Ultrastructural visualization of human bladder mucous.

Mucous within the urinary bladder appears to play a protective role in shielding the uroepithelium against pathogens. This present study employs specific anti-mucous, antisera stabilization techniques to visualize a thin, continuous layer of mucous closely adherent to the human bladder uroepithelium, in both scanning and transmission electron microscopic analyses.

Campylobacter pylori infection in Meckel's diverticula containing gastric mucosa.

A retrospective survey was undertaken of 228 resected Meckel's diverticula to determine if there was any evidence of Campylobacter pylori infection in diverticula containing gastric mucosa. Among the 65 diverticula with gastric mucosa one was heavily infected with organisms having the morphological appearances of C pylori. The specimen had been removed from a six year old Samoan boy who had been admitted with small bowel obstruction. Infection and associated mucosal inflammatory infiltrate were limited to areas of gastric mucosa only. The detection of the organism at this site remote from the gastroduodenal environment suggests the organism may be transmitted by the orofaecal route.

The intima of human coronary arteries.

Intimal thickness relative to that of the media (r) was measured in coronary and internal mammary arteries from 300 human subjects. Whereas this ratio remained low (less than 0.17) in the mammary arteries, coronary arteries showed progressive intimal thickening (r = 4.10 by 60 years). The intimal surfaces of 70 pairs of arteries were compared by light, transmission, and scanning electron microscopy. The mammary arteries had a continuous endothelial lining, but the coronary arteries showed incomplete coverage of the thickened intima. In affected vessels the endothelial cells showed loss of attachment to adjacent cells and to the underlying tissue. It was concluded that the progressive intimal thickening of the human coronary artery, which develops early in life and is associated with defects in the internal elastic lamina, is also associated with endothelial cell separation and detachment, with the formation of denuded areas on the intimal surface.

Sucralfate interactions with gastric mucus.

Sucralfate protects the stomach against a number of experimental damaging agents and is efficacious in the treatment of peptic ulcer disease. It binds with acidity to the base of an ulcer to form a protective barrier. Sucralfate also enhances prostaglandin synthesis and release in the mucosa. In this study, the rat stomach was examined to determine sucralfate's interaction with gastric mucus. Mucus in the rat stomach forms a distinct and continuous blanket. In snap-frozen samples, pretreatment with phosphate-buffered saline as a control shows a layer of mucus of homogeneous structure thinner than the homogeneous layer after pretreatment with antibodies developed against rat gastric mucus. Pretreatment with the surface protective agent sucralfate shows some increase in the thickness of mucus with a thin dense sublayer adjacent to the epithelium and a less dense-appearing outer zone of variable thickness. Analysis of x-rays generated by the electron beam on windows of mucus and epithelium showed the expected gradients of sodium, potassium, chloride, and sulfur. The percentage of aluminum and sulfur in the mucus was higher in sucralfate-treated samples than in controls. Interaction between sucralfate and gastric mucus needs further investigation.

Significance of cyclosporin-A-associated renal surface irregularities.

The purpose of this study was to investigate unusual irregularities observed on the surface of kidneys from rats which had been treated with seven 'clinically effective doses' (25 or 50 mg/kg) of cyclosporin A (CsA). Surface corrugations were obvious on gross examination and were a universal finding in our study which involved greater than 50 rats. Examination by scanning electron microscopy (SEM) showed that the surface irregularities resulted from swollen peritubular capillaries adjacent to vacuolized and partially collapsed tubules at the cortical surface. The enlarged capillaries were associated with vascular congestion, and although peritubular capillary congestion has been recognized in CsA-treated patients, it has been difficult to determine whether this reaction is associated with drug toxicity or graft rejection. Our findings, indicating that peritubular capillary congestion in addition to vacuolization and collapse of subcapsular tubules is a drug-associated phenomenon, may help give a more accurate assessment of biopsy and autopsy material from CsA-treated patients and laboratory animals.

Host defence mechanisms in the bladder. II. Disruption of the layer of mucus.

The urinary bladder wall is lined by a layer of mucus which is believed to provide an important barrier to bacterial invasion of the urinary tract. Abnormal function of this protective layer could therefore be a factor predisposing the host to urinary tract infection (UTI). This study investigated the contribution of the bladder mucus to host defence in both acute and chronic lower UTI, using a non-obstructive animal model of infection which reproduces many features of the disease in man. The ultrastructural appearance of the infected bladder mucosa was assessed in tissue in which both the layer of mucus and bacterial glycocalyces were stabilized prior to examination by scanning and transmission electron microscopy. The protective role of the mucus layer was determined by disrupting the layer immediately prior to bacterial challenge. Both ultrastructural and bacteriological analyses have shown that infection was increased in those animals where the mucus barrier was disrupted.

Host defence mechanisms in the bladder. I. Role of mechanical factors.

This study evaluated the contribution of host defence mechanisms to bacterial clearance from the urinary bladder using an animal model in which rats were infected with Escherichia coli. Factors studied included the effect of hydration status, induced ultrastructural changes to the surface of the bladder mucosa, and the relevance of bacterial replication. Clearance was divided into two phases, primary (0-4 h), and secondary (4-24 h). Ninety-nine per cent of Escherichia coli 075 was cleared during the primary phase from normal, dehydrated and polyuric animals and 93% from anuric animals. Clearance was shown to be dependent on the presence of viable tissue. Bacterial numbers continued to decrease during the secondary phase in normal and dehydrated animals but increased in polyuric and anuric groups. No such rise occurred when rats were inoculated with Escherichia coli E/2/64, a non-replicating mutant. Evidence of ultrastructural changes to the bladder associated with impaired antibacterial properties was found in polyuric and anuric animals. Clearance of particulate matter (killed Candida albicans) however was unaffected by mucus disruption. The study has shown that the clearance of microorganisms from the bladder was unrelated to the voided volume, but is closely associated with the antibacterial activity of the mucosal surface.

Transmural differences in the postischemic recovery of cardiac energy metabolism.

After 25 minutes of ischemia, in the isolated rat preparation, hearts fail to reestablish adequate contractile function. To determine whether this failure was associated with a transmural variation in the metabolic response of myocardial cells to reperfusion, the authors subjected hearts to 25 minutes of global ischemia with and without 5 or 20 minutes of reperfusion. After freeze-drying the left ventricular myocardium was divided into subepicardial (EPI) and subendocardial (ENDO) regions before estimating the lactate, total adenine pool metabolites, and creatine phosphate (CP) and phosphate concentrations in each region. Other groups of hearts were perfusion-fixed with glutaraldehyde then injected with nuclear track emulsion to demonstrate that a high proportion of capillaries in both the subendocardial (89%) and subepicardial (95%) myocardium transmitted perfusate after 5 minutes of reperfusion. Reperfusion removed lactate equally from each region. Thus the differences in the capacity of reperfusion of these regions to recover CP (ENDO, 100%; EPI, 168% of preischemic values), to elevate adenosine triphosphate (ATP) (ENDO, 32%; EPI, 63%), or to retain adenosine monophosphate (AMP) (ENDO, 625%; EPI, 277%) were unlikely to be due to regional differences in microvascular function. Despite the better preservation of both structure and metabolism in the subepicardium, there was, during reperfusion, a progressive loss of purine precursors from cells in both regions of the myocardium. These results suggest that the loss of ability of the myocardium to recover significant function after relatively short periods of ischemia is due to their inability, on reperfusion, to synthesise sufficient ATP from the available precursors. This capacity for resynthesis of ATP is lost more rapidly in the subendocardial than in the subepicardial myocardium.

Reliable diagnosis of Trichosomoides crassicauda in the urinary bladder of the rat.

Two reliable methods are described for identifying infection of laboratory rats with the nematode Trichosomoides crassicauda. The first is a rapid method where cryostat sections of the rat urinary bladder are stained with acridine orange and viewed under a fluorescence microscope. The second involves the stabilization of the bladder surface prior to examination using scanning electron microscopy (SEM).

Mucus stabilization in the urinary bladder.

Mucus in the urinary bladder may act as an important intrinsic defence mechanism by shielding the epithelial layer from pathogens. Visualization of any such layer of mucus using electron microscopy has not been possible, as preparative procedures in current use result in the loss or distortion of mucus. We evaluated methods reported to stabilize polysaccharide layers and demonstrated that pretreatment of rat bladder tissue with anti-mucus antiserum, minimized the collapse of the mucus that normally occurred during dehydration of the tissue for electron microscopic examination. Under these conditions, mucus was stabilized as a layer closely adherent to the epithelium.

Mucosal defences and gastroduodenal disease.

Peptic ulcer disease occurs when there is an imbalance between aggressive factors and mucosal resistance. Mucus plays a key role in mucosal resistance. The mucus layer is relatively resistant to peptic digestion, it provides a layer through which there is a movement of hydrogen ions from the parietal cell to the lumen but a resistance to back-diffusion of hydrogen ions in the opposite direction. It maintains a pH gradient by 'sequestering' secreted bicarbonate and by its resistance to H+ back-diffusion. Colloidal bismuth subcitrate (De-Nol) binds to mucus to stabilize the mucous layer and increase the resistance to back-diffusion of hydrogen ions without significantly modifying the ion-exchange properties of mucus.

Preservation of mucus in situ in rat colon.

Mucus, a hydrated complex consisting mainly of glycoproteins, forms a layer over the epithelial surface of the gastrointestinal tract. The usual preparative procedures for histological and scanning electron microscopic examination of the gut result in the loss or distortion of this mucus layer. Careful evaluation of two new methods reported to stabilize the mucus layer showed that acrolein vapor did not provide adequate fixation, but application of heat-inactivated antiserum raised in rabbits against rat colon mucus reliably preserved a continuous layer closely adherent to the epithelium. This stabilized layer is continuous with the mucus in the colonic crypts.

Factors affecting the development of contraction band necrosis during reperfusion of the isolated isovolumic rat heart.

The effects of time, oxygen availability and transmural fibre location on the post-ischaemic development of contraction-band necrosis was investigated in isolated isovolumic rat hearts. Anoxic reperfusion after 60 min of total ischaemia was associated with the slow development of contraction bands (8% of myocytes affected after 20 min), particularly in the circumferentially oriented mid-myocardial muscle fibres. This could be completely prevented by the presence of 3 mM amytal. Oxygenated reperfusion caused a rapid development of contraction bands (6% of myocytes affected after 5 mins) which was significantly (P less than 0.05) increased to 18% of cells affected by 20 mins reoxygenation. The mid-myocardium always contained the greatest number of contraction bands with up to 46% of cells involved after 20 mins of oxygenated reperfusion. In all groups many cells were observed to have disrupted sarcolemmae irrespective of the presence or absence of contraction bands. The predominance of contraction bands in the constrictor muscles encircling the heart suggests that their formation may be influenced by the tension or strain imposed upon myocardial fibres during the ischaemic episodes.

Location of bacteria in the mid-colon of the rat.

The distribution of microorganisms in the mid-colon of the rat was studied by light and scanning electron microscopy. An antiserum against rat colon mucus was used to stabilize the mucus in situ. In samples not incubated with antiserum, the mucus disintegrated and contracted into patchy strands only partly covering the luminal surface of the colon. Bacteria were seen within fecal pellets, tangled among the strands of mucus, and scattered on the epithelial surface. However, when incubated with antiserum, mucus almost completely filled the lumen and coated the fecal pellets. Bacteria in these stabilized preparations were limited mainly to the fecal pellets, and there were small numbers scattered in the luminal mucus, but none were observed on the epithelial surface or within the crypts. Latex particles introduced into the lumen with the antiserum or with phosphate-buffered saline showed the same distribution as the bacteria. These findings are at variance with previous reports that organisms occur in abundance in the mucous layer, adjacent to cell surfaces, and inside crypts. Our results suggest that conventional preparation for microscopy without prior stabilization of the mucus in situ may lead to artifactual redistribution of microorganisms and emphasize the importance of mucus in maintaining mucosal-floral homeostasis in the colon.

Abdominal aortic aneurysms: distribution of elastin, collagen I and III, and intermediate filament proteins desmin and vimentin--a comparison of familial and nonfamilial aneurysms.

The aortic walls of patients with abdominal aortic aneurysms (AAA) and of healthy controls were examined for elastin, collagen I and III, and the intermediate filament proteins desmin and vimentin by immunohistochemical, enzyme histochemical, and routine histological techniques. The morphology of the aneurysmatic walls varied considerably from case to case, but many pathological changes were seen in all cases, e.g., extensive atherosclerotic plaques in the intima, prominent alterations in amount and organization of the elastic lamellae in the media, and an increase of connective tissue. Both collagen I and III were present in all the aneurysmatic walls. The smooth muscle cells in all the aortic walls showed a marked heterogeneity with respect to the morphological appearance, the enzyme histochemical features, and the content of desmin and vimentin. Vimentin occurred in some intimal, medial muscle, and adventitial cells of both the controls and the AAA patients. Desmin occurred in some of the intimal, medial, and adventitial muscle cells of both the controls and the AAA patients. All the cells with desmin in the intima and media also contained vimentin. Thus, smooth muscle cells in the walls of both the normal human abdominal aorta and aneurysms contained either vimentin, desmin, or both. This variability may be explained by the presence of different phenotypes of smooth muscle cells and could be of significance for the development of atherosclerosis and aneurysms. Of special interest was the finding that 5 of the 24 AAA patients studied had blood relatives with the same disease, suggesting a hereditary influence. However, no systematic differences between the morphological appearance of the aneurysmatic walls in familial and nonfamilial AAA could be detected.

Mucosal mast cells as a component of the inflammatory response to lower-urinary tract infection.

Globule cells have been observed in mucosae for many years. Recently, a subpopulation of these globule cells in the intestinal mucosa of man and rodents have been identified as unique mast cells. In this communication, intraepithelial globule cells and some lamina-propria mast cells found in the normal rat urinary-bladder wall have been characterized as mucosal mast cells, similar to intestinal mast cells, and differentiated morphologically and histochemically from rat peritoneal mast cells. The number of mast cells in the bladder wall increased significantly during various bladder manipulations, including mechanical trauma, parasitic infestation and bacterial infection. The origin and function of the mucosal mast cells remains unknown.

The effects of altered cation balance on the fine structure of hypoxic myocardial cells.

To determine whether the changes in intracellular/extracellular cation balance which develop in ischaemic myocardium are responsible for the fine structural changes seen in such tissue, thin slices of normal canine ventricle were incubated under hypoxic conditions at 37 degrees C and physiological pH in balanced salt solution (BSS), isotonic NaCl and isotonic KCl. Slices from each solution were fixed at 10-120 min intervals and examined by light and electron microscopy. For 60 min, tissue from both NaCl and KCl showed good overall preservation of cell architecture and only mild subcellular alterations including aggregation of nuclear chromatin, disappearance of glycogen granules, and swelling of sarcoplasmic reticulum. Tissue from BSS showed early development of intramitochondrial dense inclusions together with focal contraction-band damage similar to that seen in temporarily ischaemic, re-perfused heart muscle and at the margins of infarcts. These changes thus appear to be promoted by divalent cations. The progressive reversal of monovalent cation balance in an area of permanent and severe ischaemia does not appear to be a major determinant of fine structural change.

The effect of ischaemic injury on the fine structure of canine atrial myocardium.

An experimental model for producing a uniformly and maximally ischaemic segment of atrial myocardium has been devised and used in 23 dogs to investigate the effects of severe ischaemia of up to 6 h duration on the fine structure of atrial myocardial cells. Throughout the first 30 min of ischaemia the cells maintained a fine structure comparable with that of control tissue. In the subsequent 2 h they developed morphological alteration at differing rates, despite their uniform deprivation of blood supply. Even at 90 min the ischaemic tissue showed discontinuous change, with many areas differing little from control, and it was only after 3 h that all cells showed well developed and similar alterations. These included glycogen depletion, marked clumping and margination of nuclear chromatin, and the mitochondrial degeneration typically seen in ischaemically injured ventricle. The changes were very severe after 4 and 6 h of ischaemia, when disruption of cellular membrane systems was also evident. At these times the tissue closely resembled ventricular myocardium made ischaemic for similar intervals, except for the persistence of well-preserved 'specific atrial granules'. Atrial muscle cells therefore develop the fine structural changes associated with ischaemia more slowly than ventricular muscle cells and have a more variable response to the initiation of ischaemic injury.

Changes in the contractile state, fine structure and metabolism of cardiac muscle cells during the development of rigor mortis.

Transmural slices from the left anterior papillary muscle of dog hearts were maintained for 120 min in a moist atmosphere at 37 degrees C. At 15-min intervals tissue samples were taken for estimation of adenosine triphosphate (ATP) and glucose-6-phosphate (G6P) and for electron microscopic examination. At the same time the deformability under standard load of comparable regions of an adjacent slice of tissue was measured. ATP levels fell rapidly during the first 45 to 75 min after excision of the heart. During a subsequent further decline in ATP, the mean deformability of myocardium fell from 30 to 12% indicating the development of rigor mortis. Conversely, G6P levels increased during the first decline in adenosine triphosphate but remained relatively steady thereafter. Whereas many of the myocardial cells fixed after 5 min contracted on contact with glutaraldehyde, all cells examined after 15 to 40 min were relaxed. A progressive increase in the proportion of contracted cells was observed during the rapid increase in myocardial rigidity. During this late contraction the cells showed morphological evidence of irreversible injury. These findings suggest that ischaemic myocytes contract just before actin and myosin become strongly linked to maintain the state of rigor mortis.