removal. Heart rate and mean blood pressure were monitored in all animals. Control animals underwent sham surgery (n = 12). ResultsTen of 12 normothermic animals failed to survive the reperfusion period, whereas all hypothermic animals and 11 of 12 of the hypothermic animals that were returned to normothermic conditions survived. MML was significantly increased in all animals subjected to I/R, although leakage was more marked in animals subjected to continuous normothermia. Enhanced leukocyte adhesion and decreased blood flow were observed only in normothermic animals. ConclusionsHypothermia might prove to be an effective strategy for preventing adverse side effects in clinical settings in which intestinal I/R can be predicted.

Effects of FK409 on intestinal ischemia-reperfusion injury and ischemia-induced changes in the rat mucosal villus microcirculation.

Background. The small intestine is extremely sensitive to ischemia-reperfusion (I/R) injury and a range of microcirculatory disturbances contribute to tissue damage. Nitric oxide (NO) seems to be involved in tissue protection after I/R injury. This study therefore assessed the effects of the NO donor, FK409, on intestinal I/R injury and changes induced in intestinal microcirculation. Methods. PVG rats were subjected to 30-min intestinal ischemia with a subgroup of animals receiving FK409 (10 mg/kg i.v.) 30 min before ischemia and 30 min postreperfusion. Controls underwent sham surgery. The mucosal surface was visualized via an incision made in an exteriorized ileal segment and FITC-BSA or acridine orange was used to quantitate macromolecular leak (MML) and leukocyte adhesion, respectively. MML from, and numbers of adherent leukocytes within, individual villi were determined every 15 min for 2 hr after removal of the vessel clamp. Heart rate and mean blood pressure (mBP) were monitored throughout the experiment. Results. Eleven of 12 untreated animals subjected to intestinal I/R injury failed to survive the 2 hr reperfusion period, whereas all 12 FK409-treated animals survived. MML and leukocyte adhesion were increased in untreated animals (P <0.001), and blood flow stasis eventually ensued. Although FK409 decreased mBP (P <0.001), MML and leukocyte adhesion were significantly (P <0.001) reduced, and villus blood flow was maintained throughout the observation period. Conclusions. FK409 prevented mortality after intestinal I/R, significantly reduced leukocyte adhesion, and maintained blood flow after intestinal ischemia and may therefore be of value in reducing tissue damage and improving outcome after small bowel transplantation.

Mechanisms of Helicobacter pylori-Induced Rat Gastric Mucosal Microcirculatory Disturbances In Vivo

The exact mechanisms by which Helicobacter pylori infection results in gastric mucosal injury are unclear. However, it has been demonstrated that surface protein extracts of the bacterium can induce a number of disturbances within the rat gastric mucosal microcirculation, including platelet aggregation and macromolecular leakage (MML) of labeled albumin. This study aimed to determine the mechanisms involved in inducing these events using the technique of fluorescent in vivo microscopy. Male Wistar rats were pretreated with either ketotifen, a mast cell stabilizer (1 mg/kg), pyrilamine, an H1-receptor antagonist (30 mg/kg), hexanolamine-PAF, a PAF-receptor antagonist (10 μg/kg), l-arginine, the nitric oxide precursor (300 mg/kg) or vehicle, saline. Then 0.5 ml of H. pylori extract was administered to the exteriorized gastric mucosa of the anesthetized rat. Alterations in fluorescein-labeled albumin leak, vessel diameters, and acridine red-labeled leukocyte and platelet activity were determined over a 2-hr period. Saline pretreated animals demonstrated significant MML with a peak at 5 min (11%, P < 0.02). This was prevented with ketotifen and pyrilamine, but not with hexanolamine-PAF (17.5%, P < 0.05) and l-arginine (13%, P < 0.05). Significant numbers of platelet emboli and thrombi were observed within mucosal capillaries and postcapillary venules with vehicle pretreatment; this was prevented with hexanolamine-PAF and l-arginine, but not with ketotifen and pyrilamine. In conclusion, these studies demonstrate that more than one mediator is involved in inducing the rat gastric mucosal microcirculatory disturbances associated with H. pylori administration. Mast cells and histamine are linked to MML, with PAF, probably not derived from mast cells, involved in platelet activation.

Toxigenic Helicobacter pylori induces changes in the gastric mucosal microcirculation in rats

Background and aims: One of the key components of inflammation is changes in vascular structure and function. This suggests that the microcirculation may be a key target of Helicobacter pylori released factors. It has previously been shown in vivo that pooled H pylori extracts from duodenal ulcer/gastritis patients induce platelet aggregation but no leucocyte activation within rat gastric mucosal microcirculation (GMMC). However, infection with strains associated with ulcer disease as compared with gastritis may exert greater effects on the microcirculation. This study used fluorescent in vivo microscopy to determine the acute effects of extracts of genotypically different H pylori strains on the GMMC. Methods: Three H pylori extracts, with different cagA and VacA toxigenic status, were individually administered to the gastric mucosa of anaesthetised Wistar rats. The mucosal surface was visualised via an incision made in the exteriorised stomach. Fluoroscein isothiocyanate conjugated to bovine serum albumin (FITC-BSA) or acridine orange was used to quantify macromolecular leak (MML) and leucocyte/platelet activity respectively for 120 minutes. Changes in capillary and post-capillary venule (PCV) diameters were also monitored. Results: The cagA+ VacA toxigenic strain 60190 induced significant and sustained MML by five minutes (p<0.01). Transient and less leakage was observed with its isogenic VacA− mutant and other non-toxigenic strains regardless of cagA status. Significant increases in leucocyte adhesion (p<0.05), platelet aggregation (p<0.05), and PCV vasoconstriction (p<0.05) were only observed with the cag A+ and toxigenic strain. Conclusion: Extracts of H pylori are capable of inducing marked disturbances within the rat GMMC. These disturbances seem to be dependent on the production of an active vacuolating cytotoxin. Varying effects on the GMMC may explain the clinically diverse outcomes associated with genotypically different strains.

Helicobacter pylori Prevents Proliferative Stage of Angiogenesis In Vitro: Role of Cytokines

Inhibition of angiogenesis may explain the delayed ulcer healing following Helicobacter pylori infection. We have previosuly demonstrated that H. pylori can inhibit endothelial cell proliferation. Some cytokines possess antiangiogenic properties. This study assessed a role for IL-6, IL-8, and TNF-α in H. pylori-induced endothelial cytostasis. First, 30 μl of H. pylori was coincubated with microvascular endothelial cells in the presence or absence of monoclonal antibodies to IL-6, IL-8, and TNF-α for 24, 48, 72, or 96 hr. Dual labeling with propidium iodide and Hoescht 33342 distinguished between necrosis, and apoptosis and allowed viable cell numbers to be determined. H. pylori decreased cell viability after 72 and 96 hr (P < 0.02). Neither necrosis nor apoptosis was observed. Monoclonal antibodies to IL-6 and IL-8 did not reverse cytostasis. However, significant MVEC proliferation was observed in the presence of the TNF-α monoclonal antibody. In conclusion, H. pylori induces cytokine up-regulation as part of its pathophysiological mechanism, which could prove detrimental to ulcer healing through an inhibitory effect on angiogenesis.

Effects of Genotypically Different Strains of Helicobacter pylori on Human Microvascular Endothelial Cells In Vitro

Helicobacter pylori induces a number of disturbances in rodent gastric microcirculation in vivo. These events may result from direct necrotic or apoptotic damage to endothelial cells. This study therefore aimed to investigate the effects of genotypically different H. pylori strains on microvascular endothelial cell (MVEC) viability in vitro. Four H. pylori extracts were prepared from strains with different cagA or vacA status. MVECs were plated into 96-well plates and coincubated with 50 μl of extract or vehicle for 24, 48, 72, or 96 hr. An MTT assay quantified overall MVEC viability. The dual labeling of MVECs with propidium iodide and Hoechst 33342 distinguished between necrotic and apoptotic cell death, respectively, and allowed total number of viable cells to be determined. All strains of H. pylori decreased cell viability after 72 and 96 hr. Neither necrosis or apoptosis was observed. Counting total number of viable cells revealed decreased cell proliferation with all strains when compared to controls, again reaching significance at 72 and 96 hr. In conclusion, both the MTT assay and the direct cell counting technique demonstrated that all H. pylori strains induced cytostatic but not cytotoxic effects on MVECs. This suggests that microcirculatory disturbances observed in vivo may not be the result of direct endothelial cell damage. However, inhibition of angiogenesis may explain why ulcer healing is delayed in H. pylori-infected patients.