Detection of Aerosolized Fluorescent Microspheres in the Lung Parenchyma and Air Sacs of Pigeons (Columbia livia domestica)
The objective of this study was to examine the feasibility of utilizing aerosolized fluorescent microspheres to examine particle distribution in the respiratory tract of birds following aerosol exposure. Five adult domestic pigeons (Columbia livia domestica) were utilized for this study. The birds were obtained from a local commercial squab producer and were found to be in good health and body condition. This study utilized the previously described technique for aerosolizing a monodispersed population of commercially available latex beads.1 The microsphere suspension was prepared utilizing 3-micron yellow-green Fluoresbrite™ carboxylate microspheres (Polysciences Inc., Warrington, PA, USA) which were obtained in a 2.5% solids suspension. All microspheres were coated with albumin prior to aerosolization to reduce the loss of microspheres from the lung parenchyma and air sacs during fixation and processing of tissues. The latex bead solution was aerosolized utilizing a compressed air nebulizer set at 30 PSI. The aerosol was then passed through a heated discharging column to reduce static charge and to remove water surrounding each microsphere. For aerosol exposure purposes, the birds were anesthetized with injectable anesthetics, intubated, and placed on positive pressure ventilation utilizing a mechanical ventilator. The birds were monitored and observed during the entire anesthesia and aerosol exposure process, with aerosol exposure lasting for 30 minutes. Immediately following aerosol exposure, the basilic veins were catheterized utilizing 26 ga catheters, 2,000 units of heparin were administered IV, then the birds were humanely euthanatized with an IV overdose of pentobarbital. Immediately following euthanasia, approximately 500 ml of 0.9% NaCl was infused IV utilizing a peristaltic perfusion pump at a flow rate of approximately 90 ml per minute. The avian carcasses were then fixed utilizing intravenous infusion of Karnofsky’s fixative at a pH of 7.4 and 340 mOsm. The carcasses were stored at 10°C for 24 hours then the air sacs and lung parenchyma were carefully dissected out. Initial examination of the dissected air sacs and ostia was performed using a Leica MZ12 dissecting microscope (Leica, Heerbrugg, Switzerland) with a fluorescent module. Confocal imaging was then performed utilizing a confocal microscope with a krypton argon laser. Examination of the air sacs was confined to the cranial and caudal thoracic and abdominal air sacs. The results from this study revealed that positive pressure ventilation resulted in even distribution of fluorescent beads in the cranial and caudal thoracic and abdominal air sacs of the five birds. Examination with stereoscopic and confocal microscopes allowed visualization of beads within all air sacs. The beads were found in highest concentrations within the region of the secondary bronchi and the ostia for all air sacs examined. The results from this study allow for a better understanding of particle deposition following positive pressure ventilation and aerosol exposure in birds. Ultimately, we hope to be able to utilize this technique to evaluate aerosol/nebulization regimes for treating respiratory disease in avian species.
This work was supported by a grant from the Center from Companion Animal Health, School of Veterinary Medicine, University of California-Davis, Davis, California, USA.
1. Pinkerton K, Gallen J, Mercer R, Wong V, Plopper C, Tarkington B. Aerosolized fluorescent microspheres detected in the lung using confocal scanning laser microscopy. Microsc Res Tech. 1993;26:437–443.