Intestinal ischemia-reperfusion injury mediates expression of inflammatory cytokines in rats.

The small intestine is an organ with very well developed immunological activity, responsible for synthesis of specific inflammatory mediators that participate in causing the systemic inflammation that can occur after ischemia-reperfusion injury. The aim of our work was to study mRNA expression and protein concentration of inflammatory cytokines IL-10 and TNFα in the jejunal wall after intestinal ischemia-reperfusion injury (IRI). Cytokine concentration levels confirmed the direct effect of IRI on the inflammation process. The results refer to the changes in balance between pro-inflammatory and anti-inflammatory mediators and indicate that the predominant disturbance of homeostasis after intestinal IRI is present after 1 hour of reperfusion.

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.

Trehalase as a possible marker of intestinal ischemia--reperfusion injury.

BACKGROUND: Different pathological affections of the small intestine cause corresponding morphological and functional changes. The present study was aimed to assess intestinal trehalase activities during ischemia and following reperfusion, correlate them with the pathological changes and determine whether trehalase could be used as a biochemical marker of the intestinal ischemia, ischemia - reperfusion injury. MATERIAL AND METHODS: Wistar rats, randomly divided into 5 experimental groups (IR) (each n=15), were subjected to one hour mesenteric ischemia followed by 0, 1, 4, 12 and 24 hours of reperfusion. As a control group sham operated animals were used (n=15). The activity of trehalase was determined using an adapted Dahlqwist method. The range of intestinal injury was determined using histological (histopathological injury index and goblet cell quantification) and immunohistochemical (Ki67, InSitu TUNEL) methods. RESULTS: The highest activities of trehalase were recorded in the control group (C=4.42 ± 0.373 µmol/mg/h). The most altered intestinal histology detected in group IR1 was accompanied by the lowest trehalase activity (IR1=0.97 ± 0.209 µmol/mg/h; p < 0.001 C vs. IR1). Improved histological structure in the remaining reperfusion periods correlated with increase in trehalase activity. Almost normal mucosal histological architecture and 72% of the enzymatic activity were restored after 24 hours of reperfusion (IR24=3.20 ± 0.266 µmol/mg/h; p < 0.01 IR1 vs. IR24). CONCLUSION: The correlation between intestinal histology and trehalase activities during intestinal injury has been shown. Trehalase activity is closely associated with the status of the histological architecture of the small intestine.

Intravenous administration of tetramethylpyrazine reduces intestinal ischemia-reperfusion injury in rats.

Intestinal ischemia-reperfusion injury (IIRI) is a life-threatening condition requiring prompt medical intervention. Tetramethylpyrazine (TMP) is a biologically active alkaloid isolated from Ligusticum wallichii. Previously, it was shown that TMP causes vasodilatation and inhibition of platelet aggregation as well as exhibits significant antioxidant effects. Therefore, the aim of the present study was to evaluate possible therapeutic effects of TMP in the prevention of IIRI. Wistar rats (n = 80) were randomly divided into eight experimental groups and subjected to a 1 h occlusion of cranial mesenteric artery followed by 0, 1, 12, and 24 h period of reperfusion. Thirty minutes before the IIRI animals received either TMP (30 mg/kg, i.v.) or identical volume of saline. In addition, a control group of 10 animals was not exposed to IIRI. Intestine morphology was evaluated by using histopathological injury index examination (HII), goblet and Paneth cells quantification as well as by applying immunofluorescent methods such as InSitu TUNEL and caspase-3 positivity assessment. Here we showed that preconditioning with TMP prior IIRI decreases the grade of injury. Significant reduction of HII was detected in TMP pretreated groups after 0, 1, and 12 h of reperfusion where injury reduction up to 75% was found. Lower histopathological damage in preconditioned groups was accompanied with increased number of secretory epithelial cells and decreased number of apoptotic cells. These results demonstrate the protective effect of TMP on the small intestine mucosa, suggesting administration of TMP as a molecule for pharmacological intervention against IIRI.

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.

The relationship between morphology and disaccharidase activity in ischemia- reperfusion injured intestine.

BACKGROUND: Questions regarding functional markers characterizing injured intestines remain unanswered. Brush border disaccharidases are crucial for the functioning of the intestines. AIMS: The study was designed to assess changes in disaccharidase activity (DA) following intestinal injury and to compare them with morphological changes. METHODS: Wistar rats, randomly divided into six experimental groups (each n = 6), were subjected to different ischemic/reperfusion injury. One-hour mesenteric ischemia followed by reperfusion for 0, 1, 2, 4, 12 or 24 hours was induced. As a control group sham-operated animals were used (n = 6). Intestine morphology was evaluated using histopathological injury index (HII) and goblet cell (GC) detection. DA (sucrase and maltase) was studied in mucosal scrape or in entire intestinal wall samples. RESULTS: Moderate morphological damage (HII, GC) after mesenteric ischemia was detected. Deepening of the injury was found during reperfusion with a maximum after two hours. Improved morphology with longer reperfusion confirmed reversible damage with almost normal mucosal structure after 24 hours of reperfusion. Similar pattern was observed when DA was measured. The lowest activity was detected after 2 hours of reperfusion followed by increasing activity in the subsequent reperfusion periods. Physiological values after 24 hours of reperfusion were seen only in samples of entire intestinal wall. CONCLUSIONS: Significant changes in intestinal DA were observed after intestinal ischemia/reperfusion injury. A similar pattern was seen for morphological characteristics. Although based on microscopic survey the intestine seems to be fairly regenerated, some functional limitation is expected to persist.

Intestinal ischemia-reperfusion injury - the histopathological status of remote vital organs in acute and subacute phases.

BACKGROUND: Improvement of graft recovery and function follows current trends in intestinal transplantation; however, the alteration of remote organs (RO) predicts complicated systemic rejection. This study was conceived to describe the histopathological status of RO arising in both acute and subacute stages after intestinal ischemia-reperfusion injury (IIR) injury. MATERIAL/METHODS: Wistar rats (n=54) were divided into 7 experimental groups (n=7 each). All the animals were subjected to 60 min mesenteric ischemia and subsequently to reperfusion 2 h, 4 h, 24 h, 72 h, 10 days, 20 days and 30 days following the groups IR2 h, IR4 h, IR24 h, IR72 h, IR10 d, IR20 d and IR30 d. As a control group (S; n=5) sham-operated animals were used. Histopathological scores (HPS) were evaluated in biopsies of the right kidney, heart and colon ascendens. RESULTS: Statistically significant increase in kidney HPS was seen during reperfusion, with the peak in IR4h group (p<0.01). Thereafter, improved morphology was observed; however, increased HPS was seen even in the subacute stage, and significant deterioration of HPS up to 10 days of reperfusion was detected (p<0.05). Heart biopsies also showed statistically increased HPS value in IR4h group (p<0.05). Intact morphology of the colon was detected in all reperfusion periods. CONCLUSIONS: IIR causes a systemic reaction affecting RO. The peak of alteration for kidney and heart morphology was induced by 60 min of ischemia followed by 4 h of reperfusion. Thereafter, improved morphology was observed, although latent persistence of histopathological changes was seen even in the subacute stage. The colon remained intact during the whole experiment despite its anatomical proximity, confirming its high immunological capacity.

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).

Expression of adrenergic receptors in mouse preimplantation embryos and ovulated oocytes.

Epinephrine and norepinephrine can play an important role in basic developmental processes such as embryogenesis and morphogenesis, regulating cell proliferation, differentiation and migration. We showed that beta-adrenergic receptors can mediate the effects of catecholamines on preimplantation embryos in our previous work. In the present study, we designed specific oligonucleotide primers which can distinguish among all members of the alpha-adrenergic receptor family, and showed (using RT-PCR) that the alpha2C-adrenergic receptor is transcribed in ovulated oocytes, 8- to 16-cell morulae and expanded blastocysts. We did not detect the alpha2C-adrenoceptor transcript in 4-cell embryos. Our immunohistochemical study showed the presence of alpha-2C-adrenoceptor protein in ovulated oocytes, 8- to 16- cell embryos and blastocysts, but the signal in 4-cell embryos was weak, and probably represents remaining protein of maternal origin. We did not detect any other alpha-adrenergic receptor in preimplantation embryos and oocytes. Exposure of mouse preimplantation embryos to the alpha2-adrenergic agonist UK 14 304 led to significant reduction of the embryo cell number, and the effect was dose dependent. Our results suggest that epinephrine and norepinephrine could affect the embryo development in the oviduct via adrenergic receptors directly and support the opinion that maternal stress can influence the embryo even in very early pregnancy.

Expression of beta adrenergic receptors in mouse oocytes and preimplantation embryos.

Accumulating evidence indicates the role of endogenous catecholamines in mammalian embryogenesis. We searched public databases containing nucleotide sequences derived from mouse preimplantation cDNA libraries and found a partial sequence homology between a cDNA clone from mouse blastocysts and the mouse beta 2-adrenergic receptor sequence. No significant sequence homology was found for other mouse adrenergic and dopamine receptors. Using RT-PCR, we showed that beta 2-adrenoceptor is transcribed not only at blastocyst stage but also at earlier stages of preimplantation development as well as in oocytes. Moreover, we demonstrated that transcripts encoding both isoforms of the beta 3-adrenoceptor (beta 3a- and beta 3b-) are expressed in mouse oocytes and preimplantation embryos as well. We did not detect the beta 1-adrenoceptor transcript either in oocytes or in preimplantation embryos. Using an antibody against the mouse beta 2-adrenergic receptor, we showed that the receptor protein is expressed in oocytes and preimplantation embryos; in blastocysts, the immufluorescence labeling was stronger in the inner cell mass than in throphectodermal cells. The cell number of the in vitro cultured mouse preimplantation embryos exposed to isoproterenol (a potent beta adrenoceptor agonist) was lower than in control embryos, suggesting that activation of beta adrenergic receptors by appropriate agonist concentration can influence cell proliferation in mouse pre-implantation embryos. Thus, our results indicate that beta adrenergic receptors are expressed in mouse oocytes and preimplantation embryos and that ligands for the receptors can affect the mouse embryo even in the very early stages of development.

Serotonin localization and its functional significance during mouse preimplantation embryo development.

Serotonin is a neurotransmitter functioning also as a hormone and growth factor. To further investigate the biological role of serotonin during embryo development, we analysed serotonin localization as well as the expression of specific serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos. The functional significance of serotonin during the preimplantation period was examined by studying the effects of serotonin on mouse embryo development. Embryo exposure to serotonin (1 microM) highly significantly reduced the mean cell number, whereas lower concentrations of serotonin (0.1 microM and 0.01 microM) had no significant effects on embryo cell numbers. In all serotonin-treated groups a significant increase in the number of embryos with apoptotic and secondary necrotic nuclei was observed. Expression of serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos was confirmed by in situ hybridization showing a clearly distinct punctate signal. Immunocytochemistry results revealed the localization of serotonin in oocytes and embryos to the blastocyst stage as diffuse punctate cytoplasmic labelling. It appears that endogenous and/or exogenous serotonin in preimplantation embryos could be involved in complex autocrine/paracrine regulations of embryo development and embryo-maternal interactions.