Lanthanum staining of the surface coat of cells. Its enhancement by the use of fixatives containing Alcian blue or cetylpyridinium chloride.

Among the techniques which have been reported to stain the surface coat of cells, for electron microscopy, is lanthanum staining en bloc. Similarly, the presence of the cationic dye, Alcian blue 8GX, in a primary glutaraldehyde fixative has been reported to improve the preservation of the surface coat of cells of many types; however, the preserved coat is not very electron opaque unless thin sections are counterstained. The present paper shows that for several rat tissues lanthanum staining en bloc is an effective electron stain for the cell surface, giving excellent contrast, if combined sequentially with prefixation in an aldehyde fixative containing Alcian blue. The cationic substance cetylpyridinium chloride was found to have a similar effect to that of Alcian blue in enhancing the lanthanum staining of the surface coat material of the brush border of intestinal epithelial cells. The patterns of lanthanum staining obtained for the tissues studied strikingly resemble those reported in the literature where tissues are stained by several standard methods for demonstrating mucosubstances at the ultrastructural level. This fact and the reproduction of the effect of Alcian blue by cetylpyridinium chloride constitute a persuasive empirical argument that the material visualized is a mucopolysaccharide or mucopolysaccharide-protein complex.

Endothelial contraction induced by histamine-type mediators: an electron microscopic study.

Previous work has shown that endogenous chemical mediators, of which histamine is the prototype, increase the permeability of blood vessels by causing gaps to appear between endothelial cells. In the present paper, morphologic and statistical evidence is presented, to suggest that endothelial cells contract under the influence of mediators, and that this contraction causes the formation of intercellular gaps. Histamine, serotonin, and bradykinin were injected subcutaneously into the scrotum of the rat, and the vessels of the underlying cremaster muscle were examined by electron microscopy. To eliminate the vascular collapse induced by routine fixation, in one series of animals (including controls) the root of the cremaster was constricted for 2-4 min prior to sacrifice, and the tissues were fixed under conditions of mild venous congestion. Electron micrographs were taken of 599 nuclei from the endothelium of small blood vessels representing the various experimental situations. Nuclear deformations were classified into four types of increasing tightness (notches, foldsl closing folds, and pinches. In the latter the apposed surfaces of the nuclear membrane are in contact). It was found that: (1) venous congestion tends to straighten the nuclei in al groups; (2) mediators cause a highly significant increase in the number of pinches (P < 0.001), also if the vessels are distended by venous congestion; (3) fixation without venous congestion causes vascular collapse. The degree of endothelial recoil, as measured by nuclear pinches, is very different from that caused by mediators (P < 0.001). (4) Pinched nuclei are more frequent in leaking vessels, and in cells adjacent to gaps (P < 0.001); (5) mediators also induce, in the endothelium, cytoplasmic changes suggestive of contraction, and similar to those of contracted smooth muscle; (6) there is no evidence of pericyte contraction under the conditions tested. Occasional pericytes appeared to receive fine nerve endings. Various hypotheses to explain nuclear pinching are discussed; the only satisfactory explanation is that which requires endothelial contraction.

A stereological study of the glomerular filter in the rat. Morphometry of the slit diaphragm and basement membrane.

Kidney from normal male albino rats, of body weight 170-200 g, was fixed by arterial perfusion with buffered tannic acid-glutaraldehyde, and postfixed with osmium tetroxide. Random and isotropic ultrathin sections from 23 different glomeruli from five rats were mounted on slot grids for staining and electron microscopy. Prints of whole glomeruli at a magnification of 3,909 were analyzed by stereological methods. The mean glomerular volume was (8.048 +/- 0.474) X 10(5) mum3 if the glomeruli are treated as spheres. The area of the basement membrane was 0.281 +/- 0.017 mm2 per glomerulus, of which 0.184 +/- 0.011 mm2 represents peripheral basement membrane. The aggregate epithelial slit length per glomerulus was 65.19 +/- 3.84 cm, of which 48.69 +/- 2.87 cm represents epithelial slits abutting on the peripheral basement membrane. Assuming that a slit diaphragm is 390 A wide, and that the pores of the slit diaphragm represent 26% of its area, the mean pore area is 3.96 cm2, of which 2.96 cm2 represents the area of peripheral pores. These findings are discussed in the context of the hydrodynamic theory of glomerular ultrafiltration. We conclude that the porous substructure of the glomerular slit diaphragm is significant in determining the hydraulic conductivity of the glomerulus and hence also solute flux during ultrafiltration.

B-cell activation and immunoregulation in end-stage renal disease patients receiving hemodialysis.

B-lymphocyte functions were studied in peripheral blood mononuclear cells of end-stage renal disease patients undergoing intermittent hemodialysis for longer than two years. T-cell-dependent B lymphocyte proliferation after pokeweed mitogen stimulation was low in half of the hemodialyzed patients. T cell-independent B cell response to Staphylococcus aureus, Cowan I, was also significantly reduced. Spontaneous production of immunoglobulin in cultures of peripheral blood mononuclear cells of uremic patients was comparable with that of healthy controls, but pokeweed mitogen-stimulated antibody secretion was significantly reduced with cells from patients undergoing hemodialysis. Helper T-cell functions in B-cell activation were also qualitatively deficient in uremic patients. It is concluded that B-cell activation and immunoregulation is defective in patients undergoing long-term hemodialysis.

Suppressor cells in end-stage renal disease. Functional assays and monoclonal antibody analysis.

Suppressor cell activity after concanavalin A induction was studied in peripheral blood mononuclear cells of patients undergoing long-term hemodialysis. Suppression both of the mixed lymphocyte reaction and of allogeneic cells stimulated with phytohemagglutinin was significantly higher with peripheral blood mononuclear cells from patients undergoing hemodialysis than with cells from control subjects. Expression of the Ia antigen on T lymphocytes (associated with immunologic activation) was studied by staining with monoclonal antibodies and two-color fluorescence analysis in a computer-linked cytofluorograph. In unstimulated cells, there was no significant difference between the patients and control subjects. After concanavalin A induction, the percentage of T4, and particularly of T8, cells expressing the Ia antigen was significantly higher in the group undergoing hemodialysis. The functional suppression seen after concanavalin A induction in the mixed lymphocyte reaction was significantly reduced by treatment with OKT8 monoclonal antibody and complement; in phytohemagglutinin cultures, both OKT8 and OKIa*1 antibodies were effective. The reduced in vitro response of uremic lymphocytes may thus be a consequence of increased suppressor activity associated with the T8-positive, Ia-positive subset of T cells.

T cell subsets and cellular immunity in end-stage renal disease.

The T lymphocyte population was studied by immunofluorescent staining with monoclonal antibodies and laser flow cytometry in the blood of 50 patients with end-stage renal disease undergoing long-term maintenance intermittent hemodialysis. The absolute number of T cells was lower in patients receiving dialysis for more than one year (p less than 0.001), as was the absolute count of helper T cells (p less than 0.005). In patients under 30 years of age, the absolute number of helper T cells was markedly reduced, whereas the number of suppressor/cytotoxic T lymphocytes was not changed. In patients between the ages of 30 and 60 years, both helper and suppressor cells were significantly reduced. In patients over 60 years of age, only the number of helper T cells was reduced. The in vitro response of patients' lymphocytes was reduced both in the mixed lymphocyte reaction (p less than 0.01) and after phytohemagglutinin stimulation (p less than 0.001). Natural killer cytotoxicity of patients' peripheral blood mononuclear cells, however, was unaffected.

Alpha subunit isoform influences GABA(A) receptor modulation by propofol.

We have investigated the role of the alpha subunit in the modulation of gamma-aminobutyric acid type A (GABA(A)) receptors by the general anesthetic propofol, using whole-cell patch clamp recordings made from distinct stable fibroblast cell lines which expressed only alpha1beta3gamma2 or alpha6beta3gamma2 GABA(A) receptors. At clinically relevant anesthetic concentrations, propofol potentiated submaximal GABA currents in alpha1beta3gamma2 receptors to a far greater degree than those in alpha6beta3gamma2 receptors. The alpha subunit influenced the efficacy of propofol for modulation, but not its potency. In contrast, direct gating of the ion channel by propofol, in the absence of GABA, was significantly larger in the alpha6 than the alpha1 containing receptors. The potentiation of submaximal GABA by trichloroethanol, and the potentiation and direct gating by methohexital was also studied, and showed the same relative trends as propofol.

Propofol increases agonist efficacy at the GABA(A) receptor.

Using the whole-cell patch-clamp technique, we have determined that propofol, but not midazolam, increases the efficacy of piperidine-4-sulphonic acid (P4S), a partial agonist at alpha1beta1gamma2s, GABA(A) receptors expressed in HEK 293 cells. These findings are consistent with the idea that propofol facilitates receptor gating, while midazolam increases receptor occupancy by the agonist.

Independent learning in pathology. Does it work?

The sophomore pathology course at the University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, was substantially modified with the intention of promoting guided, independent learning. The emphasis has been shifted from lectures to other modalities of instruction. This article delineates the curricular changes made, describes the instructional modalities used, and reports student performance after two year's experience with the new curriculum.

Parasites, diseases and health status of sympatric populations of sambar deer and white-tailed deer in Florida.

From December 1983 to December 1984 a study on parasites, diseases and health status was conducted on sympatric populations of sambar deer (Cervus unicolor) and white-tailed deer (Odocoileus virginianus) from St. Vincent Island, Franklin County, Florida. Ten sambar and six white-tailed deer were examined. White-tailed deer had antibodies to epizootic hemorrhagic disease virus and bluetongue virus. Serologic tests for antibodies to the etiologic agents of bovine virus diarrhea, infectious bovine rhinotracheitis, vesicular stomatitis, parainfluenza 3, brucellosis, and leptospirosis were negative in both species of deer. White-tailed deer harbored 19 species of parasites; all were typical of the parasite fauna of this species in coastal regions of the southeastern United States. Sambar deer harbored 13 species of parasites, which apparently were derived largely from white-tailed deer. The only exception was Dermacentor variabilis which occurs frequently on wild swine on the island. The general health status of sambar deer appeared to be better than that of white-tailed deer. This was hypothesized to result from the sambar deer's utilization of food resources unavailable or unacceptable to white-tailed deer and to the absence and/or lower frequency of certain pathogens in sambar deer.

Coronary artery anomalies diagnosed by magnetic resonance angiography.

Coronary artery anomalies are uncommon entities that may be associated with sudden death. Because of its 2-D projection imaging nature, conventional X-ray coronary angiography may not accurately delineate the origins and course of aberrant coronary arteries with respect to the great vessels. Non-invasive, cross-sectional imaging techniques such as coronary CT angiography and magnetic resonance angiography are increasingly used in clinical practice to diagnose coronary artery anomalies. Although this study reviews coronary artery anatomy and selected anomalies as seen with true fast imaging with steady-state precession magnetic resonance angiography, the information provided is equally applicable to electrocardiogram-gated coronary CT angiography.

Chronic reduction of renal mass: glomerular morphometry by electron microscopy.

Changes of glomerular volume in rats were measured up to 21 weeks following subtotal nephrectomy, using morphometric methods. A linear increase of glomerular volume was observed between 2 and 21 weeks after subtotal nephrectomy. This progressive increase in glomerular volume may reflect compensatory hemodynamic changes leading to an increased single nephron glomerular filtration rate.

Glomerular morphometry in the Munich Wistar rat: effects of sub-total renal ablation.

In the development of disease of the glomerular capillary bed there is a triple association between loss of nephrons, adaptive hyperfunction of residual glomeruli, and hypertrophy and sclerosis of these glomeruli. Sclerosis is closely associated with hypertrophy, and both are closely tied to loss of nephrons. This paper quantifies the response over a period of 12 weeks of superficial cortical (SC), midcortical (MC) and juxtamedullary (JM) glomeruli of the Munich Wistar rat to the ablation of approximately 5/6 of renal tissue. The mean areas of peripheral and mesangial glomerular basement membrane (GBM) were estimated by stereologic methods. While the overall estimated area of the GBM increased in response to ablation by a factor of 2.4, the peripheral GBM area increased only by a factor of 2.15, and by way of contrast, the mesangial GBM area by a factor of 3.25. The latter difference is interpreted as a measure of glomerulosclerosis. There are no marked differences between the changes in SC, MC, or JM glomeruli. These findings are consistent with the association of glomerular hypertrophy and glomerulosclerosis, and with recent suggestions that glomerular hypertrophy and sclerosis following subtotal renal ablation are driven by local or circulating growth factors.

Vesicular diffusion and thermal forces.

The concept that an endothelial vesicular shuttle serves much if not all the function of the large pores of the microcirculation for macromolecular transport has been current for 2 decades. Morphologists have expended much ingenuity in the study of plasmalemmal vesicles by the use of nonenzymatic and enzymatic tracers combined with electron microscopy. Several theoretical models of vesicular transport have been suggested, all of which assume vesicular migration by Brownian or thermal motion. Two such models based on simple diffusion are described, and more recent models in which vesicular diffusion is constrained by long-range hydrodynamic interaction with the plasmalemma are discussed. Theoretical models agree in predicting a vesicular transport time of the order of seconds. Only recently has experimental evidence appeared that tends to corroborate such predictions. Reports that frog mesenteric capillary endothelium fixed with formaldehyde-glutaraldehyde contains very few (approximately 1%) free vesicles are at variance with many in vivo tracer studies and inconsistent with the shuttle theory. It is possible that aldehyde fixation gives a poor representation of the state of the endothelium in vivo. It would seem that more instantaneous methods of fixation, such as rapid freezing, combined with tracer studies and serial sectioning, may be required to resolve this contradiction.

Glomerular hemodynamics and vascular structure in uremia: a network analysis of glomerular path lengths and maximal blood transit times computed for a microvascular model reconstructed from subserial ultrathin sections.

The dimensions of individual capillary segments were determined from a glomerular model constructed on the basis of electron micrographs of subserial ultrathin sections of kidney tissue from a rat made chronically uremic by subtotal nephrectomy. Hemodynamic calculations used computer programs for node pressure analysis and for the determination of path lengths and transit times from afferent to efferent arteriole. The afferent arteriole divided into five primary capillary segments. Three of these carried 84% of the flow through 632 paths, which accounted for 54% of the capillary endothelial area. Their mean path length was 520.97 micron, mean of segment numbers per path 17.66, and mean transit time 0.50 sec. The remaining two capillary segments carried 16% of the flow and led to paths accounting for the remaining 46% of the capillary area. The number of paths from the afferent arteriole through these two segments was 21,244. Mean path length from the initial node through these two segments was 852.18 micron, mean of segment numbers per path 29.62, and mean transit time 20.76 sec. The most striking difference between the two sets of paths was in transit time. This asymmetry would tend to reduce the filtration surface and the Kf (hydraulic conductance x filtering surface area) by causing early filtration pressure equilibrium in much of the capillary network, and suggests an intrinsic glomerular aspect of chronic renal insufficiency.

Glomerular morphometry in the Munich-Wistar rat.

Physiological studies of glomerular function in the rat have been much advanced by the accessibility of superficial glomeruli for micropuncture in a mutant strain, the Munich Wistar (MW) rat. An important parameter studied in this way is the glomerular ultrafiltration coefficient, or Kf. Kf is the product of hydraulic permeability and of the effective filtration surface area. Confounding factors are the variation in anatomical glomerular filtration surface in the rat with body weight (BW) and with the anatomical position of the glomerulus. To establish appropriate anatomical values for the interpretation of Kf in the MW rat, we have carried out a morphometric study by electron microscopy of two groups of relatively mature male MW rats, of approximately 200 g and approximately 300 g BW respectively. We find that superficial cortical (SC) and midcortical (MC) glomeruli of MW rats of approximately 200 g BW have relatively low glomerular volumes and anatomical filtration surface areas, but that MW rats of approximately 300 g BW have larger SC and MC glomeruli of similar size to juxtamedullary (JM) glomeruli, and anatomical filtration surface areas comparable to values usually assumed in physiological studies. We ascribe this contrast between SC glomeruli of MW rats of approximately 200 g and approximately 300 g BW respectively to the maturation of SC glomeruli.

A stereologic study of glomerular hypertrophy in the subtotally nephrectomized rat.

Subtotal nephrectomy in the rat is followed by glomerular hypertrophy, glomerulosclerosis, and, ultimately, renal failure. To gain more insight into this sequence, we have made a morphometric study of the glomerular changes following subtotal nephrectomy in the rat during the period of functional compensation before glomerulosclerosis appears. We found a twofold increase in glomerular volume as compared with controls 2 weeks after operation (to approximately 2 X 10(6) cu mu) followed by a linear increase to approximately 5 X 10(6) cu mu at 21 weeks. The rate of increase in glomerular volume in controls did not significantly exceed that of body weight. The volume increase after subtotal nephrectomy is a true hypertrophy, since other morphometric parameters expressed per unit glomerular volume (surface and length densities) were approximately 80% of control values at 2 weeks and constant thereafter. Glomerular hypertrophy up to 21 weeks is accompanied by a stable BUN and a gradually increasing proteinuria. We conclude that glomerular hypertrophy in this system is a pathologic rather than a compensatory process and suggest that it may represent a counter-productive response to hemodynamic changes serving to increase the glomerular filtration rate.

Ultrastructure of the glomerular basement membrane of rats with proteinuria due to subtotal nephrectomy.

Subtotal nephrectomy in the rat gives rise to progressive proteinuria, glomerular hypertrophy, and glomerulosclerosis. The proteinuria antedates significant ultrastructural lesions demonstrable by conventional techniques; its mechanism is obscure. In this article it is shown that proteinuria in this system is not accompanied by evident changes in the ultrastructural distribution of anionic sites in the glomerular basement membrane, as determined by organ perfusion with two cationic probe molecules. It is suggested that the proteinuria may reflect a change in the steric aspect of glomerular ultrafilration, in which the loss of renal mass imposes a significant rise in capillary pressure in a structure whose capillaries are peculiar in the high pressures they normally sustain and in their lack of mechanical support from the interstitium. It is suggested that a sustained rise in capillary pressure overcomes the rigidity of the glomerular basement membrane, so that its pore size increases and proteinuria results from basement membrane failure.

Humoral inhibitors of the immune response in uremia. II. Further characterization of an immunosuppressive factor in uremic serum.

The serum of Lewis rats with chronic renal insufficiency (induced by subtotal nephrectomy) contains a nondialyzable inhibitor of the mixed lymphocyte reaction. Fractionation of uremic serum by gel filtration on Sephadex G-200 shows that the inhibitory activity elutes with an apparent molecular weight of greater than 200,000 daltons. To establish the possible relationship of the uremic inhibitor to known immunosuppressive components of normal serum, uremic serum was further fractionated on a DEAE cellulose column. The inhibitory activity elutes in 10 mM sodium phosphate at pH 8.0. This fraction contains both alpha-macroglobulin and IgG. The inhibitory activity of this fraction is completely inactivated by treatment with 2-mercaptoethanol, indicating that the inhibitor is a protein. The inhibitory activity is partially inactivated by periodate treatment, suggesting that it may be a glycoprotein. To determine whether or not the inhibitory factor is an immunoregulatory alpha-macroglobulin, or an immune complex, the uremic serum was fractionated by affinity chromatography procedures, which do not induce artifactual inhibitory properties in control serum. The alpha-macroglobulin was removed by affinity chromatography on a column of Con A-Sepharose; its removal had no effect on the inhibitory activity of serum in the mixed lymphocyte reaction. To examine the possibility that immune complexes may be the uremic inhibitor, the serum was fractionated by affinity chromatography on Protein A-Sepharose or by adsorption to a suspension of Staphylococcus aureus, cowan I. Neither of the two latter procedures had any effect on the inhibitory activity of uremic serum. So far all of our findings indicate that the immunosuppressive factor of uremic serum is distinct from two major immunoregulatory factors, alpha-macroglobulin and immune complexes.

Cellular immunity and lymphocyte populations in developing uremia in the rat.

Changes in cellular immunity and in lymphocyte populations have been studied in rats developing chronic renal insufficiency following 5/6 nephrectomy. Animals remain stable for a period of six months (BUN 40-60 mg/dl); then BUN slowly increases for 2-3 months, followed by rapid deterioration and death of the animals. Skin allotransplants showed no change in survival when transplanted fifteen weeks after nephrectomy; when transplanted 22 weeks and later after surgery, their survival was prolonged. The response of splenic cells in the mixed lymphocyte reaction (MLR) was unchanged for 15 weeks after surgery but became significantly reduced after 20 weeks. At the same time we observed an increased suppressor cell activity in splenic cell suspensions and an inhibitory effect of the uremic serum in the MLR. Resistance to tumor induction by syngeneic adenovirus 12-transformed cells was decreased in the late stages of uremia as measured by tumor development in these animals. Induction of cytolytic T cells in vitro was reduced at 24 weeks after operation; at 30 weeks virtually no cytolytic T cell activity was induced. There was also a decrease in natural killer cell activity in the late stages of uremia. These changes in immune response were correlated with the analysis of the lymphocyte sub-populations by staining with monoclonal antibodies and flow cytometry. During the development of uremia no significant changes were found in the lymph nodes. The thymus underwent a severe involution 20 weeks and later after nephrectomy. In the peripheral blood there was a significant decrease in the numbers of helper T cells. The helper T cell subset was also sharply reduced in the spleen of uremic rats at 20 weeks and later after operation.