The ultrastructural localization of two basement membrane components: entactin and laminin in rat tissues

The localization of two noncollagenous components of basement membranes, laminin and entactin, was determined in rat kidney, muscle, and small intestine using electron immunohistochemistry. In the renal glomerulus anti-laminin antibodies reacted with the basement membrane of peripheral capillary loops and with mesangial matrix. In the peripheral capillary loop laminin was preferentially distributed in both laminae rarae. This was in contrast to anti-entactin that localized in peripheral capillary loops but not in mesangial matrix. Even in the peripheral capillary loops it had a different distribution than laminin. Entactin was found predominantly in the lamina rara interna. In renal tubular basement membranes both antibodies localized throughout the full thickness of the basement membranes, with laminin having a preferential distribution in the lamina rara, whereas entactin was more evenly distributed. In the basement membrane of the duodenal mucosa entactin localized in the lamina densa, whereas laminin was present in both laminae. In skeletal muscle both antibodies had similar localization in all basement membranes. These results demonstrate that entactin is an intrinsic component of basement membranes. They also demonstrate that basement membranes from different tissues have subtle variations in content and/or assembly of the different components. It is likely that these variations may be reflected in different functional properties.     

Chronic hyperglycemia in experimental diabetes mellitus of short duration does not contribute to muscle capillary basement membrane thickening

The effect of chronic hyperglycemia on the relationship of nonenzymatic glycation and capillary basement membrane thickness in muscle was studied in streptozotocin-induced diabetic rats early in the course of diabetes mellitus. Diabetic animals were placed on either standard (24%) or restricted (8%) protein diet. The animals on 8% protein diet had elevated glycated hemoglobin levels (p less than 0.01) and increased levels of nonenzymatic glycation of basement membrane (p less than 0.01) as compared to insulin-treated diabetic (euglycemic), age-matched control, and streptozotocin-injected nondiabetic animals also on 8% protein diet. In contrast, diabetic animals on restricted (8%) protein diet and those on standard (24%) protein diet showed no statistical differences between them with regards to the above parameters. Moreover, there were no statistical differences among diabetic and control animals on either 8 or 24% protein diet with respect to muscle capillary membrane thickness. Even though the peripheral muscle biopsy study of capillary basement membrane is less invasive than kidney biopsy, the results of this study suggest that neither nonenzymatic glycation nor basement membrane thickness can be utilized as predictors of renal dysfunction during early onset of diabetes mellitus.     

Origin of the glomerular basement membrane visualized after in vivo labeling of laminin in newborn rat kidneys

To examine the origin and assembly of glomerular basement membranes (GBMs), affinity purified anti-laminin IgG was directly coupled to horseradish peroxidase (HRP) and intravenously injected into newborn rats. Kidneys were then processed for peroxidase histochemistry and microscopy. Within 1 h after injection, anti-laminin bound to basement membranes of nephrons in all developmental stages (vesicle, comma, S-shaped, developing capillary loop, and maturing glomeruli). In S-shaped and capillary loop glomeruli, anti-laminin-HRP labeled a double basal lamina between the endothelium and epithelium. Sections incubated with anti-laminin in vitro showed labeling within the rough endoplasmic reticulum of endothelium and epithelium, indicating that both cell types synthesized laminin for the double basement membrane. In maturing glomeruli, injected anti-laminin-HRP bound throughout the GBMs, and double basement membranes were rarely observed. At this stage, however, numerous knobs or outpockets of basement membrane material extending far into the epithelial side of the capillary wall were identified and these were also labeled throughout their full thickness. No such outpockets were found in the endothelial cell layer of newborn rats (and they normally are completely absent in fully mature, adult glomeruli). In contrast with these results, in kidneys fixed 4-6 d after anti-laminin IgG-HRP injection, basement membranes of vesicle, comma, and S-shaped nephrons were unlabeled, indicating that they were assembled after injection. GBM labeling was seen in maturing glomeruli, however. In addition, the outpockets of basement membrane extending into the epithelium were often completely unlabeled whereas GBMs lying immediately beneath them were labeled intensely, which indicates that the outpockets were probably assembled by the epithelium. Injections of sheep anti-laminin IgG followed 8 d later with injections of biotin-rabbit anti-laminin IgG and double-label immunofluorescence microscopy confirmed that GBM formation continued during individual capillary loop expansion. GBM assembly therefore occurs by at least two different processes at separate times in development: (a) fusion of endothelial and epithelial basement membranes followed by (b) addition of new basement membrane from the epithelium into existing GBMs.     

Ontogenesis of glomerular basement membrane: structural and functional properties

Protein A-gold immunocytochemistry was applied in combination with morphometrical approaches to reveal the alpha 1(IV), alpha 2(IV), and alpha 3(IV) chains of type IV collagen as well as entactin on renal basement membranes, particularly on the glomerular one, during maturation. The results have indicated that a heterogeneity between renal basement membranes appears during the maturation process. In the glomerulus at the capillary loop stage, both the epithelial and endothelial cell basement membranes were labeled for the alpha 1(IV) and alpha 2(IV) chains of type IV collagen and entactin. After fusion, both proteins were present on the entire thickness of the typical glomerular basement membrane. At later stages, the labeling for alpha 1(IV) and alpha 2(IV) chains of type IV collagen decreased and drifted towards the endothelial side, whereas the labeling for the alpha 3(IV) chain increased and remained centrally located. Entactin remained on the entire thickness of the basement membrane during maturation and in adult stage. The distribution of endogenous serum albumin in the glomerular wall was studied during maturation, as a reference for the functional properties of the glomerular basement membrane. This distribution, dispersed through the entire thickness of the basement membrane at early stages, shifted towards the endothelial side of the lamina densa with maturation, demonstrating a progressive acquisition of the permselectivity. These results demonstrate that modifications in the content and organization of the different constituents of basement membranes occur with maturation and are required for the establishment of the filtration properties of the glomerular basement membrane.     

Increased glycosylation of glomerular basement membrane collagen in diabetes

Basement membrane was purified from glomeruli isolated from normal and streptozotocin-diabetic rats. After extraction of non-collagen protein with 8M urea, the extent of glycosylation in glomerular basement membrane collagen was determined with a specific colorimetric reaction that detects carbohydrate in ketoamine linkage with proteins. The level of glycosylation of glomerular basement membrane collagen purified from diabetic rats was significantly greater than that in non-diabetic animals. Increased basement membrane glycosylation may alter structure-function relationships of the capillary filtration barrier.     

Immunogold localization of basement membrane molecules in rat retinal capillaries

Vascular basement membrane contains laminin, fibronectin, proteoglycan and collagens. These molecules have been identified in various tissues by immunolabeling methods and biochemical analyses. We have previously localized laminin, fibronectin and type IV collagen to the basement membrane of rat retinal vessels at the ultrastructural level using an immunoperoxidase method. In this study, we use an immunogold method to re-examine the distribution of these molecules and also to study the localization of heparan sulfate proteoglycan and types I, III and V collagen in the retinal capillary basement membrane. Gold labeling for laminin, type IV collagen and proteoglycan were found diffusely on the basement membrane of the endothelium and pericyte, while that for fibronectin and type V collagen was spotty and variable and that for types I and III collagen was negligible. The segment of basement membrane between the endothelial cell and pericyte appeared less reactive to anti-laminin and anti-type IV collagen than the membrane between the pericyte and perivascular neuroretina. The immunogold method may be useful in quantitative studies of thickened basement membranes under abnormal conditions.     

Biomechanical properties of native basement membranes

Basement membranes are sheets of extracellular matrix that separate epithelia from connective tissues and outline muscle fibers and the endothelial lining of blood vessels. A major function of basement membranes is to establish and maintain stable tissue borders, exemplified by frequent vascular breaks and a disrupted pial and retinal surface in mice with mutations or deletions of basement membrane proteins. To directly measure the biomechanical properties of basement membranes, chick and mouse inner limiting membranes were examined by atomic force microscopy. The inner limiting membrane is located at the retinal-vitreal junction and its weakening due to basement membrane protein mutations leads to inner limiting membrane rupture and the invasion of retinal cells into the vitreous. Transmission electron microscopy and western blotting has shown that the inner limiting membrane has an ultrastructure and a protein composition typical for most other basement membranes and, thus, provides a suitable model for determining their biophysical properties. Atomic force microscopy measurements of native chick basement membranes revealed an increase in thickness from 137 nm at embryonic day 4 to 402 nm at embryonic day 9, several times thicker that previously determined by transmission electron microscopy. The change in basement membrane thickness was accompanied by a large increase in apparent Young's modulus from 0.95 MPa to 3.30 MPa. The apparent Young's modulus of the neonatal and adult mouse retinal basement membranes was in a similar range, with 3.81 MPa versus 4.07 MPa, respectively. These results revealed that native basement membranes are much thicker than previously determined. Their high mechanical strength explains why basement membranes are essential in stabilizing blood vessels, muscle fibers and the pial border of the central nervous system.     

Morphometric evaluation of capillary basement membrane thickness in the quadriceps muscle of diabetic and nondiabetic Chinese hamsters

Quadriceps muscle capillaries from 19-23 month old genetically diabetic (XA and AC) and nondiabetic (M) subline Chinese hamsters were morphometrically evaluated to determine if capillary basement membrane thickening (CBMT) is a quantifiable complication of diabetes. Significant CBMT was present in the diabetic XA Chinese hamsters (49.37 nm +/- 17.81, p less than 0.007) in comparison with the nondiabetic M hamsters (34.08 nm +/- 9.98). Although there was a trend towards expansion of the muscle capillary basement membranes in the diabetic AC Chinese hamsters, the value was not statistically significant. A nested analysis of variance showed that the greatest source of variation in basement membrane thickness occurred among capillaries within each animal. In addition, a positive correlation (r = 0.62; p less than 0.002) existed between blood glucose levels and CBMT in the XA subline. These data should serve as guidelines for evaluation of antimicrovascular disease compounds which will be tested to determine if they prevent or retard microangiopathy in the diabetic Chinese hamster.     

The PASDORO method for simultaneously demonstrating DNA and lipids in the brain

Conclusions and summary The PASDORO method has the advantage of allowing DNA and globular lipid to be demonstrated in frozen sections of formalin-fixed tissue. Its disadvantage is that, in most tissues, basement membrane staining interferes with the selective demonstration of DNA. However, in the central nervous system it is advantageous to be, able to trace capillary basement membrane. In this way cells related to the capillary endothelium can be easily identified. The addition of staining with Oil Red O to the basic PASD technique permits lipid-laden microglia to be readily identified around necrotic and demyelinating lesions.     

The ultrastructure of the renal corpuscle of a lizard

The few and small renal corpuscles of the lizard Podarcis (= Lacerta) taurica are composed of a tuft of three to four capillaries (glomerulus), Bowman's capsule and mesangium. The thin interdigitated capillary endothelial cells are, in most regions, in contact with the mesangium. In some regions, however, they rest on a bilaminate basement membrane with an electron-dense lamina densa and a less dense lamina rara. Bowman's capsule is composed of visceral and parietal layers. The epithelial cells (podocytes) of the visceral layer bear trabeculae connected to pedicels with microvilli. The pedicels rest on a bilaminate basement membrane which in some regions has a double-layered densa with connecting bands. Generally, this basement membrane is thicker than that of the capillary endothelial cells. The mesangium is composed mostly of irregular satellite cells with large nuclei and cytoplasmic processes, but also has smaller cells with kidney-shaped nuclei and cytoplasmic processes containing microfilaments. The mesangium cells are embedded in a collagenous matrix which extends to invade the area between the epithelial basement membrane and the capillary endothelium. These observations are discussed in relation to the structure and function of vertebrate renal corpuscles with special reference to the mesangium.     

Localization and quantitation of anionic charge sites in fetal and neonatal alveolar basement membranes

A method utilizing biopsy sized samples of lung for anionic charge site localization in alveolar and capillary basement membranes in human tissue is discussed. Tissue fixed in either paraformaldehyde-lysine-periodate or 1% paraformaldehyde with 0.05% glutaraldehyde, cut into 30 mu sections, and incubated with the cationic probe, polyethyleneimine, was processed for electron microscopic analysis using standard techniques. Anionic charge sites were identified and regularly distributed in increments of approximately 40-50 nm in the lamina rara externa of the alveolar basement membrane, with lesser amounts found in the lamina rara interna and lamina densa. Anionic charge sites were also demonstrated in the interstitial portion of the capillary basement membrane and on cell surfaces. These methods can be used to more broadly define the localization of anionic charge sites in human lung tissue in both normal and pathologic states.     

Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures

Previous studies have indicated the importance of basement membrane components both for cellular differentiation in general and for the barrier properties of cerebral microvascular endothelial cells in particular. Therefore, we have examined the expression of basement membrane proteins in primary capillary endothelial cell cultures from adult porcine brain. By indirect immunofluorescence, we could detect type IV collagen, fibronectin, and laminin both in vivo (basal lamina of cerebral capillaries) and in vitro (primary culture of cerebral capillary endothelial cells). In culture, these proteins were secreted at the subcellular matrix. Moreover, the interaction between basement membrane constituents and cerebral capillary endothelial cells was studied in adhesion assays. Type IV collagen, fibronectin, and laminin proved to be good adhesive substrata for these cells. Although the number of adherent cells did not differ significantly between the individual proteins, spreading on fibronectin was more pronounced than on type IV collagen or laminin. Our results suggest that type IV collagen, fibronectin, and laminin are not only major components of the cerebral microvascular basal lamina, but also assemble into a protein network, which resembles basement membrane, in cerebral capillary endothelial cell cultures.     

Late ultrastructural effects of heavy ions and gamma irradiation in the gastrointestinal tract of the mouse

The irradiated gastrointestinal tract of LAF1 mice was examined one year following a single dose (1000 rad) of either 12C heavy ions or 60Co gamma rays. Qualitative ultrastructural analysis of the gastrointestinal tract of mice exposed to heavy ions or gamma irradiation did not show any discernible differences. In the stomach of irradiated mice, parietal cells contained numerous lysosomes; the gastric chief cells occasionally contained myelin figures. The epithelial cells of the small intestine, especially jejunum and ileum, showed several changes: (1) increased vacuolation was seen both inter- and intra-cellularly, (2) epithelial cell projections penetrated the basal lamina and were in contact with underlying mesenchymal cells, (3) occasional Paneth cells contained intracellular vacuoles consisting of fibrillar and granular material. In the large intestine occasional signs of degeneration were observed. Qualitative analysis of stromal elements of the gut in irradiated mice indicated the presence of damage to capillary endothelial cells, smooth muscle cells and some nerve processes. The amount of basement membrane (BM) around capillaries and small vessels was increased; the same phenomenon was observed to affect the nerve processes, but with less severity. Quantitative analysis of the basement membrane thickness around capillaries in irradiated vs. control mice showed significant differences. Basement membrane thickness around capillaries in the gastric mucosa and duodenum did not differ significantly in any of the treatment groups. In jejunum, the gamma treated animals exhibited significantly higher BM thickness when compared to unirradiated controls. In ileum, only 12C-heavy ion treated animals showed thicker BM when compared to their respective controls. In colon, both 12C- and 60Co-treated animals showed increased BM thickness when compared to controls.     

Pericyte response during choriocapillaris atrophy in the rabbit

The rabbit and rat choriocapillaris atrophies in response to experimental destruction of the retinal pigment epithelium by intravenous injection of sodium iodate. This provides a convenient model of capillary atrophy. We have observed that pericytes are spared during this process; the atrophy is due to loss of endothelium only. Extensive examination of thin sections obtained 1 day to 11 weeks after administration of iodate showed that pericytes retained their normal relationship to the remnant capillary basement membrane left behind as the endothelial tube atrophied. This was most conspicuously manifested in their retention of processes longitudinally disposed along the sleeves of remnant basement membrane. The processes retained bundles of actin filaments that had dense regions along them and inserted into subplasmalemmal densities at basement membrane attachment sites, i.e. they had the characteristics of stress fibers. The pericytes did not phagocytose the debris of endothelial necrosis, in spite of their known phagocytic abilities. Necrotic endothelial cells were eliminated by sloughing into the capillary lumen. The observations support the idea that the function of pericytes in the choriocapillaris, the major source of nutrition for the retinal photoreceptors, resides in their contractility, and that pericytes do not remove necrotic endothelium during capillary atrophy.     

Fine structure of the respiratory organs of the Climbing perch, Anabas testudineus (Pisces: Anabantidae)

An electron microscopic study has been made of the three respiratory organs of climbing perch. The gill structure is similar to that of the other telcosts but the thickness of the water/blood barrier is much greater, being as great as 20 μm in some specimens. The increased thickness is due to a multilayered epithelium which is thinner (3.5–7 μm) over the marginal channel of the secondary lamellae. The other two main layers, basement membrane and pillar cell flange, are relatively thin (about 1 μm).
The pillar cells have a typical structure, but in certain regions they are contiguous with one another and line well-defined blood channels. Some of the columns of basement membrane material in such regions may be common to adjacent pillar cells.
The air-breathing organs are (a) the lining of the suprabranchial chambers , and (b) the labyrinthine plates attached to the dorsal region of branchial arches. Electron microscopy showed that their structure is well adapted for gas exchange, the air/blood barriers being only 0.12–0.3 μm, comprising an epithelial layer, basement membrane, and thin capillary endothelium. The many parallel blood channels of the respiratory islets of both organs are separated by pillar-like structures which differ from the pillar cells of the secondary lamellae. Thus the hypothesis that the air-breathing organs represent modified gills is not supported by this study.
The fine structure of the non-respiratory region of the air-breathing organs is similar to that of the skin, and includes chemoreceptor-like cells. Evidence concerning the possible homology of pillar cells with plain muscle cells is discussed.     

The connective tissue of the rat lung: electron immunohistochemical studies

The ultrastructural distribution of specific connective-tissue components in the normal rat lung was studied by electron immunohistochemistry. Three of these components were localized: type I collagen, fibronectin and laminin. Type I collagen was present not only in major airways and vascular structures, but also in alveolar septa. Laminin was found in all basement membranes, and only in basement membranes, demonstrating once more that this glycoprotein is an intrinsic component of the basement membrane. Fibronectin was found free in the interstitium and on the surfaces of collagen fibers. The basement membranes of bronchial, glandular and endothelial cells of large vessels lacked fibronectin; however, capillary endothelial and occasionally epithelial alveolar basement membranes contained some fibronectin in an irregular, spotty distribution. This localization suggests that in the lung, as in other tissues, fibronectin is not an intrinsic component of the basement membrane, but rather a stromal and plasma protein. Only basement membranes in the alveolar parenchyma contained "trapped" plasma fibronectin.     

An electron microscope study of kidney basement membrane changes in the mouse by lipoteichoic acid from Streptococcus pyogenes

Mice injected repeatedly, intraperitoneally or intravenously, for approximately 1 month with a total of 1.04 mg lipoteichoic acid from a nephritogenic strain of Streptococcus pyogenes lost weight. Analysis by electron microscopy revealed that they also exhibited extensive kidney changes in basement membrane morphology which resembled, in part, those observed in human poststreptococcal glomerulonephritis. For example, the glomerular basement membrane became electron dense and exhibited at least a twofold increase in width sporadically within the same preparation after exposure to lipoteichoic acid. Also, whereas appreciable loss or reduction in epithelial foot processes as a result of fusion was clearly evident, epithelial slits and slit membranes or diaphragms between normal foot processes were not selectively affected. In addition, another mostly thickened, highly coiled or serpentinelike basement membrane with amorphous nodules appeared in these preparations. This type membrane was not observed surrounding the capillary lumina and was the most pronounced abnormality apparent in almost all preparations from mice exposed to lipoteichoic acid. Likewise, the proximal tubular basement membrane became variable in width and increased in electron density in mice given lipoteichoic acid as compared with controls. In addition, this membrane was often punctuated with various morphological protrusions originating from only its thickened areas and which extended away from, and not into, the capillary space. This change was only associated with the basement membrane of the proximal tubular capillaries. All membrane changes persisted but gradually subsided, with normal kidney membrane morphology reappearing on the 4th day following the last injection of lipoteichoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

A fractal analysis of the radial distribution of bronchial capillaries around large airways.

We analyzed published measurements of the bronchial circulation and airway wall (Anderson JC, Bernard SL, Luchtel DL, Babb AL, and Hlastala MP. Respir Physiol Neurobiol 132: 329-339, 2002) and determined that the radial distribution of bronchial capillary cross-sectional area was fractal. We limited our analysis to bronchial capillaries, diameter < or =10 mum, that resided between the epithelial basement membrane and adventitia-alveolar boundary, the airway wall tissue. Thirteen different radial distributions of capillary-to-tissue area were constructed simply by changing the number of annuli (i.e., the annular size) used to form each distribution. For the 13 distributions created, these annuli ranged in size from to of the size of the airway wall area. Radial distributions were excluded from the fractal analysis if the sectioning procedure resulted in an annulus with a radial thickness less than the diameter of a capillary. To determine the fractal dimension for a given airway, the coefficient of variation (CV) for each distribution was calculated, and ln(CV) was plotted against the logarithm of the relative piece area. For airways with diameter >2.4 mm, this relationship was linear, which indicated the radial distribution of bronchial capillary cross-sectional area was fractal with an average fractal dimension of 1.27. The radial distribution of bronchial capillary cross-sectional area was not fractal around airways with diameter <1.5 mm. We speculated on how the fractal nature of this circulation impacts the distribution of bronchial blood flow and the efficiency of mass transport during health and disease. A fractal analysis can be used as a tool to quantify and summarize investigations of the bronchial circulation.     

Measurement of capillary basement membrane thickness by magnification on a rear opaque projection screen

2190 measurements of capillary basement membrane thickness were obtained from the images of magnified micrographs using a rear opaque projection screen. This method proved to be reproducible and reliable. The electron microscope magnification used, 2500x, permits sharp definition of structures in the resulting micrographs. Projection on the opaque rear screen magnified the image 16x, yielding a final magnification of 40,000x. This high final magnification of micrographs of good quality minimized the error in measurement. The use of a special dial caliper with pins attached allows an open field and decreases the subjective components of measurements. Moreover, this technique results in less stress for the persons performing the measurements.