Microvascular transport and endothelial cell alterations preceding skeletal muscle damage in ischemia and reperfusion injury.

We determined the leakage of macromolecules using FITC-dextran-150 as a tracer and measured the extent of no-reflow phenomenon by video field analysis. The cremaster muscle of anesthetized rats was fashioned as a single layer, splayed on a lucite chamber and suffused with bicarbonate solution at 35 degrees C. After a 1 hour period of baseline data collection, ischemia was produced by cross-clamping the cremasteric vascular pedicle for periods of 30 minutes and 2 hours in separate experiments. Macromolecular leakage was visualized after reinstitution of perfusion. Leakage occurred at postcapillary venules 15 to 50 micron in diameter and quickly spread to the interstitium. The magnitude of leakage decreased as a function of time with continuous buffer suffusion, but remained higher than in the control period. No reflow occurred in approximately 30 percent of the muscle microvasculature upon reperfusion. The no-reflow values at 30 minute and 2 hour periods of ischemia were significantly different from the control values but were not from each other. Electron micrographs demonstrated endothelial cell swelling and migration of leukocytes and normal myocytes after 1 hour of reperfusion following 2 hours of ischemia. Our results demonstrate that permeability changes, occurrence of no reflow, and leukocyte migration precede the onset of damage to skeletal muscle in ischemia and reperfusion injury.

An ultrastructural morphometric comparison of the peripheral with the hilar air-blood barrier of dog lung.

Excised and isolated perfused lungs are frequently used as models for studies of fluid flow and membrane transport. Regional variations of the air and blood vascular compartments of the lung have been recognized to result from gravitational effects. This ultrastructural morphometric study considered the components of the air-blood barriers of the peripheral and hilar regions of excised dog lungs. The lungs of three mongrel dogs were fixed by endotracheal instillation of glutaraldehyde and immersion in fixative. Stratified random sampling, point counting volumetry, and line intercept counts were used to determine the thicknesses of the air-blood barrier and the epithelial, interstitial, and endothelial compartments. Point counting volumetry also established the volume density of the alveolar spaces. The morphometric values for structures within the periphery were statistically compared to those within the hilar region by Student's t-test. Endotracheal and immersion fixation as used in this study, combined with stratified random sampling, equalized regional differences in the alveolar lumen volume and air-blood barrier thickness. With these conditions no significant differences in the volume densities of the epithelial and endothelial cells and the interstitium of the air-blood barrier were identified when those of the hilum were compared to those of the periphery.

Morphological changes in lipid droplets within cells of the duodenal transition zone in suckling rats.

This ultrastructural study compares the lipid droplets within epithelial cells at the bases of the duodenal villi in the transition zone between crypts and the lateral portions of the adjacent villi in newborn rats suckled 24 h, 10 days and 15 days and contrasts with cells in similar locations in newborn unsuckled and weaned rats. The mean diameters and numbers of lipid droplets per field decreased when cells from rats at 10 and 15 days of age were compared to those at 24 h of age. The range of sizes also narrowed. There was cell-to-cell variability in number and in opacity of the lipid droplets. Multiple membranous laminae enclosed the droplets. Cells in similar locations in newborn unsuckled and in weaned rats did not contain lipid droplets. It is concluded that the numbers and sizes of the large lipid droplets within cells in the transition zones at the bases of the villi decrease as cells at the same relative locations mature during suckling and as the infant rats mature.

An ultrastructural morphometric comparison of the right and left sides and lobes of the dog lung.

Regional variations of the components of lobes of the lung have been recognized to result from gravitational effects. Excised and isolated perfused lungs are frequently used as objects for studies of fluid flow. This ultrastructural study considered differences between the right and left sides and lobes of the excised lung. The lungs of three mongrel dogs were fixed by simultaneous endotracheal instillation of glutaraldehyde and immersion in fixative. Stratified random sampling, point-counting volumetry, and line intercept counts were utilized to determine the volume densities of alveolar space, endothelial vesicles, interstitial cells, fibers, and matrix. Thicknesses of the air-blood barrier, epithelial, interstitial, and endothelial compartments were measured. Values for the right side were compared to values for the left by t-test. Values for lobes were compared statistically by a two-way analysis of variance. There was no statistically significant column effect, no row effect, and no interaction between the mean measurements of the factors computed for the right and left lungs and the lobes within the lungs. It is concluded that stratified random sampling combined with endotracheal and immersion fixation as used in this study equalize regional differences in the alveolar lumen volume and air-blood barrier thickness. With these conditions there are no significant differences by sides or lobes in the volume densities of the epithelial and endothelial cells and components of the interstitium of the air-blood barrier.

Ultrastructure of casein in the stomach and duodenum of infant rats at different ages of suckling.

Casein coagulate in stomachs of suckling rats at 12 and 24 h and 5, 10, and 15 days of age was examined by transmission electron microscopy. The ingested casein particles in the layered gastric milk curd were linked to produce clumps and chains of protein. This coagulation probably was caused by low pH. At 12 and 24 h of age the gastric milk curd also contained large masses of 4 to 8 nm granules. These large masses were unreported and may represent a specialization of colostrum in this species. Both particulate and granular forms disperse in the duodenum, probably as a result of enzyme action.

Milk lipid absorption and chylomicron production in the suckling rat.

Structural correlates of milk lipid absorption and chylomicron production were studied in 10-day-old suckled rats. The gastric and duodenal contents and duodenal mucosae were examined with the light and electron microscopes. In the gastric lumen the milk lipid globule cores were smooth, circular and uniformly electron opaque. Many membranes and lamellar structures with a trilaminar and multilamellar appearance were adherent to the peripheries of the cores. In the central duodenal lumen the milk lipid globule cores were also smooth, circular and uniformly electron opaque. Very few milk lipid globules in the duodenal lumen showed adherent membranes or lamellae. Membrane fragments and lamellae were present in the lumen separate from the milk lipid globules. In the duodenal lumen between villi the milk lipid globules had multiple electron lucent indentations of the core. It is believed that the irregular peripheries of the milk lipid globule cores are the result of lipolysis within the duodenal lumen acting at the milk lipid globule surface. This lipolysis of triacylglycerol would produce amphiphilic lipids which may result in the electron lucent spaces at the milk lipid globule periphery. The absorptive epithelial cells along the length of the duodenal villus varied in structure relative to their position at the tip, middle, or base of the villus. Typical mid-villus epithelial cells contained lipid droplets averaging 0.3-micrometer diameter in the smooth and rough endoplasmic reticulum and in Golgi complexes in the apical cytoplasm. Villus tip and villus base cells contained large lipid droplets between 7-16 micrometers. Only a few 0.3-micrometer lipid droplets were present within these cells. These large lipid droplets appeared to be accumulations of triacylglycerol present in the apical cytoplasm associated with lamellar and membranous structures. Numerous chylomicrons were present between epithelial cells located in the middle region of the villus while significantly fewer chylomicrons were seen between epithelial cells at the tip and base of the villus. These observations suggest that the cells at the middle of the duodenal villus of suckling rats were more efficient in the production of chylomicron triacylglycerol derived from incoming milk triacylglycerol than cells at the tip and base of the villus.

Phagocytic inability of Kurloff cells in the lung and spleen of the guinea pig.

Adult female guinea pigs received subcutaneous implants of diethylstilbestrol-cholestrol pellets which produced splenomegaly and increased numbers of splenic Kurloff cells. Latex spheres subsequently injected intravenously were not phagocytized by Kurloff cells within the lungs and spleen as examined with the electron microscope. This is considered as evidence that Kurloff cells are probably not phagocytic. The origin of these cells is discussed.

Morphological changes in isolated perfused dog lungs after acute hydrostatic edema.

Morphometric data from stable (nonedematous) isolated dog lungs, perfused with nearly cell-free perfusates, were compared to similar stereological evaluations of isolated dog lungs after induction of severe acute hydrostatic edema. In the edematous lungs, capillary surface and volume densities were substantially increased. Alveolar surface density was also increased. Thicknesses of the endothelial and type I epithelial cellular compartments of the air-blood barrier were unchanged. Thickness of the interstitial compartmemt of the air-blood barrier was substantially increased and this, in turn, caused an overall increase in mean thickness of the barrier. Volume densities of the nonparenchymal connective tissue spaces surrounding the extra-alveolar vessels and airways were also increased. In both the endothelial and type I epithelial cells cytoplasmic volume densities of pinocytotic vesicles were increased. In addition, the number of vesicles opening onto the luminal and albuminal cellular surfaces increased signficantly. Transendothelial vesicular passage may contribute to interstitial edema formation, and transepithelial vesicular transport may contribute to alveolar flooding in isolated perfused dog lungs.

Isolated perfused dog lungs: a comparative stereologic comparison with normal dog lungs.

Established stereologic techniques were used to evaluate the morphologic integrity of isolated dog lungs perfused with plasma for periods of 30 minutes, 1 hour and 2 hours. The morphometric data from the isolated lung preparations were compared to similar morphometric evaluations of dog lungs fixed immediately after removal from the thorax. In the isolated lungs capillary surface and volume densities were both substantially decreased. These estimations of capillary surface density provide a morphologic definition of capillary surface area which should be useful in the estimations of endothelial permeability in isolated lungs. This morphometrically defined decrease in capillary volume density was attributed, in part, to swelling of the endothelial cells. Alveolar surface density was also decreased and the type-I epithelial cells were increased in thickness. In both the endothelial and epithelial cells, cytoplasmic volume densities of the mitochondria and smooth endoplasmic reticulum were increased, while those of the rough endoplasmic reticulum and micropinocytotic vesicles were unchanged. The thickness of the interstitial compartment of the air-blood barrier and the volume densities of the peribronchial, peribronchiolar, and perivascular connective tissue sleeves were unchanged; there was no evidence of interstitial edema in the isolated lungs. These morphologic change must be considered in the interpretation of physiologic studies which employ isolated perfused dog lung preparations.

A morphometric examination of type II alveolar epithelial cells in normal and isolated-perfused dog lungs.

The volume densities of type II alveolar cell cytoplasmic organelles and alveolar surface densities were estimated by established stereologic procedures. The morphometric measurements were obtained from normal dog lungs (in situ) and isolated dog lungs perfused for 30-minute, 1-hour, and 2-hour periods. The type II cell lamellar body volume densities and the alveolar surface densities progressively decreased as the times of perfusion were increased. The volume densities of the granular and agranular endoplasmic reticulum progressively increased during the periods of perfusion. These morphometric parameters from lungs in situ and isolated lungs suggest that changes occur in pulmonary surfactant synthesis and activity during perfusion. It is further postulated that progressive increases in the rates of surfactant removal and/or inactivation during perfusion may contribute to spontaneous edema in lungs isolated for periods exceeding two hours. The morphologic and physiologic integrity of isolated perfused lung preparations, widely used as models of lungs in vivo, in situ requires further evaluation.