Agar-Screen Specimen Carrier for Bulk Processing of Biopsy Materials for Electron Microscopy

A specimen carrier for processing large numbers of biopsy materials for epoxy embedding and electron microscopy is described. Commercially available 18-mesh stainless steel or 16-mesh aluminum wire screening is used. The screening is cut into 1 × 3-inch strips. One corner is snipped off for orientation purposes. Four drops of warm 4% agar is placed on a prewarmed standard microscopic glass slide. A thin agar support film is formed on the bottom side of the horizontally held wire screen by lightly running it against the agar. Tissue blocks trimmed to 1 mm³ are blotted on filter paper and placed in a prearranged order on the top surface of the support film. A thin top coating of agar is applied on the specimen by touching it with the tip of a pasteur pipette containing warm 4% agar. The agar-screen unit with the mounted specimens is stabilized in 4% buffered formalin and rinsed with Sorenson's phosphate buffer, pH 7.4, with 6.8% sucrose. It is then processed as a unit through routine osmium tetroxide postfixation, alcohol dehydration, and Epon 812 infiltration. The tissue blocks are plucked off the agar support film with fine-tipped tweezers and embedded in individual capsules. No difficulty in thin sectioning was encountered and examination of the sections under the electron microscope showed good infiltration by the epoxy resin.     

Airway-parenchymal interdependence after airway contraction in rat lung explants

The constrictionof pulmonary airways is limited by the tethering effect exerted byparenchymal attachments. To characterize this tethering effect at thescale of intraparenchymal airways, we studied the pattern ofparenchymal distortion due to bronchoconstriction in a rat lung explantsystem. First, we measured the elastic modulus under tension for 2%(wt/vol) agarose alone (37.6 ± 1.5 kPa) and for agarose-filled lung(5.7 ± 1.3 kPa). The latter is similar to the elastic modulus ofair-filled lung at total lung capacity (4.5-6 kPa) (S. J. Lai-Fook, T. A. Wilson, R. E. Hyatt, and J. R. Rodarte.J. Appl. Physiol. 40: 508-513,1976), suggesting that explants can be used as a model of lung tissuedistortion. Subsequently, confocal microscopic images of fluorescentlylabeled 0.5-mm-thick explants prepared from agarose-filled rat lungsinflated to total lung capacity (48 ml/kg) were acquired. Images weretaken before and after airway constriction was induced by directapplication of 10 mM methacholine, and the pattern of parenchymaldistortion was measured from the displacement of tissue landmarksidentified in each image for 14 explants. The magnitude of the radialcomponent of tissue displacement was calculated as a function ofdistance from the airway wall and characterized by a parameter,b, describing the rate at which tissuemovement decreased with radial distance. The parameterb was 0.994 ± 0.19 (SE), which isclose to the prediction of b = 1 ofmicromechanical modeling (T. A. Wilson. J. Appl.Physiol. 33: 472-478, 1972). There was significantvariability in b, however, which wascorrelated with the fractional reduction in airway diameter (r = 0.496). Additionally, parenchymaldistortion showed significant torsion with respect to the radialdirection. This torsion was similar in concentric zones around theairway, suggesting that it originates from inhomogeneity in theparenchyma rather than inhomogeneous airway constriction. Our resultsdemonstrate the significance of the nonlinear mechanical properties ofalveolar walls and the anisotropy of the parenchyma in determining the nature of airway-parenchymal interdependence.

     

Ultrastructural characterization of the pulmonary cellular defences in the lung of a bird,the rock dove,Columba livia

Free (surface) avian respiratory macrophages (FARMs) were harvested by lavage of the lung/air-sac system of the rock dove, Columba livia. The presence of FARMs in the atria and infundibula was confirmed by scanning electron microscopy. The respiratory system has developed several cellular defence lines that include surface macrophages, epithelial, subepithelial and interstitial phagocytes, and pulmonary intravascular macrophages (PIMs). Hence, C. livia appears to have a multiple pulmonary cellular protective armoury. Ultrastructurally, the FARMs and the PIMs were similar to the corresponding cells of mammals. The purported high susceptibility of birds to respiratory diseases, a state that has largely been deduced from morbidities and mortalities of commercial birds, and which has chiefly been attributed to paucity of the FARMs, is not supported by the present observations.