Prepared in ten DMSO and injected intravenously 1 h prior to ischemia, respectively.
Prepared in ten DMSO and injected intravenously 1 h prior to ischemia, respectively. For all experiments, the drug concentrations had been calculated such that all animals received equal volumes of DMSO. All experimental groups are outlined in Table I. So as to establish the impact of I/R on hepatic metastasis, mice from the sham and manage groups (n=10 per group) had been sacrificed by cervical dislocation 12 days just after surgery. Metastasis of the LacI Protein medchemexpress ischemic and non-ischemic lobes was scored as the hepatic replacement location (HRA) (20). HRA was defined as the percentage of liver tissue replaced by tumor tissue, depending on 4 non-sequential hematoxylin and eosin (H E)-stained sections. The images have been analyzed applying a Leica microscope camera and Biosens Digital Imaging Method evaluation technique, version 1.6 (Leica Microsystems, Beijing, China). Survival time was recorded until 12 days immediately after the surgery. The second experiment was designed to establish the impact of the drugs on hepatic metastasis in mice. Mice from the Ro and Ro + GW groups (n=10 per group) had been sacrificed 12 days after surgery. Liver samples had been obtained and the metastasis and survival time had been scored as described above. The third experiment was made to quantify the expression of numerous metastasis-associated proteins. Mice have been sacrificed at 2, eight and 24 h following the initiation of reperfusion (n=6 per group at every single time-point). Liver samples had been obtained for evaluation by light microscopy or storage at 80 until tissue analysis.EXPERIMENTAL AND THERAPEUTIC MEDICINE 11: 387-396,Table I. Description of experimental groups. Experiment 1 Rationale Establish the effect of I/R on hepatic metastasis Group Sham Control two Identify the effect of drugs on hepatic metastasis Quantify the expression of metastasis-associated proteins Ro Ro + GW Sham Ro Control Ro + GW Procedure Laparotomy, liver manipulation, intraportal injection of H22 tumor cells and closure. Sacrificed 12 days soon after surgery Intraportal injection of H22 tumor cells just after partial hepatic ischemia. Sacrificed 12 days just after surgery As within the manage group, but treated with rosiglitazone 1 h prior to ischemia As in the manage group, but treated with rosiglitazone + GM-CSF Protein Biological Activity GW9662 1 h before ischemia Samples collected soon after 45 min sham ischemia and 2, 8 and 24 h reperfusion Treated with rosiglitazone 1 h prior to ischemia. Samples collected after 45 min ischemia and 2, eight and 24 h reperfusion Samples collected just after 45 min ischemia and 2, 8 and 24 h reperfusion Treated with rosiglitazone + GW9662 1 h before ischemia. Samples collected just after 45 min ischemia and 2, 8 and 24 h reperfusion n 10 ten 10 ten 6a 6a 6a 6aaAt each and every time-point. I/R, ischemia/reperfusion; Ro, rosiglitazone; GW, GW9662.Histochemistry and immunohistochemistry. For light microscopy, sections on the left lobe from the liver have been fixed in 10 phosphate-buffered formalin for 5 days. The resulting paraffin-embedded sections (5 ) were stained with H E for routine histological examination in accordance with standard procedures. Vascular cell adhesion molecule (VCAM)-1 protein was stained using immunohistochemical procedures (streptavidin peroxidase). In short, deparaffinized sections had been incubated with three H 2O2 to block endogenous peroxidases and with 0.five goat regular serum to block nonspecific binding web-sites. Polyclonal mouse anti-VCAM-1 antibodies (1:50) had been applied as primary antibodies. Biotinylated anti-goat rabbit immunoglobulin antibodies had been used as secondary antibodi.
