Tracheal Replacement by Allogenic Aorta in the Pig: Histologic Examination

Follow-up and Evaluation: Pulse oximetry was measured throughout the surgical procedure. Clinical examination was performed daily thereafter. Follow-up bronchoscopies were performed as previously described on a monthly basis and at any other time if the animals showed signs of respiratory distress. Animal deaths were planned at 3, 6, and 12 months for macroscopic and microscopic evaluation and genetic study. Euthanasia was performed under general anesthesia using embutramide, mebezonium, and tetracain (T61; Intervet; Beaucouze, France). Animals showing respiratory signs before the predetermined time of death were euthanized and underwent systematic bronchoscopy and autopsy.
The trachea and main bronchi were carefully excised. Transverse and longitudinal sections were cut at the level of the graft following 2 days of formalin fixation. Additional sections sampled proximal and distal to the grafted trachea served as controls. Specimens were embedded in paraffin, cut into 3-^m slides, and stained with hematoxylin-eosin-saffron (HES), eosin/Alcian blue (for acidic proteoglycans), and elastic fiber stain (orcein) for microscopic examination.
Digoxigenin-11-uridine triphosphate-labeled single-strand RNA probes were prepared using a DIG RNA labeling kit (Roche Applied Science; Mannheim, Germany) according to the instructions of the manufacturer. Mouse a1 (II) collagen complementary DNA, kindly provided by Y. Yamada (National Institute of Dental Research, National Institutes of Health, Bethesda, MD), was used to generate antisense and sense probes. After deparaffinization, the sections were treated with 10 |j,g/mL of proteinase K for 15 min at room temperature, and subjected to 0.2 N HCl to inactivate endogenous alkaline phosphatase. Hybridization was performed at 50°C in 50% formamide, and washes were carried out at a stringency of 2 X saline sodium citrate buffer containing 50% formamide at 55°C. Then, slides were subjected to 10 |j,g/mL of ribonuclease A in TNE buffer (10 mmol/L Tris-HCl, pH 8.0, 500 mmol/L NaCl, 1 mmol/L ethyl-enediamine tetra-acetic acid) at 37°C for 30 min for digestion of nonhybridized transcripts, and washed. The samples were incubated overnight with anti-Dig-alkaline phosphatase. We detected the Dig probe with nitro-blue tetrazolium chloride/5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt as a substrate to give a purple color. We stopped the reaction with three changes of buffer containing 1 mmol/L ethylenediamine tetra-acetic acid, fixed the samples with 4% paraformaldehyde/0.2% glutaralde-hyde, and counterstained with eosin. These samples were mounted (Glycergel; Dakocytomation; Milan, Italy) and observed under a microscope (Axioskop; Carl Zeiss; le Pecq, France).

This entry was posted in Allogenic Aorta and tagged airway, lung cancer, transplants.