Morphometrics of rapidly frozen goose lungs.

To understand the structural basis of avian gas exchange better, we made a morphometric study of domestic and Canada goose lungs. The volume of glutaraldehyde-fixed domestic goose lungs (30 cm3/kg body weight) was similar to that determined from silicone casts of Canada goose lungs by Duncker (33 cm3/kg). To examine finer structures, we rapidly froze goose lungs under physiologic conditions, fixed tissue samples by a freeze substitution procedure and analyzed samples with stereological methods. From light micrographs we determined that about 55% of the lung is parabronchi in both species. Volume densities of air capillaries, blood capillaries and tissue and surface:volume ratios of these same structures were determined from electron micrographs. Our measurements agree with those from glutaraldehyde-fixed Canada goose lungs from other laboratories. Gas exchange surface area was largest in the good flier (Canada goose) but both birds had larger surface areas than comparably sized mammals. The harmonic mean blood-gas barrier thickness is smaller in both species of birds (0.3 microns) than in mammals. Thus, membrane diffusing capacities for gases should be larger in birds than in mammals. Pulmonary blood capillary transit time, as calculated from blood capillary volume and normal levels of cardiac output, are longer in birds than in mammals and should allow more time for blood-gas equilibrium. Pleats and folds were frequently observed in air and blood capillaries, suggesting that the avian lung may not be as rigid as was previously thought and that capillary volumes and surface areas may change under physiologic conditions.

Influence of vascular and transpulmonary pressures on endothelial vesicles in nonedematous and edematous lungs.

We examined the effects of vascular and transpulmonary (Ptp) pressures on endothelial vesicles in nonedematous and edematous dog lungs by transmission electron microscopy (TEM). Edema was defined as a 30% increase in lung weight. Lungs were prepared for TEM by rapid freezing followed by freeze substitution. Using a random sampling procedure, the size and numerical density (Nv) of vesicles was obtained by standard morphometric techniques and the percentage of cytoplasm occupied by vesicles (Vv) calculated. Results show that at Ptp = 5 cm H2O, vascular pressure has no influence on the variables examined in nonedematous and edematous lungs. At Ptp = 25 cm H2O, increases in vascular pressure were associated with significant decreases in the percent cytoplasm occupied by vesicles for both series of experiments. When nonedematous lungs were compared to edematous lungs, we found increases in vesicle size, Nv, and the percentage of endothelial cell cytoplasm occupied by vesicles. The results confirm the increase in vesicles associated with edema. This result does not appear to be due to increased vascular pressure per se, although time related influences could be involved.

Size of pores of Kohn: influence of transpulmonary and vascular pressures.

We investigated the influence of transpulmonary (Ptp) and vascular pressures on the size of the pores of Kohn in primary alveolar septa. Dogs lungs, perfused and ventilated in situ, were rapidly frozen with Freon 22 in zone II or III conditions following deflation to Ptp of 5, 15, or 25 cmH2O. Frozen samples were freeze-substituted for transmission electron microscopy. Five fields containing at least one pore each were selected randomly from each section of tissue, and the minimum diameter visible in the cut section was measured. For both zone II and III conditions, as Ptp increased, mean pore size increased. The mean pore size under zone III conditions was 1.2015, 1.788, and 2.249 micrometer for Ptp of 5, 15, and 25 cmH2O, respectively. For zone 2 conditions, the corresponding values were 1.1438, 1,8757, and 2.08 micrometer. For both zones II and III, increasing capillary hydrostatic pressure had no significant effect on pore size. The results support the notion that alveolar pores can increase collateral ventilation by dynamically stretching as Ptp increases. Capillary pressure does not influence pore size probably because of collagen fibers, which surround the pore lumen. Presumably, these fibers resist encroachment of capillaries on the pore lumen as vascular pressures increase.

Interaction of convection and diffusion in pulmonary gas transport.

Normal human subjects inspired various volumes of a normoxic argon mixture containing low concentrations of several biologically inert tracer gases with markedly different diffusivities (helium, neon, and sulfur hexafluoride). The behavior of Ne, Ar, and SF6 could be predicted on the basis of axial dispersion due to differences in diffusivity. For example, neon, having the highest diffusivity of the three, was more uniformly distributed within the bronchial tree than either argon or SF6. The behavior of helium, however, was not consistent with predictions based solely on axial diffusion. Contrary to expectation, the early portion of expiration was helium enriched while gas assumed to come from the alveolar regions contained relatively less helium than the other gases. Results of this study suggest that radial diffusion during convective bulk flow may play a significant role in intrapulmonary gas transport if relative diffusivity is extremely large. We conclude that diffusion gradients do exist within the bronchial tree during normal quiet breathing and that these gradients become less significant as inspired volume increases.

Shrinkage of lung after chemical fixation for analysis of pulmonary structure-function relations.

Glutaraldehyde is widely used to chemically fix lungs for analysis of pulmonary structure-function relations. Accurate interpretation of observations on fixed tissue requires a clear definition of any artifacts, such as tissue shrinkage, resulting from fixation with glutaraldehyde. Two experimental procedures were used in this study to examine possible shrinkage artifacts resulting from fixation of lung by glutaraldehyde. In the first, isolated perfused dog lungs were rapidly frozen at different transpulmonary pressures. Samples were then freeze substituted at -50 degrees C using 70% ethylene glycol with and without fixatives present. In the second series of experiments, the left lungs of mongrel dogs were fixed by vascular perfusion with glutaraldehyde at different transpulmonary pressures. In both series of experiments any changes in linear dimensions resulting from the fixation procedure were measured. Also, the presence of aldehyde was demonstrated by a positive reaction with Schiff reagent. The results demonstrate that lung tissue fixed either by vascular perfusion or freeze substitution tends to shrink to about the same extent. This shrinkage is reasonably constant at about 9% for transpulmonary pressures of 5 and 15 cmH2O and increases to about 15% when the transpulmonary pressure reaches 25 cmH20.

Pulmonary vascular resistance during unilateral pulmonary arterial occlusion in ducks.

We measured mean pulmonary arterial pressure (Ppa) during temporary unilateral pulmonary arterial occlusion (TUPAO) in 10 ducks. Ppa increased from 11.4 +/- 0.8 mmHg during control conditions to 18.8 +/- 1.8 during TUPAO. In 5 of the 10 ducks we also measured mean left atrial pressure (Pla) and cardiac output (Q). In these ducks Ppa significantly increased with TUPAO from 13.9 +/- 0.4 to 22.0 +/- 1.2 mmHg, whereas Pla and Q did not change significantly. Pulmonary vascular resistance (PVR) increased from 10.6 +/- 1.3 to 24.1 +/- 5.3 mmHg X min X 1(-1) on TUPAO. By assuming equal vascular resistance in either lung it can be calculated that the vascular resistance in only one lung was 22.5 +/- 3.5 mmHg X min X 1(-1) during control conditions. Thus doubling flow resulted in no significant change in one lung's vascular resistance. A morphometric study of both lungs of a domestic goose that were rapidly frozen during TUPAO indicated very little compliance in pulmonary blood capillaries. The relative volume of exchange tissue occupied by blood capillaries was 0.28 in the occluded lung and 0.36 in the perfused lung. Surface-to-volume ratios of blood capillaries were 12,524 cm-1 in the occluded lung and 11,056 cm-1 in the perfused lung. We conclude that PVR in birds is relatively insensitive to changes in Q, in contrast to mammals.

Mixing in flowing gas.

We used a mass spectrometer to analyze continuously from a flow stream after changeover of one gas to a mixture of other gases. At the end of a straight cylinder (length = 100 cm, inner diameter = 1/2 inch), light-weight gases appeared later but reached full-scale deflection earlier than heavy gases. Apparently gases with high molecular diffusivity tended to diffuse radially so that they were not carried forward axially in rapidly moving streams. This blunting of the profile of mean concentration vs distance as fresh gas move down a tube, plus spreading of a gradient region of transition between fresh gas and stale gas, are the fundamental processes of diffusion/convection interactions in the lung. Turbulence and molecular diffusion are similar in that they limit the penetration of inspired gases that could occur if laminar flow were the only process acting. However, turbulence and molecular diffusion cause true irreversible mixing of fresh and stale gases, whereas dispersion by laminar flow does not.

Ventilation-perfusion relationships during hemorrhagic hypotension and reinfusion in the dog.

The inert gas elimination technique was used to estimate pulmonary ventilation-perfusion (VA/Q) mismatching in heparinized, ventilated, anesthetized dogs during a 90-min period of hemorrhagic hypotension (mean arterial pressure 40 Torr) and subsequent reinfusion of the shed blood. Systemic and pulmonary arterial pressures, as well as cardiac output, were similar to those in previously reported studies using this protocol. Mean arterial O2 partial pressure (PO2) fell from 86 to 75 Torr after hemorrhage and rose to a mean value of 78 Torr after reinfusion. The VA/Q distributions showed that a mean of 56.7% of the ventilation was associated with unperfused or poorly perfused (VA/Q greater than 10) regions during hypotension (control 33.7%). After reinfusion, a mean of 47.8% of the ventilation was still directed to lung with little or no perfusion. This could not be explained on a hydrostatic basis, since pulmonary arterial pressure after reinfusion was greater than the control value. Shunt or blood flow to low VA/Q regions did not increase at any time during hemorrhagic hypotension or reinfusion. Microscopic examination of lung tissue revealed extensive leukocyte aggregation that was not seen in control animals. The mean diameter of obstructed pulmonary vessels was 35 microns (range 13.8-59.8 microns). Storing the shed blood in acid-citrate-dextrose instead of heparin had no significant effect on the extent of leukocyte aggregation. We suggest that leukocyte aggregation and margination may be related to the high VA/Q regions seen in these animals.

Identification of functional lung unit in the dog by graded vascular embolization.

To study the dimensions of the functional gas exchange unit, spherical polystyrene beads (diam 50-500 micrometers) were injected intravenously into 12 normal anesthetized paralyzed dogs (15-24 kg wt). We argued that beads small enough to lodge within gas exchange units would not give rise to a population of high ventilation-perfusion ratio (VA/Q) areas, whereas embolization of larger vessels supplying these units would. Each dog received only one bead size in cumulative 0.25-g doses up to a maximum of 2.25 g. Multiple inert gas elimination data were obtained after each dose to monitor the development of high VA/Q regions. Injection of 50- and 100-micrometers beads never gave rise to high VA/Q regions, whereas 150-, 250-, and 500-micrometers beads consistently induced a high VA/Q mode comprising up to 45% of the ventilation. Histological examination of lungs from five additional dogs injected with small (approximately 0.5 g) doses revealed that beads rarely formed clusters and appeared in vessels of their own diameter in over 90% of instances. By the above criterion, the functional gas exchange unit in these lungs is that volume of tissue subtended by 150- micrometers-diam arteries (vessels associated with respiratory bronchioles).

Electron micropscopic appearances of rapidly frozen lung.

Analysis of pulmonary structure-function relationships by microscopy requires that the lung be fixed under carefully controlled physiological conditions, since lung structure is extremely responsive to the relationship between airway and vascular pressures. Unfortunately, standard post-mortem fixation techniques leave some doubt as to the exact relationship between these pressures during fixation. This problem can be circumvented by stabilizing lung structure by rapid freezing under carefully controlled physiologic conditions. Using ethylene glycol in a freeze substitution technique we have developed procedures which yield a degree of preservation compatible with the high degree of resolution of the electron microscope. These can be used to obtain a more detailed understanding of pulmonary structure-function relationships under well-defined physiological conditions.

Electron microscopy of lung rapidly frozen under controlled physiological conditions.

Light microscopy of lung rapidly frozen under controlled physiological conditions has been very successful in correlating pulmonary structure and function. However, to study some aspects of pulmonary capillary morphology, the higher resolution of electron microscopy (EM) is necessary. To date, most EM of lung has involed the instillation of a fixative through the airways or vascular system, techniques that probably alter the normal pressure relationships of the capillaries and therefore their morphology. We describe here a technique for rapidly freezing lung to a depth of 1--2 mm below the pleural surface and preparing sections for EM. Lungs from open-chest rats were frozen at various transpulmonary pressures with cold (--80 degrees C) 70% ethylene glycol. Small pieces were then fixed with a solution containing glutaraldehyde and paraformaldehyde for 24 h at --50 degrees C. Staining was with osmium tetroxide and uranyl acetate. Lung frozen at high volumes showed marked stretching of the alveolar septa with severe deformation of the capillaries. Lung frozen at low inflation pressures revealed open capillaries containing numerous red blood cells; in addition, infolding of the alveolar wall was frequently seen. We conclude that this technique gives a level of preservation of rapidly frozen lung suitable for electron microscopy.