Susan L. Bernard

Histological Methods to Determine Blood Flow Distribution with Fluorescent Microspheres

We evaluated several histological methods and determined their advantages and disadvantages for histological studies of tissues and organs perfused with fluorescent microspheres. Microspheres retained their fluorescence in 7-10 microm serial sections with a change in the antimedium from toluene when samples were fixed in formalin and embedded in paraffin. Several antimedia allowed both wax infiltration of tissue and preservation of microsphere fluorescence. Histoclear II was the best substitute for toluene. When samples were fixed in formalin and embedded in glycol methacrylate, thinner (3-5 microm) sections provided greater histological detail but had fewer microspheres per section. Air dried lung tissue followed by Vibratome sectioning provided thick sections (100 microm) that facilitated rapid survey of large volumes of tissue for microspheres but limited histological detail, and the air drying procedure was restricted to lung tissue. Samples fixed in formalin followed by Vibratome sectioning of unembedded tissue provided better histological detail of lung tissue and was also useful for other organs. These sections were more difficult to handle and to mount on slides compared to air dried tissue, whereas fixed tissue embedded in gelatin provided better tissue support for Vibratome sectioning. Rapid freezing followed by cryo-microtome sectioning resulted in frozen sections that were relatively difficult to handle compared to embedded or unembedded tissue; they also deteriorated relatively rapidly with time. Paraffin sections were stained with hematoxylin and eosin or with aqueous methyl green, although tissue autofluorescence by itself was usually sufficient to identify histological features. Methacrylate sections quenched tissue autofluorescence, and Lees stain or Richardsons stain were used for staining sections. Toluene based mountants such as Cytoseal quenched fluorescence, particularly the red fluorescent microspheres. Aqueous based mountants such as Aquamount, Crystal/Mount, Fluoromount-G were substituted, although such preparations were not as permanent as Cytoseal mounted coverglasses and tended to cause fading of stained sections.

Quantifying the genetic influence on mammalian vascular tree structure.

The ubiquity of fractal vascular trees throughout the plant and animal kingdoms is postulated to be due to evolutionary advantages conferred through efficient distribution of nutrients to multicellular organisms. The implicit, and untested, assertion in this theory is that the geometry of vascular trees is heritable. Because vascular trees are constructed through the iterative use of signaling pathways modified by local factors at each step of the branching process, we sought to investigate how genetic and nongenetic influences are balanced to create vascular trees and the regional distribution of nutrients through them. We studied the spatial distribution of organ blood flow in armadillos because they have genetically identical littermates, allowing us to quantify the genetic influence. We determined that the regional distribution of blood flow is strongly correlated between littermates (r2 = 0.56) and less correlated between unrelated animals (r2 = 0.36). Using an ANOVA model, we estimate that 67% of the regional variability in organ blood flow is genetically controlled. We also used fractal analysis to characterize the distribution of organ blood flow and found shared patterns within the lungs and hearts of related animals, suggesting common control over the vascular development of these two organs. We conclude that the geometries of fractal vascular trees are heritable and could be selected through evolutionary pressures. Furthermore, considerable postgenetic modifications may allow vascular trees to adapt to local factors and provide a flexibility that would not be possible in a rigid system.

Regional blood flow measurements from fluorescent microsphere images using an Imaging CryoMicrotome

An automated image acquisition and analysis system has been developed that rapidly determines regional blood flow by using the locations of fluorescent microspheres deposited in tissue. A motor-driven microtome removes sections of frozen tissues in steps variable between 10 and 100 μm. Filtered light excites microsphere fluorescence from the exposed surface of the remaining tissue block. A charge coupled device camera records fluorescence images from the tissue block surface after removal of each slice. Approximately 450 images are analyzed from perfused rat hearts providing precise x, y, and z locations of about 10 000 microspheres. Image analysis of fluorescent microspheres is much faster and less labor intensive than traditional indirect microsphere-based flow measurements while providing higher quality data.

Axial and radial distribution of the bronchial vasculature in sheep

A morphometric analysis was made on the bronchial vasculature of intrapulmonary airways in sheep lungs. This study provides the parameters to calculate the quantity of soluble gas diffusion between the vasculature and airways for use in a mathematical model describing heat and mass exchange in the lungs. To achieve these results, the lungs of four adult sheep (30-36 kg.) were excised, fixed, dissected and microtomed to obtain airway cross-sections for measurement. Blood vessel size and airway proximity was measured using a microscope interfaced with a computer. Distance from airway lumen to most airway vessels ranged from 30 to 270 microm. It was found that the bronchial vessels surrounding intraparenchymal airways can be described by a right-skewed distribution. Most importantly, a practical description of the bronchial capillary size and airway proximity as a function of airway diameter was found using a weighed average. This analysis facilitates calculation of soluble gas flux from the bronchial vasculature to the airway for use in a mathematical model.

Bronchial circulation in the marsupial opossum, Didelphis marsupialis

This study characterizes the existence of a bronchial circulation in a marsupial, an animal which does not undergo placental development and does not have a ductus arteriosus. Direct perfusion of the lung by the pulmonary vasculature during the fetal development of opossums may occur, potentially eliminating the need for a bronchial circulation. We used radio- and fluorescent-labeled microspheres in conjunction with postmortem intravascular casting to determine if opossums have a systemic (bronchial) blood supply to the lung (n = 9). Gross postmortem examination of the intravascular casts showed a well-developed common bronchial artery. The histological distribution pattern of fluorescent microspheres was primarily to the airways. A few fluorescent microspheres were observed in the alveolar capillaries, indicating that a precapillary bronchial-to-pulmonary anastomosis exists in the opossum. Using the reference flow technique, total bronchial blood flow to the left lung averaged 0.95 +/- 0.58 SE ml/min. The presence of a bronchial circulation in the opossum suggests that it is more than a vestigial structure from embryonic development, potentially supporting its functional importance for carrying nutrients to the airway.

Positive End Expiratory Pressure Reduces Bronchial Blood Flow after Aspiration Injury

We hypothesized that since added airway pressure compresses bronchial vessels, the airway hyperemia found following airway injury would be reduced by positive end-expiratory pressure (PEEP). Accordingly, we measured the effect of 15 cm H2O PEEP on bronchial and pulmonary blood flows by the radioactive microsphere reference flow technique in closed chested goats (n = 7) before and after aspiration injury to the left lung with 0.1 N HCl. Thirty minutes after aspiration, the pulmonary blood flow to the injured left lung was reduced by one third, whereas the total bronchial blood flow to the left lung (normalized to mean systemic pressure of 100 torr) doubled (11.3 +/- 2.2 to 20.6 +/- 1.0 ml/min 100 torr; p < 0.01). Increasing PEEP from 5 to 15 cm H2O decreased total bronchial blood flow by about half both before (11.3 +/- 2.2 falling to 5.7 +/- 1.4 ml/min/100 torr) and after injury (20.6 +/- 1.0 falling to 10.3 +/- 2.7 ml/min/100 torr). The airway portion (down to 2-3 mm airways) of the total bronchial blood flow of the injured lung increased more than three-fold (1.4 +/- 0.5 rising to 5.5 +/- 1.3 ml/min/100 torr; p < 0.01). This increased flow after aspiration was less affected by PEEP of 15 cm H2O (5.5 +/- 1.3 to 2.8 +/- 0.7 ml/min/100 torr, p = 0.09) than before injury (1.4 +/- 0.5 falling to 0.5 +/- 0.1 ml/min/100 torr; p < 0.05). The increase of the parenchymal portion of the bronchial blood flow after injury, although apparent (9.9 +/- 1.8 increasing to 15.1 +/- 1.2 ml/min/100 torr), was not significant (p = 0.08).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of low-dose endotoxin on pulmonary vascular permeability following acute hemorrhagic shock

The purpose of the current study was to determine the effect of low-dose Escherichia coli lipopolysaccharide (LPS) on pulmonary vascular permeability when administered after hemorrhagic shock (40% of baseline cardiac output) followed by resuscitation. Animals were monitored for 3-4 h after LPS infusion. Thirty minutes prior to termination of the experiment, 3 mCi of 125I-human serum albumin was injected intravenously to calculate a permeability index from the left lung lavage and plasma 125I ratios. The two control groups were (1) shock only (no LPS, n = 4), and (2) LPS only (no shock, n = 8). The permeability index for the shock-only group was 0.0015 +/- 0.0007 (mean +/- SE) and that for the LPS-only group was 0.0035 +/- 0.0014. The permeability index for the experimental group (shock followed by LPS, n = 10) was 0.0071 +/- 0.0030 (p > 0.05). Similarly, there was no difference in the wet-to-dry ratios between the three groups. The shock+LPS group required more intravenous fluids to maintain mean arterial blood pressure at control values than the LPS-only group (p < 0.003). We conclude that hemorrhagic shock and resuscitation do not lead to an acute increased permeability of the lung when it is subsequently challenged by a low dose of bacterial LPS.

Characterization of Bronchial-to-Pulmonary Communications

complex, both anatomically and physiologically. Bronchial arterial blood can travel through bronchial capillaries or anastomose with pulmonary vessels in lung parenchyma. Using serial computed tomography and histology, Wagner et al. (1999) have shown that ‘functional’ intraparenchymal bronchial vasculature drainage is predominantly into postcapillary pulmonary vessels. However, there was considerable variability between animals. We used two different techniques to characterize anastomotic vessels. First, we used fluorescent microspheres (FMS) to see where different size microspheres are trapped in the lung. In a second series of animals, we used an intra-vascular casting material, Microfil, to visualize the vessels in cleared lung tissue. For the microsphere study, we created an aortic pouch isolating the origin of the broncho-oesophageal artery in four anaesthetized sheep. Microspheres were injected through a mixing chamber in line with the aortic pouch. One million 15 mm blue-green FMS were injected, followed by 100,000–200,000 100mm orange FMS. After the study, animals were deeply anaesthetized, heparinized and killed. Lungs were flushed free of blood, removed from the chest, inflated to 30cm of water, and air dried. They were then encased in polyurethane foam, sliced in a transverse plane and systematically sampled using a grid system. The samples were sliced with a vibratome and the FMS were counted in anatomical locations (parenchyma or airways) using a fluorescent microscope. A total of 3474 100 mm orange FMS were counted in 4214 sections. Of these, 250 (6.2 ± 1.3%) were located in lung parenchyma and 3224 were located in the airways. A total of forty-one 15mm FMS were counted in the parenchyma. Large numbers of 15 mm FMS were seen in airways but were not counted. These studies using fluorescent microscopy in sheep suggested that precapillary bronchial-to-pulmonary anastomoses larger than 100 mm existed. Based on the number of FMS injected, proportionally more 15 mm FMS are found in airway walls than in parenchyma. We speculate that anatomical differences (branching angles and vessel diameters) between anastomotic and airway mucosal vessels may account for this observation. In order to better characterize these anastomotic vessels, three sheep were heparinized and killed with an overdose of sodium pentobarbital. The lungs were inflated to TLC with a continuous flow of air. The aorta was cross-clamped on both sides of the origin of the bronchial artery and the aortic segment was cannulated for infusion of Microfil. 60ml of Microfil was then injected into the ‘aortic pouch’. Pouch pressure was monitored during injection and did not exceed normal systemic pressure. The lungs were removed and fixed with neutral buffered formalin at total lung capacity. Pieces of airways were cut out as blocks and cleared with methyl salicylate. We observed bronchial-to-pulmonary anastomoses in vessels ranging from 30–240 mm in diameter. These vessels arose from the bronchial arterioles on the airways and directly connected to the alveolar capillaries. In some instances bronchial-to-pulmonary artery/vein communications were seen where the Microfil created a pool into the pulmonary vessels. These studies confirm the existence of precapillary bronchial-to-pulmonary anastomotic vessels greater than 100mm in sheep. SHORT COMMUNICATION