After biopsy the third week, the pigs in group one and two again received i. v. fluids via a new Hickman catheter placed in the left jugular vein. The same amount of fluids and medication was given at the same time each day as after primary operation, but full read only for three days postoperatively. Oral medication was con tinued with 5 mgkg Erytromycin daily and 20 mg Esomeprazol two times per day, until sacrificing the sixth week. Blood sampling For pre PHx reference values, blood was sampled from the jugular vein at the time of laparotomy. After surgery, we sampled regularly from the jugular vein for analysis of 1 Cytokines IL 1, IL 6, IL 10 2 Humoral growth regulating factors TNF, TGF B Sin gle Plex, Troms, Norway.
Other analysis ASAT, ALAT, GT Bilirubin, Albumin Inhibitors,Modulators,Libraries INR Statistics Time, group and group time interaction of blood ana lyses was examined using General Linear Model with Repeated Measures in SPSS version 15, with p 0. 05 considered significant. We defined time as a fixed factor and subject as a random effect. An autoregressive AR1 covariance matrix was used. All curves for all animals in all groups are drawn as group averages 1 SD. Biopsies A reference sample was taken from all animals in all groups upon laparotomy, before PHx, at time points three weeks post PHx and six weeks post PHx. Biopsies were immersed immediately in RNAlater, and preserved at 70 C until RNA extraction and microarray analysis. Microarray methods Two colour microarray experiments were conducted to identify genes being significantly differentially expressed due to resection over time adjusting for effects by using the expression profiles obtained from the control ani mals and the sham operated animals.
The microarray experiment was conducted as a com mon reference design using a reference consisting of equal Inhibitors,Modulators,Libraries amounts of total RNA from all samples. Total RNA was extracted from each sample and DNase treated using RNeasy Maxi Kit. Quantities were mea sured using a NanoDrop ND 1000 Spectrophotometer and qualities were examined by the 28S 18S rRNA ratio using the RNA 6000 Nano LabChipW Kit on 2100 Bioanalyzer. Alexa Flour labeled cDNA was synthesized from 20 ug of total RNA using Superscript Plus Direct Inhibitors,Modulators,Libraries cDNA Labeling System and purified using the NucleoSpin 96 Extract II PCR Clean up kit. The reference samples were labelled with Alexa 555 and the Inhibitors,Modulators,Libraries individual samples were labelled with Alexa 647.
The labelled and purified reference samples were mixed and divided into aliquots before combining it with a labelled sample. Each of the 36 labelled samples were co hybridized with an aliquot of the labelled reference sample and a hybridization blocker containing polydA and Yeast tRNA to 27k pig oligonucleotide microarrays representing Inhibitors,Modulators,Libraries approximately 20k porcine genes using inhibitor order us a Discovery XT hybridisation station.