Rats were maintained in standard con ditions, and received rodent laboratory chow and water ad libitum. All experimental animals were treated in accordance with all the guidelines of the ethics commission in the Polish Academy of Science. CC531 cancer cells CC531 is really a moderately differentiated and weakly im munogenic adenocarcinoma of the colon which is in duced by 1,2 dimetylhydrazine and is syngenic to WAG Rij rats. CC531 cells had been cultured in RPMI medium supplemented with 10% FCS, penicillin, streptomycin, fungizon and ceftriaxone. The cultures were maintained in plastic tissue culture flasks, and incubated in 5% CO2 at 37 C within a humidified incubator. Tumor cells have been harvested from sub confluent cultures by brief exposure to trypsin diluted 1,10 in PBS with no Ca2 or Mg2.
Cells had been suspended in PBS without Ca2 or Mg2 supplemented with 10% FCS, and centrifuged at 400 g for 10 minutes, then resus pended in serum cost-free PBS with out Ca2 or Mg2, and centrifuged as prior to. Cell viability was determined by the Trypan blue exclusion method, and was always higher than 90%. Inoculation of CC531 cells Rats have been anesthetized with ether. A mid line incision two cm lengthy was selleckchem created within the abdominal wall. A suspension of two ? 106 CC531 cells in 0. 5 ml 0. 9% NaCl was ready for every animal, and injected into the portal vein. Abdominal wounds were sutured. Liver and periton eal metastatic sort tumors developed four to six weeks immediately after CC531 inoculation. Out of your 20 rats, four did not develop any metastasis, 2 had person tumor foci inside the liver, and two had advanced cancer with peritonitis.
These 8 out of 20 rats that didn’t match the study criteria have been excluded in the study and euthanized by decapitation. For the study, we applied a homogeneous group description of 12 rats with at least four tumor metastases 4 mm in size towards the liver, plus a couple of person peritoneal tumors but no peritonitis. Tumor stage was assessed throughout the intes tinal resection. Intestinal anastomoses We performed excisions of a short segment from the distal intestine with subse quent end to finish restoration of intestinal continuity in two groups of rats. Group 1 consisted of 12 rats with CC531 colon cancer metastatic tumors, although group 2 consisted of 12 regular healthful rats. Bromdeoxyuridine administration Six rats had been randomly chosen from each the cancer and manage groups.
Intraperitoneal injection of bromodeox yuridine 10 mg was administered day-to-day to each and every rat on days from 1 to 3 through the observation period. Immunohistochemistry of intestinal wounds On days three, 7 and 14, samples of anastomotic wounds had been taken. Samples have been reduce on a cryostat into sections five um thick, which had been mounted onto polylysine treated slides. Cryosections had been fixed in alcohol for hematoxylin and eosin and for trichrome staining.