Evidence of Innervation in Talc-Induced Pleural Adhesions: Study Design and Procedures

Ten white, male, New Zealand rabbits weighing 1.5 to 2.0 kg were used in the present study. Right thoracotomy was performed at the seventh intercostal space, as previously described in detail, and 200 mg/kg of talc (mean ± SEM maximum diameter, 8.36 ± 0.20 |j,m; Distalc; Barcelona, Spain) suspended in 2 mL of endotoxin-free saline solution were instilled into the pleural cavity. This dose was chosen because 200 mg/kg is the minimum dose that produces an effective pleurodesis in this animal model. Two groups of five rabbits were killed with a lethal injection of pentobarbital at 1 week and 1 month after instillation, respectively. At autopsy, a total of 60 adhesions were excised from opposing surfaces of the visceral and parietal pleura, including costal, mediastinal and diaphragmatic parietal pleura. The adhesions were removed with part of the adjacent organs, and the specimens were processed for histopathologic, immuno-cytochemical, and ultrastructural examination. The study was approved by our Ethics Committee on Animal Experimentation. canadian pharmacy
For histopathologic analysis, pleural adhesion samples were fixed in 4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline solution (PBS), embedded in paraffin, and sectioned at a nominal thickness of 6 |j,m. Sections were then dewaxed with xylol, hydrated, and stained with hematoxylin-eosin. Examination was carried out by light field and polarizing microscopy.
Pleural adhesion specimens were fixed in 4% paraformaldehyde in PBS for 2 h at room temperature. After washing with PBS, the samples were infiltrated with 30% sucrose at 4°C, embedded in optimum-cutting temperature compound (OCT; Miles Laboratories; Naperville, IL), quickly frozen in dry ice, and stored at — 20°C. Cryostat sections were cut at 6 |j,m, thaw mounted onto gelatin-coated slides, air dried, and stored desiccated at — 20°C. Sections were then incubated with a mouse anti-human platelet endothelial cell adhesion molecule-1 (PE-CAM-1) [Dako; Glostrup, Denmark] overnight at 4°C at 1:25 dilution. Bound monoclonal antibody was visualized by incubation with fluorescein isothiocyanate-conjugated goat anti-mouse Ig (Dako) for 1 h at room temperature. After extensive washes, sections were mounted (Fluoromount G; Electron Microscopy Science; Washington, PA) and observed with an epifluorescence microscope (Polyvar 2; Reichert-Jung; Vienna, Austria).
Additional adhesion samples were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in PBS for 2 h at room temperature. Primary fixation was followed by postfixation with 1% osmium tetroxide in PBS for 1 h. The samples were dehydrated in acetone and embedded in Spurr resin following standard procedures. Semithin sections were stained with methylene blue and observed with an optical microscope (Polyvar 2; Reichert-Jung). Ultrathin sections were stained with uranyl acetate and Reynold’s lead citrate and examined with a transmission electron microscope (H-600 AB; Hitachi; Tokyo, Japan).

This entry was posted in Pulmonary Function and tagged adhesion, innervation, lymphangiogenesis, neovascularization, pleurodesis, talc, ultrastructure.