The effect of Betanin parenteral pretreatment on Jejunal and pulmonary tissue histological architecture and inflammatory response after Jejunal ischemia-reperfusion injury.

Intestinal ischemic-reperfusion (IR) injury has detrimental effects on both local and distant organs in the body. Betanin is known for its antioxidant properties, and it is found mostly in vegetables. Therefore, the aim of the present study was to test the hypothesis that betanin administration prior intestinal IR, may be beneficial in protecting jejunal mucosa and lung parenchyma against IR damage. Male specific pathogen-free Charles River Wistar rats were used (n = 42). Betanin (50 mg/kg) was administered intraperitoneally 30 min before ischemia of the superior mesenteric artery lasting 1 h, followed by 1, 4 and 24 h of reperfusion. Immunohistochemical as well as histomorphometrical analysis indicated a protective effect of betanin pretreatment on jejunal tissue. Regarding morphometrical analysis betanin significantly (p < 0.01) augments intestinal villus height after 24 of reperfusion comparing to early stages. Betanin application reduced number of mast cells population in early reperfusion periods (p < 0.05). The protective effect of betanin on lung parenchyma, was detected in late reperfusion period (24 h) with improvement of histopathological injury index and morphometric analysis (p < 0.001 for both). The improvement of histopathological injury index (p < 0.001) and morphometric analysis (p < 0.001) during the late reperfusion period, suggests a protective effect of betanin on lung parenchyma. Moreover, suppression of the inflammatory response was mirrored by the reduction of myeloperoxidase (MPO) positive cells within lung parenchyma after 1 and 4 h of reperfusion (p < 0.001). Especially, during the first 4 h of reperfusion after betanin administration, a reduction of 74% of the polymorphonuclear neutrophils infiltration (MPO positive cell population) and of a nearly 46% of active MCs was observed. Upon morphometric examination, the lung histological architecture after 24 h of reperfusion appeared to be almost 100% better following betanin treatment, with 25% thinner interalveolar septa and 20% larger alveolar surface for respiratory gas exchange. The results suggest that betanin pretreatment protects the jejunal mucosa and the lung parenchyma, as well as reduces the inflammatory cell density after intestinal IR injury.

Quercetin protects jejunal mucosa from experimental intestinal ischemia reperfusion injury by activation of CD68 positive cells.

The aim of our study was to analyse the possible protective effect of quercetin application during the jejunal ischemia-reperfusion injury (IRI) in rats. Quercetin was administered intraperitoneally 30min before 1h ischemia of superior mesenteric artery with following 24h lasting reperfusion period. The male specific pathogen-free (SPF) Charles River Wistar rats were used. In the group with applied quercetin, the significantly increased (p<0.001) levels of anti-inflammatory cytokine IL10 were observed both in the blood serum and jejunal tissue. The improvement of the mucosal tissue morphology and proliferating and DNA repairing cell number measured by PCNA activity were recorded by more than 30% higher in the quercetin group. Simultaneously, significant elongation of the intestinal glands (p<0.001) and increase in the number of CD68-positive cells in the lamina propria mucosae (p<0.001) in comparison with control group were found. Based on our results, the preventive application of quercetin before induction of jejunal IRI stimulates faster jejunal mucosa restoration and it seems to have immunomodulatory and anti-inflammatory effects as well. CD68-positive macrophages could have crucial role in this process since they work as both growth factor and cytokine producers.

Quercetin attenuates the ischemia reperfusion induced COX-2 and MPO expression in the small intestine mucosa.

Quercetin, the active substance of tea, fruits and vegetables, exerts a broad spectrum of pharmacological activities and is considered to have potential therapeutic application. The present study was designed to investigate the beneficial effect of quercetin against experimental ischemia- reperfusion (IR) injury of the small intestine in rats. Quercetin was administrated intraperitoneally 30min before 1h ischemia of superior mesenteric artery with following reperfusion periods lasting 1, 4 and 24h. The male specific pathogen-free Charles River Wistar rats were used (n=45). In acute phase, 4h after start of reperfusion, the quercetin induced a significant decrease in mucosal injury index (p<0.05) accompanied by a significant decrease in cyclooxygenase-2 (COX-2) expression in the epithelial lining of the intestinal villi in comparison with the control group (p<0.01). In the epithelium of the intestinal glands, COX-2 expression resulting from IR injury significantly increased regardless quercetin application (in control group p<0.001; in quercetin group p<0.05), but in quercetin group, significant decrease in it during 24h of reperfusion in a late phase of IR injury was detected (p<0.001). Based on morphology of COX-2 positive cells, the COX-2 positivity was found particularly in goblet cells of the intestinal villi epithelium and enteroendocrine cells respectively, in the glandular epithelium. We concluded that quercetin application attenuated mucosal damage from IR injury by inhibiting neutrophil infiltration which was demonstrated by a lower number of myeloperoxidase positive cells in the lamina propria during both phases of IR injury and the significant decrease in that in a late phase after 24h of reperfusion (p<0.05).

Protective effect of ischemic preconditioning on the jejunal graft mucosa injury during cold preservation.

Protection of intestinal graft mucosa during cold preservation is still an unmet need in clinical practice, thus affecting the success of transplantation. The present study investigates the ability of two ischemic preconditioning (IPC) procedures to limit cold preservation injury. Three groups of Sprague-Dawley rats were recruited (n=11 each) as follows: the short IPC (SIPC) performed through 4 cycles of mesenteric ischemia of 4 min each followed by 10 min of reperfusion, the long IPC (LIPC) obtained by 2 ischemic cycles of 12 min each followed by 10 min of reperfusion, and the control group (C) without IPC. Grafts were then stored in cold histidine-tryptophan-ketoglutarate solution and samples were taken at 0, 3, 6 and 9 h lasting preservation. Both IPC groups showed an advanced degree of preservation with delayed development of graft mucosa damage, mainly in the crypt region. At the beginning of preservation, the graft mucosa in both IPC groups showed lower degree of mucosal injury index (MII) by 50% in comparison with C group. Specifically, a significant improvement of MII was observed after 3h of preservation in the LIPC group (p<0.05) in comparison with untreated C grafts. Significant atrophy of the intestinal mucosa in C group was found after 3h of preservation (p<0.01), in SIPC group the progress of atrophy was delayed to 6 h (p<0.001), and in LIPC group only moderate decrease in that was found. A parallel increase of laminin expression with the MII rate after 6 and 9h of preservation in comparison with the level at time 0 was observed in all grafts (p<0.001 and p<0.01, respectively). In both IPC groups the apoptotic cell (AC) rate was significantly reduced at the beginning of cold preservation (p<0.05 both). Moreover, in both the SIPC and C groups, the progressive increase in MII rate connected with AC rate decrease was due to a predominance of necrosis. By contrast in the LIPC group, after an increase of nearly 50% in the AC rate at the 3rd hour, its level remained fairly constant during the further 6 h of preservation, thus probably preventing necrosis and improving graft viability.

Influence of dietary supplementation with flaxseed and lactobacilli on the mucosal morphology and proliferative cell rate in the jejunal mucosa of piglets after weaning.

The aim of the study was to investigate the influence of flaxseed and lactobacilli supplementation to the diet of piglets during the time period between 10 days before and 21 days after weaning. The morphometry of the jejunal mucosa and proliferative ratio of both epithelial and lamina propria cells were compared with those found in a group of piglets fed with the usual diet added with sunflower oil during the same time period. The addition of flaxseed oil to the diet significantly increased the crypt depth in comparison with both groups supplemented with sunflower (P < 0.05 and 0.001 respectively) on the weaning day. Moreover, the flaxseed addition caused a significant decrease in villus height (P < 0.01) and crypt depth (P < 0.01) 21 days postweaning in comparison with the sunflower group. The proliferative ratio of the epithelial cells in the sunflower group on the weaning day was significantly higher than in both flaxseed groups (P < 0.01). Paradoxically, significantly higher proliferative activity in the mucosal connective tissue in the group with flaxseed supplementation in comparison with the sunflower group was observed on the day of weaning, as well as 3 days later (P < 0.05 both). A combination of flaxseed with lactobacilli showed significantly lower proliferative activity in the connective tissue cells from weaning up to 7 days after weaning (P < 0.05 all) in comparison with the flaxseed group.

Influence of dietary supplementation with flaxseed and lactobacilli on the cells of local innate immunity response in the jejunal mucosa in piglets after weaning.

A histological study was designed to determine the influence of flaxseed and/or lactobacilli inclusion in the diet of piglets from 10 days before to 21 days after weaning. The selected inflammatory cell population incidence in the piglet jejunal mucosa was investigated. Significantly higher numbers of myeloperoxidase-positive (P<0.01) and CD163-positive (P<0.001) cells in the jejunal mucosa were recorded on the weaning day and for 7 days after (P<0.001 and P<0.01, respectively) in the flaxseed group compared with the basal diet. The number of intraepithelial lymphocytes was also significantly increased until 3 days after weaning (P<0.001). A prolonged significant increase in the myeloperoxidase-positive cells and intraepithelial lymphocyte numbers in the flaxseed+lactobacilli group was detected. In contrast, the number of CD163-positive cells in the flaxseed+lactobacilli group was significantly lower on the day of weaning (P<0.05) and 3 days after (P<0.01). The same effect was observed in the group with lactobacilli alone during the first 3 days after weaning (P<0.05 and P<0.01, respectively) and these findings indicate down-regulation of CD163 expression in the jejunal mucosa by lactobacilli. The presence of lactobacilli in the diet had a stimulatory effect on goblet cell quantity in the epithelium (P<0.001) and a distinct 50% reduction in the flaxseed group (P<0.01) compared with the basal diet was observed on the weaning day. A significant increase in myeloperoxidase-positive cell number in the jejunal mucosa in the flaxseed+lactobacilli group was the only significant difference (P<0.05 and P<0.01, respectively) found 21 days after weaning in comparison with all the other groups, indicating the pro-inflammatory effect of this feed additive combination. We conclude that dietary supplementation with flaxseed and lactobacilli on the cells of local innate immunity response in the jejunal mucosa in piglets after weaning might be linked with significant anti-inflammatory effects in the jejunal mucosa.

Immunohistochemical expression of MPO, CD163 and VEGF in inflammatory cells in acute respiratory distress syndrome: a case report.

Acute respiratory distress syndrome (ARDS) is a serious medical condition occurring in patients with polytrauma, pulmonary or non-pulmonary sepsis, pneumonia and many other circumstances. It causes inflammation of the lung parenchyma leading to impaired gas exchange with a systemic release of inflammatory mediators, causing consequential lung tissue injury, hypoxemia and frequently multiple organ failure. The aim of current study was to describe expression of inflammatory markers (myeloperoxidase, CD163 and vascular endothelial growth factor) by the cells in acute phase of ARDS. The lung samples of a 20-year-old man who had suffered a serious motorbike accident were obtained for histological examination. He died on the seventh day as a consequence of respiratory failure. Our results imply that expression of CD163 was restricted to activated alveolar macrophages and monocytes. Immunopositivityof MPO was observed in neutrophil granulocytes within lung alveoli and lung blood vessels. Myeloperoxidase positivity was observed in alveolar macrophages, too. Vascular endothelial growth factor was expressed in cytoplasm of neutrophil granulocytes, monocytes, small-sized alveolar macrophages and type II pneumocytes localized mostly inside lung alveoli. On the contrary, no positivity was observed in lung endothelial cells of blood vessels.

Impact of alanyl-glutamine dipeptide on proliferative and inflammatory changes in jejunal mucosa after acute mesenteric ischemia.

PURPOSE: The aim of our study was to determinate the impact of dipeptide (alanyl-glutamine) administration on inflammatory and proliferative changes in jejunal mucosa after acute mesenteric ischemia. METHODS: Male Wistar rats (n=30) were divided into three groups: ischemia/reperfusion (IR) group which undergoes 60min of mesenteric ischemia and 1 or 24h of reperfusion (IR1, IR24, n=12). Groups with dipeptide administration (D+IR1, D+IR24, Dipeptiven con inf., i.v., 0.75 g/kg) prior to IR injury were followed by 1 and 24h of reperfusion. At the end of reperfusion period jejunal bioptic samples were obtained for histological (H&E), histochemical (Alcian blue) and immunohistochemical (anti-PCNA, anti-MPO) evaluations. RESULTS: Our results pointed out a significant (p<0.001) increase of histopathological injury score in IR1 group compared to D+IR1 group. Immunohistochemical evaluation showed that MPO-positivity was significantly increased in IR groups after 1 (p<0.001) as well as 24h of reperfusion (p<0.01) compared to dipeptide pretreated groups. Proliferative/reparatory rate was assessed using anti-PCNA antibody and showed a significant increase (p<0.01) in PCNA cell positivity in lamina propria in dipeptide treated group compared to IR group. CONCLUSION: In conclusion we may suggest that administration of alanyl-glutamine dipeptide prior to IR injury may help to protect small intestine and its mucous membrane integrity against insult such as intestinal ischemic/reperfusion injury presents.

Immunohistochemical study of jejunal graft mucosa cell populations during the initial adaptation phase in the host body in rats.

The character of the changes in cell populations within the jejunal graft mucosa during the initial adaptation phase in the host body was investigated. 24 adult male Wistar rats underwent intestinal heterotopic allotransplantation. Aorto-aortal and porto-caval anastomoses were performed using the end-to-side microsurgery technique. Graft tissues were compared to the intestinal tissues of the recipients. This study demonstrates that: (1) Distinct injury to the graft mucosa 1h after transplantation was accompanied by significant reduction in numbers of epithelial secretory cell populations. The injury was more intense in the mesenteric portion. Six hours after transplantation the graft mucosa was covered by a continuous epithelium, but the number of goblet and Paneth cells was found to be less than 30% of that in the recipient epithelium. (2) In comparison with recipients, myeloperoxidase-positive cell numbers increased significantly in the graft mucosa 1 h after transplantation. In the epithelial layer, denudation and destruction of villi was associated with a significant reduction in intraepithelial lymphocyte numbers. A significant decrease in mucosal mast cell numbers was detected 6 h after transplantation. They attained only 10% of the number found in the recipients. (3) Time-dependent changes in the graft mucosa revealed that CD163-positive cells increased significantly in the graft mucosa during 6 h after transplantation and reached the level found in the recipients. In contrast, the myeloperoxidase-positive cell population significantly decreased in the graft mucosa within the initial 6 h.

Mesenteric ischemia-reperfusion injury: specific impact on different cell populations within the jejunal wall in rats.

The progress of jejunal damage and recovery in the course of mesenteric ischemia-reperfusion injury in rats at different time periods was investigated. Mesenteric ischemia lasting 1h followed by 1h of reperfusion caused a significant disintegration of the mucosa, reduction of the muscular layer and diminution of the wall thickness. The loss of epithelium included enterocytes, goblet cells and Paneth cells. Paradoxically, increasing numbers of serotonin-producing cells and the beginning of regenerative processes, expressed by significantly higher proliferation, were recorded in the epithelium during this period. Disintegration of connective tissue and massive degranulation of serotonin-positive cells were found in the lamina propria. After 24h of reperfusion, restitution of the mucosa was found, expressed by normal villous morphology and re-epithelialization. However, some parameters were still significantly affected even more than in the acute phase of reperfusion. In the epithelium, decreased numbers of Paneth cells and increased population of serotonin-producing cells were found. The greatest proliferation of connective tissue cells and intensified reduction of the muscular layer were also detected in this reperfusion period. After 30 days of reperfusion, moderate damage remained, but only the increased number of Paneth cells and decreased number of serotonin-producing cells in the lamina propria were significant.

Different ischemic preconditioning regimens affecting preservation injury of intestines.

Decreasing ischemia-reperfusion injury in intestinal transplantation is of paramount importance for improving graft recovery and function. This study explores the ability of two ischemic preconditioning (IPC) regimens to reduce preservation injury. Sprague-Dawley rats were divided into 3 groups (n = 11 each). In the controls (group C), intestinal grafts were harvested and preserved. IPC was performed either through 4 cycles of mesenteric ischemia of 4 min each followed by 10 min of reperfusion (group BIPC) or 2 ischemic cycles of 12 min each followed by 10 min of reperfusion (group LIPC). Grafts were stored in histidine-tryptophan-ketoglutarate, and samples were taken 0, 3, 6, 9, 12, 18, and 24 h after preservation. Preservation injury was scored using the Park/Chiu scale. Goblet cells (GC), enteroendocrine cells (EEC) and serotonin-producing EEC (SPEEC) were studied for evaluation of the graft conditions. Group C had the most advanced preservation injury followed by group BIPC. GC count was lowest in group C, followed by BIPC. Comparison between groups BIPC and LIPC showed superior parameters (preservation injury, GC, EEC, and SPEEC) in LIPC. In conclusion, an IPC regimen of 2 ischemic cycles of 12 min each followed by 10 min of reperfusion distinctly decreased the preservation injury of intestinal grafts compared with non-manipulated grafts.

Morphological and apoptotic changes in the intestinal mucosa and lung parenchyma after ischaemic/reperfusion injury of the jejunum.

Ischaemic/reperfusion (IR) injury of the small intestine may lead to the development of multiple organ failure. Little is known about the morphological changes occurring in the organs during the subacute course of this syndrome. The objective of this study was to observe histopathological features and the role of apoptosis in the jejunal mucosa and lung parenchyma after intestinal IR injury in a long-term experiment. Wistar rats (n = 36) were divided into 4 experimental groups (IR(10), IR(20), IR(30), S). Groups IR(10), IR(20) and IR(30) (each n = 10) were subjected to 1-hour ischaemia of the cranial mesenteric artery followed by 10, 20 or 30 days of reperfusion, respectively. The control group S (n = 6) was not subjected to ischaemia. The jejunal mucosa remained intact after all periods of reperfusion. Apoptotic cells were found particularly in the lamina propria, with the most significant difference observed in the IR(30) group (P < 0.01). The lung parenchyma had lower regenerative capacity, which was confirmed by a high index of histological damage after 30 days of reperfusion (P < 0.01) and by the presence of an increased number of apoptotic cells, especially in the pulmonary interstitium. The number of apoptotic cells was ten times higher than in the control group (P < 0.001).

Enterococcus faecium EK13--an enterocin a-producing strain with probiotic character and its effect in piglets.

The experiment was conducted to determine the effects of the inoculation of the probiotic and enterocin A-producing strain Enterococcus faecium EK13 on selected parameters of metabolic profile, gut microflora, growth, and health in newborn piglets of Slovak White Improved. Piglets for study were divided into two groups: one group (EK13 group, n=8) received strain EK13 per os once daily for 7 days (2ml per piglet, 10(9)CFU/mL of saline buffer). The control group of piglets (n=7) was given placebo-saline buffer. The experiment lasted 14 days. After 7 days, strain EK13 reached 9.8 log(10) CFU/g in faeces of E. faecium EK13 treated piglets while counts of Escherichia coli were significantly lower (P<0.01) than in piglets of the control group. The concentrations of total serum protein, calcium, haemoglobin, haematocrit, red blood cell count and index of phagocytic activity of leukocytes were significantly higher after application of strain EK13. On the other hand, cholesterol was significantly lower in the EK13 group of animals. On day 14, piglets were killed and samples of intestinal contents were taken. Total counts of bacteria in the intestinal contents (jejunum, ileum, caecum, colon) were not significantly influenced. The pH value was significantly lower (P<0.05) only in duodenum of piglets receiving E. faecium EK13. There was a significant higher concentration of lactic acid (P<0.01) and propionic acid in the colon (P<0.001) of the EK13 group. Application of E. faecium EK13 did not influence the daily body weight gain significantly.

Testing two Lactobacillus plantarum and Lactobacillus acidophilus strains for their suitability as a lipoid probiotic.

Two strains of lactobacilli (Lactobacillus acidophilus T-135 and Lactobacillus plantarum 4/97) were selected in order to study their inhibitory properties against frequent udder pathogens (Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Salmonella enteritidis and Bacillus pumilus), their production of organic acids as well as their ability to survive on the teat skin, the teat duct mucosa and in a lipoid emulsion. Both strains inhibited the tested pathogenic microbes and survived on the investigated surfaces and in an emulsion for more than 6 hours and 11 days, respectively.

The influence of omega-3 polyunsaturated fatty acids (omega-3 pufa) on lactobacilli adhesion to the intestinal mucosa and on immunity in gnotobiotic piglets.

The effect of application of omega-3 polyunsaturated fatty acids (omega-3 PUFA) on intestinal colonization by Lactobacillus paracasei and on cellular immunity has been investigated in gnotobiotic pigs. The administration of polyunsaturated fatty acids positively affected the adhesion of Lactobacillus paracasei to the jejunal mucosa of gnotobiotic piglets. When compared to the control group, the number of Lactobacillus paracasei adhering to the jejunal mucosa was by 12% higher in piglets of the experimental group (5.10 log 10/cm2 vs. 4.55 log 10/cm2). The respective counts of Lactobacillus paracasei adhering to the ileal and colonic mucosa of 28 day old gnotobiotic piglets reached 4.45 and 5.05 log 10/cm2 in group C and 4.44 and 4.95 log 10/cm2 in group E. Omega-3 PUFA supplementation increased the phagocytic activity of neutrophils by almost 100% on day 28 of life as well as the subpopulations of lymphocytes (CD8) in the peripheral blood of germ-free piglets on day 21 of life. Our results indicate that the action of probiotics in the gut may be modulated by dietary PUFA. The stimulatory effect of PUFA upon adhesion of lactobacilli could be used for enhancing the effectiveness of probiotics in inhibiting digestive tract pathogens.