Jarmila Veselá

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.

Detection of early stages of apoptosis in experimental intestinal ischemia-reperfusion injury

Apoptosis is a form of programmed cell death that plays an important role in small intestine ischemia-reperfusion (IR) injury. The aim of this study was to determine the total proportion of apoptotic cell death (apoptotic index) following injury induced by ischemia and during various subsequent reperfusion periods, total histopathological status and the intestine regeneration dynamics after the IR injury. Experimental animals, Wistar rats (n = 45) were divided into three experimental and one control groups. In the experimental groups 1 h ischemia was followed by 1, 4 and 24 h reperfusion. Intestinal ischemia was induced by superior mesenteric artery (SMA) occlusion. Segments of jejunum were stained with hematoxylin and eosin and studied immunohistochemically using M30 CytoDEATH and in situ TUNEL methods for apoptosis detection. Our experimental data showed that: (i) apoptosis is an important form of cell death in the small intestine after IR injury induced by SMA occlusion; (ii) maximum levels of histopathological damage and apoptotic index of mucosa occurred after 1 h ischemia and 1 h of reperfusion; and (iii) mucosa possesses great regeneration ability. The lowest levels of histopathological damage and apoptotic index were observed in the group with 1 h ischemia and 24 h reperfusion where, however, the highest mitotic index was present.

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.

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).

The immediate response of jejunal mucosa to small bowel heterotopic allotransplatation in rats.

The course of histopathological alterations within jejunal graft architecture during the initial adaptation phase in the host body was investigated. Graft tissues were compared to the intestinal tissues of the recipients. This study demonstrates: (1) renewal of intestinal epithelial lining in the graft biopsies during initial hours after transplantation is more likely caused by migration and extension of remaining epithelial cells than by their increased mitotic division. (2) Distinct decrease in histopathological injury was observed in transplanted grafts after 6h, but the morphometrical parameters, particularly villus height and wall thickness, remained altered. (3) Significant decrease in apoptotic cell death in the epithelial lining within 6h of graft recirculation was accompanied by no effect on apoptosis levels of the cells in lamina propria connective tissue. (4) Although the apoptosis level in the connective tissue cells was not modulated in the grafts within the first hour after transplantation, caspase-3 dependent apoptosis was decreased significantly.

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.

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 changes in basement membrane associated with jejunal graft injury

Jejunal mucosa injury and morphometric continuity of the intestinal epithelial basement membrane (BM) in experimental groups after 1 h and 6 h of reperfusion following intestinal heterotopic allotransplation in male Wistar rats were observed. Intestinal graft tissue was compared to the jejunum of recipients. Significant damage to the jejunal mucosa was detected and followed by complete intestinal villi destruction, damage to epithelial intestinal crypts and the surrounding lamina propria mucosae. There was a local interruption of BM continuity, as well as its absence in particular areas. The percentage of continuous, clearly visible and undamaged BM detected using the periodic acid and Schiff reagent (PAS) method was 22.4% in the graft samples taken 1 h after transplantation. In the same samples, 43.1% undamaged BM was visualised by silver impregnation. Disappearance of the BM was significant in comparison with recipient samples using both staining methods (both p < 0.001) Biopsies of the intestinal mucosa graft 6 h after transplantation showed significant reduction of the damage to the jejunal mucosa (p < 0.05 and p < 0.01, respectively). The percentage of continuous, clearly visible and undamaged PAS-positive BM was 54%, and argyrophilic BM rose to approximately 83%. The difference between BM continuity detected by PAS method was significant when comparing graft and recipient samples (p < 0.001), but impregnation method did not revealed significant distinction. Based on our results, the silver impregnation method seems to be more sensitive to damage assessment of the jejunal graft.

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.

Detection of Intestinal Ischemia-Reperfusion Injury by Fluorescence Analysis of Intestinal Samples

ABSTRACT The aim of this study was to evaluate endogenous fluorescence of small intestine samples and compare them with the histopathological results. The autofluorescence of intestinal homogenates and histopathological injury index after 1h of mesenteric or portal ischemia, and 1 or 24 hours of reperfusion was analysed. Our results revealed two maximum peaks on the EEM. Increase of the intestinal histopathological injury index was accompanied with significant increase of fluorescence intensities in both zones. Close correlation between autofluorescence and histopathological results was found. Autofluorescence can be used was revealed suggesting use of autofluorescence as a new diagnostic method of intestinal injury.