Heterogeneity of the composition and thickness of tracheal mucus in rats.

Inability to preserve airway mucus in situ has limited our understanding of its structure and function. This light- and transmission electron-microscopic study of rat tracheal mucus used a nonaqueous fixative that retains mucus (epiphase) over a lucent layer (hypophase). The fixative is a 1% solution of osmium tetroxide dissolved in a perfluorocarbon. The mean thickness of rat tracheal epiphase was 5 microns, with significant variation (0.1-50 microns) around the tracheal circumference. Tracheal mucus was thickest at the trachealis muscle region and contained cells, cellular debris, and a variable amount of surfactant and lipid, estimated at 4-16% of the total epiphase in five rats, with a mean composition of 9%. Lipid was observed on the surface of the epiphase, embedded within mucus, and at the epiphase-hypophase interface. Refined study of developmental, physiological, and pathological alterations to the airway coat may benefit from this approach.

Interleukin-2 alters the positions of capillary and venule pericytes in rat cremaster muscle.

Interleukin-2 (IL-2) mediates regression of metastatic cancer, but its therapeutic efficacy has been limited by toxicities which occur secondarily to IL-2 induced macromolecular leakage from microvessels. Previous studies have indicated that pericytes function in an endothelial cell junction-dependent manner in lung capillaries and cremaster muscle venules, possibly to limit cellular and macromolecular leakage. The purpose of this investigation was to determine if pericyte positioning on skeletal muscle capillaries was junction-related, and if it was altered by IL-2. Anesthetized rats received intravenous injections of fluoroscein isothiocyanate conjugated to albumin. The cremaster muscle model of microcirculation was used in conjunction with intravital fluorescence microscopy to monitor changes in interstitial fluorescence which was used as an index of macromolecular leakage. IL-2 induced a progressive increase in interstitial albumin which started within 30 min of application and continued to increase until the end of the experiment at two hours. Cremaster muscle tissue was fixed and prepared for examination by transmission electron microscopy. A digitizing platter and morphometric software were used to quantify pericytes near vs away from endothelial cell junctions of capillaries and venules. In microvessels from animals in the control group, pericytes were randomly positioned on capillaries, and concentrated at endothelial cell junctions of venules. After treatment with IL-2, capillary pericytes were concentrated at junctions. Venular pericyte density also increased in endothelial junctional regions. IL-2 appears to alter the distribution of pericytes in the microcirculation, perhaps by induction of contraction.

Heterogeneity of pericyte populations in equine skeletal muscle and dermal microvessels: a quantitative study.

The objective of this ultrastructural investigation was to determine if populations of pericytes in equine dermal and skeletal muscle capillaries increase in a head-to-foot direction, as has been reported in human skeletal muscles. Samples of equine microvessels were obtained from the longissimus dorsi skeletal muscle 150 cm. from the ground, from the dermis above this muscle, from the extensor carpi radiali muscle at 55 cm. from the ground, from the dermis adjacent to that muscle, and from dermis 15 cm. from the ground, just above the hoof wall. Tissues were processed for transmission electron microscopy. Electron micrographs were analyzed with a digitizing tablet and computer, to determine the ratios of endothelial cell outer circumference and pericyte inner lengths. Pericytes were separated into two classes; those closest to the endothelial cells were defined as covering capillaries. Those separated from endothelial cells by another layer of pericytes were termed enveloping pericytes. There was much greater coverage and envelopment of dermal capillaries (85% and 135%) than skeletal muscle capillaries (27% and 31%). Regression analysis of the pericyte coverage and envelopment of dermal capillaries revealed a significant increase in pericytes toward the ground. Similarly, the two skeletal muscle tissues differed significantly in their pericyte coverage and envelopment (25/27% at 150 cm., 31/35 at 55 cm.). The data indicate that, as in humans, capillary pericytes are not homogeneously distributed within the same tissues, but are more numerous closer to the ground. Differences in pericyte populations could affect studies of microvessel function.

Attempts to modify reperfusion injury of equine jejunal mucosa using dimethylsulfoxide, allopurinol, and intraluminal oxygen.

This study compared the severity of ischemic injury to the equine jejunal mucosa caused by arteriovenous obstruction (AVO) or venous obstruction (VO) with that caused by reperfusion after ischemia. The degree of mucosal damage and regeneration was scored according to a modified version of an established light microscopic classification for ischemic injury. Biopsy specimens taken after 3 and 4 hours of obstruction, and after 3 hours of obstruction and 1 hour of reperfusion, were compared. There were no changes in the severity of mucosal injury (characterized by epithelial sloughing, loss of villus architecture, and necrosis of crypt cells) at 4 hours of ischemia when compared with 3 hours of ischemia. The mucosal injury score increased by one grade in three of six and five of eight segments during reperfusion for the VO and AVO models, respectively; however, only the scores for the AVO model were significantly different from the injury caused by ischemia alone. Modification of reperfusion injury was attempted by the administration of intravenous (IV) allopurinol, dimethyl sulfoxide (DMSO), or intraluminal oxygen insufflation at the time of release of the AVO and VO. Treatments did not significantly alter either the severity of injury noted after 1 hour of reperfusion or the degree of mucosal regeneration after 48 hours of reperfusion. In this group of ponies, the severity of mucosal damage was greater after 1 hour of reperfusion for both AVO and VO.

Non-aqueous fixative preserves macromolecules on the endothelial cell surface: an in situ study.

A fixative composed of 1% w/v osmium tetroxide dissolved in a tluorocarbon (FC-72) was used to perfusion-fix rat blood vessels for this ultrastructural study. With non-aqueous fixation, the lumenal surfaces of endothelial cells in cardiac and skeletal muscles retained an extracellular coat which was morphologically consistent with glycocalyx. Venules displayed the greatest density and depth (up to 870 nm thick) of glycocalyx, but all vessel types had at least 70 nm of surface coat. The results indicate that non-aqueous fixatives will be useful in studies of the endothelial cell lumenal surface, while offering excellent preservation of general ultrastructure.

Non-aqueous fixative preserves macromolecules on the endothelial cell surface: an in situ study.

A fixative composed of 1% w/v osmium tetroxide dissolved in a fluorocarbon (FC-72) was used to perfusion-fix rat blood vessels for this ultrastructural study. With non-aqueous fixation, the lumenal surfaces of endothelial cells in cardiac and skeletal muscles retained an extracellular coat which was morphologically consistent with glycocalyx. Venules displayed the greatest density and depth (up to 870 nm thick) of glycocalyx, but all vessel types had at least 70 nm of surface coat. The results indicate that non-aqueous fixatives will be useful in studies of the endothelial cell lumenal surface, while offering excellent preservation of general ultrastructure.

Preservation of tracheal mucus by nonaqueous fixative.

Two nonaqueous fixatives, composed of fluorocarbon solvents with dissolved osmium tetroxide, were used to determine the feasibility of preserving the mucous coat in bovine and rat trachea for light and electron microscopy. Aqueous fixatives, while providing excellent cytological preservation, wash away the mucous lining, precluding ultrastructural analysis. Inclusion of ruthenium red or alcian blue within aqueous fixative improved retention of mucus, but provided incomplete, patchy results. Fixation with nonaqueous fluorocarbon solvent and dissolved osmium tetroxide preserved a continuous mucous epiphase layer above a clear hypophase layer. Subcomponents of the mucus included an electron dense surface layer, interrupted patches of mucus above the surface layer and electron dense membrane-like material within the mucus. This method of fixation will preserve mucus for light, scanning and transmission electron microscopy, using either intratracheal or immersion methods of fixation. The latter would enable use of materials from large animal models, autopsy or an abattoir.