Mast cells in peritoneal fluid in rats with experimentally induced peritoneal adhesions.

Mast cells (MC) produce, store and release many biologically active substances, especially inflammatory factors, chemotactic substances for neutrophiles, cytokines and prostaglandins. They play very important role in fibrinosis and they are an important factor in peritoneal adhesions formation and lysis. In this study we tried to evaluate role of mast cells in peritoneal adhesions formation. We estimated number of mast cells in peritoneal fluid in rats with experimentally developed peritoneal adhesions. The number of mast cells per ml was counted in flow cytometry in specimens of peritoneal fluid taken from operated rats. The fluid was taken in standardized conditions the same for each group at the first operation and during reoperation. Peritoneal cavity was washed with 0.9% Saline solution. MC were visualized using indirect immunohistochemical method LSAB with mouse antibody. The animals were divided into 4 groups. 1 st group was control group (n=20) on which the abdomen was opened and closed without any manipulations, and the reoperation was done after 72 hours. The other groups (2, 3, 4; n=20 for each group) were operated and scarification of the partial peritoneum and serosa was performed. The rats were brought back to conscious and then were reoperated respectively after 24, 72 and 168 hours after first surgery. After the laparotomy and damage of the peritoneum we observed formation of the peritoneal adhesions between intestine loops and between intestines and damaged parietal peritoneum. Also the higher number of mast cells was observed in the groups of animals with damaged peritoneum. The highest number of peritoneal adhesions was observed in the group of animals reoperated after 72 hours. After 72 and 168 hours the higher number of MC and neutrophils was observed. The difference was statistically significant. The percentage of mast cells was increasing during the experiment duration. It was different from other cells populations which decreased after 168 hours. The MC and neutrophils were cell population which changed significantly after manipulations in peritoneal cavity. It is very probable that they play an important role in peritoneal adhesions formation.

Use of synaptophysin immunohistochemistry in intestinal motility disorders.

The purpose of this study was to describe synaptophysin (SY) immunoreactivity in colonic specimens from patients with Hirschsprung's disease (HD), chronic constipation (CC), or anal atresia (AA). This membrane protein is specific for the synaptic vesicles in the central and peripheral nervous system and responsible for neurotransmission. Biopsy specimens of the intestinal wall were obtained from 18 patients (age range, 2 days to 7 years). Immunohistochemistry was performed using rabbit anti-human antibodies specific for synaptophysin (DAKO). In the ganglionic colon of HD patients and others the immunoreactivity of SY-positive synapses was abundantly present in the smooth muscle layers. Distinct immunoreactivity showed ganglion cells and nerve fibers inside circular and longitudinal muscle layers. In some non-HD patients' colonic specimens SY-positive synapses were present in the muscularis mucosae. In the aganglionic colonic segment of HD-patients no immunoreactivity of synapses and ganglions was seen. In the transition zone, where ganglion cells appeared sporadically, synapses were very rarely present. In two patients from the CC group the amount of visualized synapses was clearly smaller and the concentration of ganglion cells within ganglions in these cases was much lower than usual (but still within normal ranges). In the AA group in the distal part of the atretic rectum (at the place where the fistula was cut) SY-positive synapses were present in smooth muscle layers and small dysplastic ganglions were seen in the submucosal and muscular region, but not in large numbers. These patients had a normal distribution of ganglion cells and synapses at the place of colostomy. Synaptophysin immunohistochemistry is an indirect labeling method with a high detection rate for intestinal ganglion cells by demonstrating their synapses. Changed intestinal distributions of SY-positive synaptic vesicles usually accompany colonic ganglion cell disorders. The pattern of SY-positive synapses distribution in circular and longitudinal colonic muscles and intermuscular ganglions can reflect functional disturbances of large bowel motility and could be helpful in the description of the innervation status of colonic specimens in HD patients.

Oxyuriasis-induced intestinal obstruction in a child--case report.

PURPOSE: The presentation of an unusual case of the tumor of ileum wall induced by pinworm infection in a 5-years-old child. MATERIAL AND METHODS: The record of a 4-years-old boy treated in the department of pediatric surgery was analyzed concerning the diagnostic difficulties. After 6 month from an episode of ileo-cecal intusussception successfully treated with a barium colon enema, the diagnosis of lymphoma was made and the resection of distant segment of small intestine was performed. RESULTS: No clinical and laboratory features of oxyuriasis could be stated before the onset of disease, during hospitalization and in the follow-up period. The hypertrophied and activated lymphatic tissue with a non-specific inflammatory reaction to the pinworms were seen in the wall of ileum, appendix and mesenteric lymph nodes. No neoplastic cells were found in the microscopic study of ileum, appendix, mesenteric lymph nodes and peritoneal lavage fluid. CONCLUSIONS: The proper diagnosis of oxyuriasis may be difficult when the course is atypical. The enterobius vermicularis infestation as an etiologic factor should be taken into account in any case of abdominal pathology. However, the methods routinely used in "acute abdomen" including examinations of blood, urine and stool, repeated ultrasound and CT, are not reliable. As the infestation may mimic neoplasm, the surgical treatment and microscopic examination can be necessary for the final diagnosis in some cases.

Suitability of selected markers for identification of elements of the Intestinal Nervous System (INS).

The possibility of identifying and characterising elements of the enteric nervous system based on their contents of cathepsin D, chromogranin A, neuronal specific enolase and S-100 protein was studied in colorectal specimens (operative full-thickness, seromuscular and mucosomuscular biopsies) obtained from 15 children, aged 2 days to 10 years. Nine patients suffered from Hirschsprung's disease, and two from chronic constipation. Four neonates with imperforate anus or meconium ileus composed the control group. All markers were identified immunohistochemically by antibodies against human antigens with appropriate detection methods. Chromogranin A staining was not always adequate to identify all neuronal cell bodies and other nervous elements. However, it proved superior to the other methods in the depiction of neuroendocrine cells in the intestinal mucosa. Cathepsin D antibodies stained normal and abnormal neural cells with different intensity; nerve fibres were not stained. This marker did not allow an unequivocal differentiation of ganglion cells from macrophages within the submucosa; the latter exhibited exceptionally strong marking and in some cases represented the predominant elements in this area. Neuronal specific enolase was distinctly expressed in nerve cells and fibres of the intestinal wall. Atrophic and hypoplastic features could be identified, suggesting that this method may give some insight into functional aspects. Continuous connections between ganglions were also observed. S-100 protein antibodies resulted in a negative image of unstained ganglion cells surrounded by extensively marked Schwann cells and neural fibres. With respect to clinical application, all these markers may provide supplementary information for the differential diagnosis of intestinal motility disorders.

Hemorrhagic shock-induced changes in the cathepsin D activity in the intestinal wall and blood serum in rats.

Hemorrhagic shock causes release of lysosomal proteolytic enzymes which contribute to intestinal wall destruction and can be moved into the circulation as well as into the gut lumen. The aim of the study was to examine the activity of cathepsin D in relation to the intestinal wall injury after 60 minutes of untreated hemorrhagic shock in rats. The total cathepsin D activity was investigated in duodenum, jejunum, ileum, cecum and colon, as well as in systemic and mesenteric blood serum, and the biochemical results were compared with morphological changes in the respective segments including immunohistochemical expression of cathepsin D. We found an increase in cathepsin D activity in duodenum and significant decrease in other parts of the gut in shocked rats. The enzyme activity increased also in blood serum, especially systemic (p < 0.05) and insignificantly in mesenteric blood. However, sham-operated animals (one-side carotid artery occlusion) revealed a significant increase in cathepsin D activity measured in mesenteric blood. The shock resulted in lowering protein concentration in the intestinal wall and its increase in mesenteric blood. The contents of peptides and amino nitrogen, as potential proteolytic reaction products, changed in different ways in various segments of intestine. Morphologically, the most intensive destruction was observed in ileum, duodenum and jejunum. Lifting of epithelial layer from lamina propria was the most frequently observed lesion of the intestinal wall after 60 minutes of shock. More advanced lesions, such as denuded mucosa with disintegration of lamina propria, occurred rarely and were not observed in colon and rectum. By means of polyclonal antibodies against cathepsin D, we found that the strong expression of this enzyme was in epithelial layer--the part of intestinal wall which was partially detached into gut lumen due to hemorrhagic shock. The changes of cathepsin D activity after 60 minutes of hemorrhagic shock were correlated with signs of morphological damage to the intestinal wall. Cathepsin D liberation in the intestinal wall during shock indicates the lysosomal membranes impairment and can confirm involvement of proteases in the damage to the intestinal tissue. We conclude that liberation of intestinal cathepsin D is an early phenomenon during hemorrhagic shock which may contribute to the local wall disintegration and activation of systemic inflammatory response.

Cathepsin D activity in the intestinal wall in experimental untreated hemorrhagic shock.

The total cathepsin D activities in the intestinal wall and in venous mesenteric and arterial systemic blood were investigated on the rats in untreated hemorrhagic shock lasting 60 minutes. We observed a decrease in cathepsin D activity in homogenates of respective segments of small and large bowels and an increase in the enzyme activity in blood serum of both origin. The shock resulted in lowering protein concentration in the intestinal wall and its increase in the mesenteric blood. We found a negative correlation between cathepsin D activity in the intestinal wall and its morphological destruction. Molecules of the enzyme, after liberation from lysosomes due to hemorrhagic shock, are translocated to the circulation and probably to the gut lumen. Liberation of the intestinal cathepsin D may contribute to the local damage and multiorgan failure in hemorrhagic shock.