Oxygen Transport to Tissue XXXV by Sabine Huffel Gunnar Naulaers Alexander Caicedo Duane F. Bruley & David K. Harrison

Author:Sabine Huffel, Gunnar Naulaers, Alexander Caicedo, Duane F. Bruley & David K. Harrison
Language: eng
Format: epub
Publisher: Springer New York, New York, NY

33.2 Materials and Methods

As previously described [3], WSU-DLCL2 cells were subcutaneously inoculated in the flanks of 5–7-week-old female nude mice to induce tumor xenografts. Tumor-bearing mice were randomly chosen and divided into the treatment group and the control group. CHOP treatment is as follows: cyclophosphamide, 40 mg/kg i.v., day 1; hydroxydoxorubicin, 3.3 mg/kg i.v., day 1; Oncovin, 0.5 mg/kg i.v., day 1; and prednisone, 0.2 mg/kg p.o., day 1–5. Sham treatment (saline) was performed on the control group. All animal studies were performed in accordance with the guidelines of the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC).

Anesthetized tumor-bearing mice were snap-frozen in liquid N2 1 day after the last treatment. The frozen tumors (three from the treated group and three from the control group) were quickly removed from the mouse body with a handsaw and embedded in homemade mounting media followed by redox scanning as previously described [5, 7]. The PC-collected scanning data were analyzed with a customized Matlab® program that uses the reference standards to convert the intensity-based images into concentration-based images and generates redox ratio images.

Each xenograft was scanned with three to five sections spacing 800–1,000 μm with the first section approximately starting at about 1,000–1,500 μm beneath the skin. Three tumors in each group of a total of six tumors were scanned. The redox indices (Fp, NADH, and Fp redox ratio, i.e., Fp/(Fp + NADH)) were averaged for each tissue section. Univariate analysis was conducted on these redox indices using IBM SPSS Statistics (version 20) with tissue depth as the covariate and p < 0.05 taken as statistically significant. Data were finally reported as mean ± standard error of mean (SEM).

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