Ultrastructural changes in the pulmonary arterioles in acute hypoxic pulmonary hypertension in the rat.

This study was planned to obtain ultrastructural details of the early changes in intra-acinar arterioles in acute hypoxic pulmonary hypertension that could lead to understanding the mechanisms in the development of chronic hypoxic hypertension. In the anesthetized rat, using 5-10% normobaric O2, within minutes after hypoxia, there are changes in endothelial cells characteristic of activation: prominence of cell body and protuberance of the nucleus, electron-dense membrane-bound bodies adluminally, increasing pseudopodia of the adluminal cell membrane, edema within (vacuoles) and beneath the endothelial cells with separation of the endothelial cells from the basal lamina. There is activation of platelets and leucocytes in the lumen and accumulation of platelets at the endothelium. Arteriolar wall edema rapidly increases, is excessive within 1 h, with dissection of the basal lamina and wall and cytolysis of wall components. At 24 h edema is reduced, the number of platelets is increased at the endothelium and fibroblasts are newly aligned within the arteriolar wall. At 48 h platelets further increase, a basal lamina develops in fibroblasts termed transitional cells and myofibrils occur subsequently to form smooth muscle. These findings suggest that activation of the endothelial cell is the initial event in a cellular cascade in the arteriolar hypoxic responses with fibroblast-to-smooth muscle transformation, which results in pulmonary arteriolar hyperplasia and vascular remodeling in hypoxic chronic pulmonary hypertension.

Age related alterations in the response of the pial arterioles to adenosine in the rat.

To evaluate the effects of aging on vasoreactivity of pial arterioles to adenosine and barium chloride, an hydraulically intact cranial window preparation was developed in the rat. The microvasculature of anesthetized 3- and 24-month-old Fischer-344 rats was studied during superfusion with artificial cerebrospinal fluid with and without test agents and results determined by videomicroscopy techniques. In both cohorts, the response of pial arterioles to adenosine was both dose and vessel size dependent: arteriolar dilation increased with increasing concentrations of adenosine and at any given concentration the percent increase in diameter was greater in the smaller vessels. During adenosine superfusion the absolute changes and percent increase in vessel caliber were greater in the young rats. Arteriolar vasoconstriction due to barium chloride was vessel size dependent but there were no significant differences in response between young and aged rats. The results indicated an attenuated cerebrovascular response in aged rats to adenosine, but not to barium chloride. This may be due to a difference in the mode of action in these two compounds. Venules did not respond to adenosine at any concentration.

Acute wound repair in an aged animal: a model for accelerated aging of the microvasculature?

This study of wound repair in the aged rat is based on increased carbohydrate content of various proteins which occurs with aging and is readily seen in the microvasculature (MV). We have used the periodic acid-Schiff (PAS) reaction to identify histochemically the carbohydrate moiety of the glycoproteins found in these blood vessels. In the young rat, as in other young vertebrates, elements of the MV are PAS negative and become increasingly PAS+ beyond the half life span. During acute wound repair in an old animal, the new capillaries and venules are PAS- 2 weeks after injury, moderately PAS+ at 4 weeks, and intensely PAS+ at 8 weeks. Arterioles are present and PAS+ at 6 weeks, and intensively positive at 8 weeks, comparable to vessels remote from the wound site. The MV in wound repair in a young animal remains PAS- throughout healing. Rapid aging of the microvasculature in wound repair in an old animal reproduces histochemically the aging which occurs progressively during the prior 24 months. These histochemical changes may result from successive enzymatic and nonenzymatic glycosylation of the various basement membrane proteins in the microvasculature in both normal aging and wound repair in the aged animal. The latter may serve as a model for study of accelerated aging.

Histochemical characterization of the aging microvasculature in the human and other mammalian and non-mammalian vertebrates by the periodic acid-Schiff reaction.

Prior histochemical studies with the periodic acid-Schiff (PAS) reaction have shown altered biochemical composition in a limited part of the microvasculature (MV) in aging in two species of laboratory animals. We therefore studied, with the PAS reaction, all the components of the MV in multiple tissues from various immature, adult and aged mammals, including human, and immature and aged nonmammalian vertebrates. We now demonstrate that there is an altered biochemical composition of capillaries, arterioles and venules in various tissues with aging. These are first detectable somewhat beyond half the life-span in man (greater than 45 years), marmoset (5 years) and dog (8 years) and seen in old fish, reptiles and birds. The capillary wall is increasingly PAS+; in arterioles there are focal PAS+ areas in the media which increase in size and number with age and become hyalinized masses. The non-muscular venules are increasingly PAS+ apparently due to a polysaccharide staining of connective tissue elements. These histochemical changes in the MV with aging are in the extracellular matrix and appear to be a specific manifestation of aging in vertebrates. The consequences of such changes in MV aging may be important physiologically.

Demonstration of uniform capillary basal lamina thickness by computer technology.

Blood capillaries are assumed to be circular cylinders composed of an inner layer of endothelial cells surrounded by an acellular uniformly thick basal lamina. The capillary basal lamina is the structural and functional interface between the capillary endothelial cells and the adjacent extracellular matrix, important in growth and control mechanisms of the endothelial cell. Capillaries examined in electron micrographs are cut randomly and a projected image of a capillary other than perpendicular to its long axis will produce artifactual thickening of the assumed uniformly thick basal lamina. We have developed an interactive computer program to determine the thickness of the capillary basal lamina that corrects the thickness resulting from the sectioning artifact. We have applied this methodology to demonstrate that the basal lamina of the pancreatic capillaries of the rat are uniformly thick.

Collagen and elastin fibers in human pulmonary alveolar walls.

The morphology and morphometric data of collagen and elastin fibers in the pulmonary alveolar walls are presented. Specimens were obtained from postmortem lungs quick-frozen at specified transpulmonary pressures. Collagen was stained by silver, and elastin was stained by orcein. Photomicrographs were composed by computer. Young lungs typically show small collagen fibers that radiate from the "posts," whereas larger fiber bundles traverse the septum irrespective of capillary blood vessels. In older lungs, rings of collagen around the posts appear enlarged. Elastin bundles do not show obvious variation in pattern with age and inflation pressure. Statistical frequency distributions of the fiber width and curvature are both skewed, but the square root of the width and the cube root of the curvature have approximate normal distributions. Typically, for young lungs at transpulmonary pressure of 4 cmH2O, the mean of (width)1/2 (in micron1/2) for collagen fibers is 0.952 +/- 0.242 (SD), that of (curvature)1/3 (in micron-1/3) is 0.349 +/- 0.094. The corresponding values for elastin are 0.986 +/- 0.255 and 0.395 +/- 0.094.

Elasticity of arterioles and venules in postmortem human lungs.

The elasticity and branching order of noncapillary microscopic blood vessels less than 100 microns diam were studied in human lungs obtained 7-30 h postmortem, using a silicone elastomer method that selectively filled pulmonary arterioles or venules. The lungs were inflated to 10 cmH2O pressure and a gradient of transmural vascular pressure of 0-17 cm H2O, from lobe base to apex, was established in the silicone-filled vascular system. Histological materials were obtained after airway fixation by formaldehyde solution and analyzed for vessel diameter in the branching order of 1, 2, and 3, with the smallest noncapillary vessel designated as order 1, in accord with the Strahler system. The change in vessel diameter within a branching order at different levels of transmural pressure is a derived measure of vascular elasticity expressed as compliance coefficient alpha, alpha Values are 0.128, 0.164, and 0.210 micron/cmH2O or 0.682, 0.472, and 0.354%/cmH2O, respectively, of orders 1-3 for arterioles and 0.187, 0.215, and 0.250 micron/cmH2O or 0.992, 0.612, and 0.424%/cmH2O, respectively, of orders 1-3 for venules. The percent is normalized with D0, which is the value of diameter (D) when the transmural pressure is zero. These data are compared with those for the cat where alpha = 0.274 for similar juxta-alveolar vessels.

Collagen and elastin fibers in human pulmonary alveolar mouths and ducts.

To provide a quantitative basis for the understanding of the mechanical properties of the lung tissue, the morphology of the pulmonary alveolar ducts was studied and the dimensions of the collagen and elastin fiber bundles in the alveolar mouths were measured. Statistical data are presented in this report. It was found that the probability frequency functions of the widths of both the collagen and elastin fibers are skewed to the right, but the fourth roots of the widths are normally distributed. Hence, knowing the mean and standard deviation of (width)1/4, the probability of finding fiber bundles of width D is known. On the other hand, we already know the analytic expressions of the strain energy per unit length of fibers of given width. With the probability distribution of width and an estimation of the length of alveolar mouths, the strain energy of the alveolar mouths can be computed. Then the contribution of the alveolar mouths to the stress in the lung parenchyma due to any strain can be obtained by a differentiation of the strain energy function with respect to that strain.

How many pulmonary alveoli are supplied by a single arteriole and drained by a single venule?

A detailed measurement of histological specimens of the lungs of the cat shows that each terminal precapillary vessel (arteriole) supplies, on the average, 24.5 pulmonary alveoli; each terminal postcapillary vessel (venule) drains, on the average, 17.8 alveoli. These numbers link pulmonary alveolar blood flow in capillary sheets with the flow in pulmonary arteries and veins which are cylindrical tubes. They are key numbers needed for hemodynamic analysis. In the literature, these numbers are variously speculated to be 1 or smaller; thus our results correct, even though only for the cat, an important concept.

Morphometry of cat's pulmonary arterial tree.

Morphometic data of the pulmonary artery in the cat's right lung are presented. Silicone elastomer casts of cat's right lung were made, and measured, counted and analyzed. The Strahler system is used to describe the branching pattern of the arterial vascular tree. These data are needed for any quantitative approach to the study of the pulmonary circulation. For all the pulmonary blood vessels of the cat lying between the main pulmonary artery and the capillary beds, there are a total of 10 orders of vessels in the right upper lobe, 9 orders of vessels in the right middle lobe and 11 orders of vessels in the right lower lobe. The ratio of the number of branches in successive orders of vessels or the branching ratio, is 3.58. The corresponding average diameter ratio is 1.72, whereas the average length ratio is 1.81.

Changes in arteriole in acute and chronic hypoxic pulmonary hypertension and recovery in rat.

After 1 h of exposure to 0.5 atm of pressure, the electron microscopy of intra-acinar arterioles of the young female adult rat showed edema and subendothelial blebs. Pulmonary hypertension developed rapidly with an increase in hemoglobin, hematocrit, and right ventricular weight. By 24 h, there was a threefold increase in the number of fibroblasts within the arteriolar wall, followed during the next 2 days by transformation of the fibroblast through a transitional cell form to a smooth muscle cell. By 1 wk, the neomuscularization was essentially complete. There was further minor thickening and increase in density of the wall over the next 9 mo. On return to 1 atm after prolonged hypoxia, within 4 wk, the smooth muscle of neomuscularized arterioles dedifferentiated but did not disappear. There was a concurrent rapid fall in the pulmonary arterial pressure, hemoglobin, hematocrit, and right ventricular weight. Veins, capillaries, and arteries remained normal. Parallel studies in the male rat during 14 days of hypoxia demonstrated the same phenomena except slightly accelerated over the female. The rapid sequential changes in the arteriole, beginning with subendothelial blebs and wall edema, followed by fibroblast recruitment and transformation into smooth muscle through a transitional cell form, suggest a cascade. The anatomic and physiological responses to hypoxia are not sex related.

Morphometry of cat pulmonary venous tree.

Morphometric data of the pulmonary veins in the cat right lung are presented. Silicone elastomer casts of the right lungs of five cats were made, measured, counted, and analyzed. The Strahler system is used to describe the branching pattern of the vascular tree. These data are needed for the physicomathematical approach to pulmonary circulation. For all the pulmonary blood vessels lying between the left atrium and the capillary beds, there are a total of 10 orders of vessels in the right upper lobe, 9 orders of vessels in the right middle lobe, and 11 orders of vessels in the right lower lobe. The ratios of the diameters, lengths, and the number of branches in successive orders of vessels are called the diameter, length, and branching ratios, respectively. For the cat pulmonary venous tree, the average branching ratio is 3.521, the average diameter ratio is 1.727, and the average length ratio is 2.402 for vessels of orders 1-3 and 1.532 for vessels of orders 4-10.

Patency and compliance of pulmonary veins when airway pressure exceeds blood pressure.

Our measurements on cat's lung show that pulmonary veins and venules are not collapsible, but remain open when the alveolar gas pressure (PA) exceeds the local blood pressure (Pv). Their compliance constants show no discontinuity as Pv falls below PA. The capillaries, however, do collapse when PA greater than Pv. The explanation of the patency of the veins when PA greater than Pv is the pulling on the blood vessels by tension in the interalveolar septa. Photomicrographs show that each venule (or vein) is pulled radially by three or more interalveolar septa. Capillary sheets, however, are exposed to gas on the lateral sides and can readily collapse when PA greater than Pv. These facts provide the key to the analysis of pulmonary blood flow in zone 2. The "sluicing" gate, i.e., the site of flow limitation, must be located at the junctions of capillary sheets and the first generation of venules. Further, data on the branching pattern and compliance of small pulmonary veins, which are needed in quantitative analysis of pulmonary circulation, are presented.

Computer-derived image compositing.

A computer-derived composite image is obtained of the details contained in successive photomicrographs taken by optical sectioning of the collagen networks in the pulmonary interalveolar wall. Three-dimensional information is therefore contained in a two-dimensional portrayal, providing a basis for computer digital analysis otherwise not readily available.

Redistribution of canine left ventricular myocardial blood flow in unloaded systole.

The left coronary arteries of dogs were cannulated and perfused with blood from support dogs. The experimental hearts were unloaded by severing the aortas to maximize strains and minimize fiber stress. In each heart we compared the transmural distribution of blood flow in two states: (1) provision of perfusion pressure (40 mm Hg) only during systole and then (2) provision of perfusion pressure throughout the cardiac cycle. The distribution of flow in each of these perfusion states was labeled with a diffusible radioisotope (42K or 86Rb, one labeling the first state; the other labeling the second). Quantitative, paired autoradiography was used to visualize the two flow distributions. The differences between the two distributions after standardization was plotted as differences between activity vs. depth in the myocardium (r = 0.91). This was fitted with a line by least squares, the slope of which was significantly different from zero at the 0.005 level. The magnitude of the gradient of the systolic flow was represented by the ratio of deep to shallow flow. The mean of these ratios was 0.54 +/- 0.12 (95% confidence interval). A graphical analysis shows that the data are consistent with a gradient of extravascular compression across the left ventricular wall.