Immunohistochemical differentiation of Schwann and epithelial basal lamina in the rabbit cornea

Transmission electron microscopy has shown that at the point of entry of axons into corneal epithelium, Schwann cell lamina densa merges with lamina densa of epithelial origin. Staining properties and the thickness of lamina densa from these sources are similar. Immunostaining with monoclonal antibodies to laminin and type IV collagen revealed that while Schwann cell basal lamina fluoresced to both of these probes, epithelial basal lamina was visualized only with anti-laminin. Monoclonal antibodies to neurofilament protein (70 and 200 kDa) were used to visualize the neural tissue. Segments of the deep stromal and subepithelial innervation appeared similar to those seen following gold chloride impregnation. The results suggest that intercalation of lamina densa from these tissue sources and regulation of the production of basal lamina components by both the epithelial and the Schwann cells probably occur.     

Degeneration and regeneration of the lip mucosal epithelium after cryo treatment in mice

The process of degeneration and regeneration of the lip mucosal epithelium after cryo treatment was observed by transmission electron microscopy. The epithelial cells were degenerated by the formation of ice crystals and subsequently detached from the basement membrane, forming a blister cavity. The separation occurred between the epithelial cells and the lamina densa, leaving a small amount of cell debris on the lamina densa. The surviving cells at the periphery of the blister cavity, especially the cells in the basal half of the epithelium, provided the regeneration cells. They migrated over the cell debris, attached to the lamina densa and gradually phagocytozed it. Finally, they formed hemidesmosomes with the old lamina densa. The connections between the epithelial cells by desmosomes were so tight that desmosomes were preserved even between dead cells and between dead and living cells. Regenerating cells were moving in a multilayered form, remaining connected to each other by the dosmosomes. They were seen to divide by mitosis and thereby increase the number of the cell layer, whilst maintaining their connections with the neighbouring cells.     

Observation on the basal lamina of duodenal mesothelial cells during metamorphosis of Xenopus laevis

Morphologic changes in the basal lamina of duodenal mesothelial cells during metamorphosis of Xenopus laevis were observed. In the prometamorphosis stage (Stage 56-59), the basal lamina was almost completely flat; the lamina densa of the basal lamina was a 50 nm layer of high electron density. In the early stages of metamorphic climax (Stage 60-62), the basal lamina showed occasional slight folding (stage 60), with the lapse of time, the folding became continuous and deeper. The development of an additional thin basal lamina was observed in areas where the folded basal lamina was separated from mesothelial cells, viz. on the side adjacent to the mesothelium. The lamina densa in this stage was approximately twice the thickness of the prometamorphosis stage and exhibited high electron density. In the later stages of metamorphic climax (stage 63-66), the basal lamina just under mesothelium became more apparent. The folded basal lamina shifted from the mesothelium into the subserosa and gradually disappeared, and the basal lamina became a single layer. The thickness of the lamina densa was almost the same as in the prometamorphosis stage. Since the timing of the folding of the basal lamina coincides with the shortening of the digestive tract and the marked narrowing of the lumen, we suggest that physical changes in the digestive tract during metamorphosis may play an important role in these morphologic changes of the basal lamina.     

Genesis of hadacidin-induced cleft palate in hamster: Morphogenesis,electron microscopy,and determination of DNA synthesis,cAMP, and enzyme acid phosphatase

A morphological, electron microscopic, and biochemical study was undertaken to analyze the genesis of hadacidin-induced cleft palate in hamster fetuses. Gross and light microscopic observations indicated that hadacidin affected the growth of vertical palatal shelves to induce cleft palate. Electron microscopic observations showed that initial hadacidin-induced changes were seen in the mesenchymal cells. Within 12 hr of drug administration, the perinuclear space was swollen and a lysosomal response injury was evident in the mesenchymal cells. Subsequently, 24 hr after hadacidin treatment, lysosomes appeared in the epithelial cells; changes were also seen in the basal lamina which included separation of the lamina densa from the basal cells, duplication of lamina densa, and complete loss of basal lamina. Between 36 and 42 hr post-treatment, the cellular and basal lamina changes subsided, and the epithelium of vertical shelves underwent stratification. Biochemical determination of enzyme acid phosphatase indicated that the levels of enzyme activity in both the control and treated palatal tissues corresponded to the appearance of lysosomes. Measurement of cAMP levels suggested that the peak activity of cAMP corresponded to that of enzyme acid phosphatase and cell injury. The cAMP activity in hadacidin-injured cells, however, was significantly lower in comparison to that of the dying cells of control palates. Hadacidin treatment also affected DNA synthesis in the developing primordia of the palate. It was suggested that hadacidin injures the precursor cells of the palate prior to the appearance of the primordia, and subsequently affects their proliferative behavior, stunting the vertical growth of the palatal shelves and inducing a cleft palate.     

Differences in the width of the intercellular spaces in the epithelial basal infolding and the renal glomerular filtration site between freeze-substitution and conventional fixation

After aldehyde prefixation, pretreatment with cryoprotectant and subsequent freeze-substitution with OsO₄ in acetone (AC-FS), extensive gap junction-like close membrane appositions are frequently found in the basal infolding of the salivary gland epithelium, although the desmosomal intercellular space had the same width as with conventional electron microscopy. The intercellular space between podocyte pedicles and endothelial cells at the renal glomerular filtration site was narrower by the total width of 2 laminae lucidae following AC-FS than with conventional electron microscopy and was occupied by a homogeneous lamina densa without a lamina lucida, although no marked difference was discernable in the thickness of the lamina densa itself between the 2 preparative procedures. In addition, a decrease in the thickness of the glycocalyx was evident in the intestinal epithelial microvilli following AC-FS. It is thus likely that osmication in acetone at freezing temperatures remove the glycocalyx and related structures to a variable extent, and that this loss is responsible for reducing the intercellular spaces at some of the simple appositions narrower to the dimensions of the gap junction. It is also responsible for disappearance of the lamina lucida of the basement membrane.     

Regeneration of mouse lip epidermis after cryo treatment. Hemidesmosome formation and HSPG (heparan sulfate proteoglycan) distribution in basement membrane

The processes of degeneration and the regeneration of the lip epidermal cells was observed by electron microscopy, focussing on the substance and the structure of the lamina lucida, on which regenerating cells migrated. After the repetitive freezing and thawing treatment, epidermal cells degenerated and detached from the dermis. The separation occurred between the epidermal cells and the basement membrane, leaving a small amount of cell debris on the lamina densa. After the separation of the epidermis, there were some thick parts in the lamina densa which appeared to be the part below hemidesmosomes. Regenerating epidermal cells migrated from the nondegenerated area along the cellular surface of the old lamina densa. They migrated over the cell debris which was gradually phagocitized, and formed new hemidesmosomes with the old lamina densa. Regenerating epidermal cells did not make close contact with the old lamina densa during their migration, but there was a clear space in between, indicating that some of the materials and the structure of the lamina lucida of the old basement membrane was preserved. By immunoelectron microscopy using anti-HSPG (heparan sulfate proteoglycan) antibody, it became clear that after the epidermal separation, HSPG was preserved in the basement membrane to some extent, especially in the thick parts of the lamina densa located below. The immunoelectron micrographs support the view that hemidesmosomes may reform at the previous locations at the old lamina densa.     

Dedifferentiation of iris epithelium during lens regeneration in newt larvae

Early stages in lens regeneration from the pigmented epithelium of the dorsal iris were studied in larval Notophthaimus viridescens by means of transmission electron microscopy. Normal iris epithelium is composed of two layers of low cuboidal cells, packed with melanosomes and surrounded by a basal lamina. Scattered desmosomes attach adjacent cells. Following lens removal, the intercellular spaces enlarge and the epithelial cells increase in size. Some irregular microvilli from these cells extend into the intercellular spaces. Macrophages invade the iris epithelium and phagocytize melanosomes discharged from the pigmented cells. These invading macrophages have numerous microprojections and are often separated from the surface by a very thin layer of iris epithelial cell cytoplasm. In the iris cells, nucleoli become more prominent and granular, polyribosomes increase greatly in number, melanosomes gradually disappear, mitochondria become more numerous, and mitotic activity is greatly augmented. Fine cell processes of adjacent cells interdigitate near the external surface, where numerous micropinocytotic vesicles can be seen. Over the external surface, the basal lamina may be disrupted or duplicated in places where pseudopodia project from iris cells or a macrophage has entered an intercellular space. It is lacking on the lumenal surface. Sloughed membranes are often found in these intercellular spaces.     

Changes in basement membrane thickness in the human endometrium during the luteal phase of the menstrual cycle

We have examined aspects of the fine structure of the basal laminae associated with the luminal and glandular epithelium and small blood vessels in the human endometrium. Four short studies are presented and reviewed. Study 1 examined biopsies from 20 fertile women taken on days after the luteinizing hormone surge (LH): LH +2, 4, 6, 8 and 10. The basal lamina (both lamina densa and lucida) increased in thickness over the period studied. Study 2 again studied the glandular epithelium and examined the effect of RU486 (a progesterone receptor blocker) administered on day LH +3 and biopsied on day LH +6. The basal laminae were found to be the same as LH +2 control group but thinner than LH +6 control. Study 3 documented increased thickness of the basal laminae between LH +6, 8 and 13 in the luminal epithelium. The within-group coefficient of variation was 16% and 27% for LH +6 and LH +13 groups but only 2 % for LH +8. Study 4 demonstrated an increase in basal lamina thickness associated with small blood vessels between LH +6 and LH +10 in normal fertile women. The basal lamina provides the interface between epithelial and mesenchymal environments; changes in its structure can alter the phenotypic expression of the epithelia. It is one of the maternal barriers that must be transgressed by the trophoblast during implantation. Together, these combined studies provide quantitative baseline structural information on the electron microscopical appearance of the basal lamina during the luteal phase of the menstrual cycle.     

Ultrastructural and cytochemical observations on 5-fluorouracil-induced cleft-palate development in hamster

Sequential alterations in 5-fluorouracil-treated hamster fetal palate were studied by light and electron microscopy and by acid phosphatase cytochemistry. At an early stage in 5-fluorouracil-treated fetuses, when the palatal shelves were vertical, lysosomes first appeared in cells of the prospective fusion epithelium and then in the cells of subjacent mesenchyme. In contrast to controls, increasing numbers of both the epithelial and mesenchymal cells of the vertical palate showed lysosomal injury in 5-fluorouracil-treated fetuses as development progressed. Subsequently, the basal lamina in the vertical palate showed alterations, characterized initially by disturbances in lamina lucida, by fingerlike extensions of lamina densa, and ultimately by its complete breakdown. At a later stage, when shelves became horizontal, the lysosomes were absent in both the epithelial and mesenchymal cells, and the basal lamina continuity was restored. Unlike controls, however, 5-fluorouracil-treated horizontal shelves never contacted one another. Instead, the epithelia of the horizontal shelves underwent stratification. It appears that premature formation of lysosomes in palatal epithelial and mesenchymal cells following 5-fluorouracil treatment disrupts normal cytodifferentiation and affects the integrity of the basal lamina; both effects are associated with cleft-palate development.     

Regeneration of the epidermis and mucosal epithelium on the basement membranes

The processes of degeneration and regeneration of the lip epidermis and mucosal epithelium after cryo treatment were observed by transmission electron microscopy. Epidermal and mucosal epithelial cells degenerated due to the formation of ice crystals and detached from the basement membranes, leaving a small amount of cell debris. Regenerating cells migrated over the cell debris, which was gradually phagocitized, and formed new hemidesmosomes with the preexisting lamina densa. Regenerating epidermal cells migrated from the undamaged areas and the hair follicles. Regenerating mucosal epithelial cells originated from surviving cells in the basal half of the epithelium, at the periphery of the blister cavity. They migrated in a multilayered fashion. Desmosomes were preserved even between dead cells and between dead and living cells.     

Ultrastructure of basement membranes in monkey and shark teeth at an early stage of development

The basement membrane, which separates the inner enamel epithelium from the dental papilla in the early stages of tooth development, is known to play a significant role in odontogenesis. In this review article, this basement membrane was described in detail based on our recent findings with the use of high-resolution electron microscopy. Tooth germs of a monkey (Macaca fuscata) and a shark (Cephaloscyllium umbratile) were processed for thin-section observations. During the early stage of development, the basement membrane of the inner enamel (dental) epithelium was composed of a lamina lucida, lamina densa, and much wider lamina fibroreticularis. At higher magnification, the lamina densa in both species was made up of a fine network of cords, which are generally the main constituents of the basement membranes. In the monkey tooth, the lamina fibroreticularis was rich in fibrils, which were now characterized as basotubules, 10-nm-wide microfibril-like structures. The space between the basotubules was filled with a cord network that extended from the lamina densa. Dental papilla cell processes were inserted into the lamina fibroreticularis, and their surface was closely associated with numerous parallel basotubules via 1.5- to 3-nm-wide filaments. In the shark tooth during its early stage of development, the basotubules were absent in the lamina fibroreticularis and only narrow extensions, 60–90nm wide and 1–2µm long, of the cord network of the lamina densa were present. The dental papilla cells were immobilized by means of the binding of their processes to the extensions. These results indicate that basement membranes in both monkey and shark teeth at early stage of development are specialized for functions as anchoring and firm binding, which are essential for the successful differentiation of the odontoblasts.     

Structure of the complex basement membrane underlying the epithelium of the vas deferens in the rat

Underlying the epithelium of the vas deferens there is a complex basement membrane showing a thick lamina densa separated from the plasma membrane of epithelial cells by a lamina lucida. On the connective tissue side of the lamina densa, there are plaques composed of a material that is similar to that of the lamina densa but is more compact and has a greater electron density. This material also forms plaques at a short distance from the lamina densa, where it appears as irregular nodular masses. The plaques are bridged by striated anchoring fibrils (SAF) that are variable in structure. Some SAF are long (0.5-0.6 micron) and bilaterally symmetrical, with a central fusiform segment and, on each side, coarsely banded segments. While the fusiform segment presents 5 or 6 diffuse cross striations, the coarsely banded segments show distinct bands labeled B1-B4. Shorter SAF show a coarsely banded segment alone or a coarsely banded segment plus a fusiform segment. Some SAF also branch at the level of the fusiform segments, in which case they form star-shaped structures with three or more branches that have their extremities inserted into plaques. The plaques, as well as the lamina densa, are immunohistochemically reactive to type IV collagen, laminin, and heparan sulfate proteoglycan, whereas the SAF are not immunoreactive to these substances. SAF and plaques, considered as integral components of this basement membrane, form a series of arches or open tunnels traversed by collagen fibrils. It is thus apparent that these elements contribute to the attachment of the basement membrane and the overlying epithelium to the underlying dense connective tissue of the lamina propria.     

Polycystic kidney disease in the CBA/N immunodeficient mouse.

We describe a polycystic lesion of the kidney in the CBA/N mouse with an X-linked recessive immunodeficient syndrome. There is progressive cystic dilatation affecting all parts of the nephron. The cyst lining is composed of a single layered epithelium with focal nuclear crowding and the formation of micropapillary structures. The cystic epithelial cells show subnuclear vacuolation. Focal basement membrane thickening is also a feature. There is no significant inflammatory infiltrate present within these kidneys. Electron microscopic examination reveals that the subnuclear vacuolation is due to loss of the membrane infoldings at the basal pole of the epithelial cell with fluid accumulation within the extracellular space. The basement membrane thickening is due to expansion of the lamina densa. These changes are not present at birth but develop progressively with age. The finding of a polycystic kidney lesion in these mice offers an opportunity to investigate the relationship between the immune system and renal cyst formation.     

The ultrastructural histochemistry of the basement membranes of the exocrine pancreas

The basement membranes in the exocrine pancreas were examined by routine electron microscopy, by fixation for electron microscopy with a glutaraldehyde-tannic acid solution and by staining for ultrastructural demonstration of mucosubstances with the dialyzed iron, high iron diamine, ruthenium red and Concanavalin A-horseradish peroxidase procedures. The basement membranes are considered from morphologic and histochemical observations to consist of an inner lamina lucida, an intermediate lamina densa and an outer lamina diffusa. Sulfated mucosubstance was found in the lamina diffusa of the basement membrane of all epithelial cells and capillary endothelial cells but was encountered in the lamina lucida of the duct cells exclusively. Bridging structures, presumably polypeptides, were also seen connecting the lamina densa and the basal plasma membrane in specimens fixed with the glutaraldehyde-tannic acid solution. The findings demonstrated that the histochemical and morphological qualities of the basement membrane are uniform for a given cell type but differ considerably among the several cell types in the pancreas.     

The ultrastructural histochemistry of the basement membranes of the exocrine pancreas.

The basement membranes in the exocrine pancreas were examined by routine electron microscopy, by fixation for electron microscopy with a glutaraldehyde-tannic acid solution and by staining for ultrastructural demonstration of mucosubstances with the dialyzed iron, high iron diamine, ruthenium red and Concanavalin A-horseradish peroxidase procedures. The basement membranes are considered from morphologic and histochemical observations to consist of an inner lamina lucida, an intermediate lamina densa and an outer lamina diffusa. Sulfated mucosubstance was found in the lamina diffusa of the basement membrane of all epithelial cells and capillary endothelial cells but was encountered in the lamina lucida of the duct cells exclusively. Bridging structures, presumably polypeptides, were also seen connecting the lamina densa and the basal plasma membrane in specimens fixed with the glutaraldehydetannic acid solution. The findings demonstrated that the histochemical and morphological qualities of the basement membrane are uniform of a given cell type but differ considerably among the several cell types in the pancreas.     

Penetration of the basal lamina of the uterine luminal epithelium during implantation in the rat

During early stages of implantation in the rat, as in other species that form a hemochorial placenta, there is a progressive increase in intimacy between blastocyst and endometrium. After initial invasion of the uterine luminal epithelium by trophoblast cells and displacement of epithelial cells, the trophoblast comes to lie adjacent to the residual basal lamina of the displaced epithelium but does not penetrate it. After a pause at the basal lamina, this temporary barrier is breached. To study the interrelations of trophoblast, uterine epithelium, and decidual cells with the epithelial basal lamina during the time of penetration of the basal lamina, implantation sites collected on day 7 of pregnancy were oriented so that the implantation chamber could be sectioned either longitudinally or transversely. Neither trophoblast nor uterine epithelial cells have processes that extend through the basal lamina. However, flange-like processes from the decidual cells penetrate the basal lamina and underlie both trophoblast and, more rarely, epithelium. Smaller folds of the surface of decidual cells partially surround bundles of collagen fibrils oriented parallel to the long axis of the implantation chamber. Initially the area of penetration of basal lamina by decidual cell processes is quite restricted; as implantation proceeds the basal lamina becomes displaced and is sometimes not discernible, extracellular materials accumulate, and the relationships become more difficult to follow. It is concluded that the initial breaching of the basal lamina is an activity of the decidual cells, and that contact of basal lamina with trophoblast is not necessary to permit this penetration.     

Light and electron microscopic study of the degeneration and early regeneration of olfactory epithelium in the mouse

Degeneration and early regeneration of olfactory epithelium from two strains of mice was studied at the light and electron microscopic levels from 12 hours to 3 days following nasal irrigation with 1% aqueous solution of zinc sulfate (ZnSO₄) (a compound known to selectively damage olfactory epithelium). Distinct patterns of degeneration and stages of regeneration were evident following treatment. During the first 24 hours after treatment three progressive manifestations of the degenerative process were seen: (1) a relatively mild condition which was characterized by surface irregularities produced by cell protrusions, highly vacuolated cytoplasm, presence of large lysosome-like bodies and prominent intercellular spaces, (2) a more severe condition in which large areas of the epithelium were detached from the basement membrane and cellular debris was present in the nasal chamber, and (3) a condition of total or near-total denudation of the epithelium of olfactory mucosa. The basal lamina was continuous and intact in most regions and the integrity of the subjacent connective tissue was mostly well-preserved. Nerve bundles of the fila olfactoria were noted in varying degrees of degeneration during the course of the experiment. The most advanced neural degeneration was seen 24 to 72 hours after treatment. Onset of regeneration was suggested by the appearance of a simple squamous layer of cells above the basement membrane 48 to 72 hours after treatment. In addition to the simple epithelium a stratified epithelium consisting of two to four cell layers was also observed at this time. Glandular cells, containing secretory granules identical to those in Bowman's glandular cells, were noted in an apparent process of migration from the lamina propria into the stratified epithelial layer. The last mentioned observation supports the proposition that new supportive epithelial cells originate from cells of Bowman's gland.     

Functional units in rainbow trout (Salmo gairdneri,Richardson) liver: II. The biliary system

The intrahepatic biliary system was studied in the rainbow trout (Salmo gairdneri), a teleost known to form liver neoplasms after exposure to various carcinogens. Normal adults (N = 25) were examined using light microscopic, enzyme histochemical, and transmission and scanning electron microscopic methods. In light micrographs, longitudinal arrays of hepatocytes appeared as double rows incompletely divided by elongated darkly stained cells. Electron micrographs showed tubules of five to nine pyramidally shaped hepatocytes with their apices directed toward a central biliary passageway and their bases directed toward sinusoids. Sequentially, beginning with hepatocytes, biliary passageways included canaliculi, preductules, ductules, and ducts. Canaliculi were short and joined transitional passageways (preductules) formed by junctional complexes between plasma membranes of hepatocytes and small, electron-dense cells with a high nuclear to cytoplasmic ratio. Ductules, completely lined by biliary epithelial cells, occupied central regions of hepatic tubules. Relatively elongated, ductular cells were intimately associated with surrounding hepatocytes, separated from them by only a thin extracellular space devoid of a basal lamina. Epithelium of bile ducts included cuboidal through mucus-laden columnar cells, surrounded by basal lamina and, in larger ducts, by fibroblasts, smooth muscle cells, and a capillary plexus. Bile ducts and hepatic arterioles, but not venules, were distributed together. The ultrastructure of biliary epithelium, periductular, and periductal cells is presented.     

Distribution and characterization of anionic sites in the basal lamina of developing human amniotic epithelium

We have studied the distribution of anionic sites in the basal lamina of developing human amniotic epithelium by using the cationic stain ruthenium red. Amnions at 7-12 weeks of gestation and at term contained ruthenium red-positive granules in a quasi-regular array on both the cellular and interstitial sides of the lamina densa. In order to characterize the anionic sites, small pieces of amnion were incubated in the presence or absence of either chondroitinase ABC, neuraminidase, Streptomyces hyaluronidase, or heparitinase in appropriate buffer systems. Incubation in the presence of heparitinase resulted in the complete disappearance of the basal lamina-associated granules, but other enzymes tested had no demonstrable effect on these granules. We conclude that the anionic sites associated with amnion basal lamina, and demonstrable with ruthenium red, consist of glycosaminoglycans rich in heparan sulfate, probably present as heparan sulfate proteoglycan. Because amniotic fluid has a low protein content and amniotic epithelium (at least at term) lacks tight junctions, we postulate that the heparan sulfate proteoglycan associated with the amnion basal lamina may have an important function as a permeability barrier to anionic macromolecules.     

Colloidal gold localization of type IV collagen in the extracellular matrix of rat gastric mucosa: influence of alcohol and prostaglandin

The effect of acute alcohol exposure on the gastric mucosal basal lamina, and its major structural protein type IV collagen, was assessed by transmission electron microscopy (TEM) and immunogold (IG) labeling of this collagenous material. Fasted rats orally received either 50% or 100% ethanol. Five or 60 minutes later animals were sacrificed and mucosal samples were obtained from the glandular epithelium for TEM or IG localization of type IV collagen. For IG studies, the number of gold particles/area lamina densa was quantified in interfoveolar, pit, and gland regions as an index of the molecular integrity of type IV collagen. Both ethanol concentrations induced epithelial exfoliation with pleating of the denuded lamina densa. Absolute ethanol, and to a lesser extent 50% ethanol, caused frequent rupture of a thickened, precipitated lamina densa. Immunolabeling of type IV collagen varied with the experimental protocol. In control tissues exposed to oral saline, binding was greatest in the interfoveolar zone. Low binding occurred with 100% ethanol in all regions when compared with controls, but 50% ethanol evoked significantly higher binding in interfoveolar regions, in a similar fashion to controls. In additional studies in which 16,16 dimethyl prostaglandin E2 (PGE2) (10 micrograms/kg) was injected subcutaneously prior to oral ethanol exposure, PGE2 pretreatment prevented the large decrease in IG binding induced by absolute ethanol, but the level still remained significantly less than with corresponding controls. In contrast, pretreatment with PGE2 prior to 50% ethanol exposure restored type IV collagen immunolabeling to control levels. These results indicate that ethanol induces a concentration-dependent lowering of IG binding to type IV collagen which also effects its reversibility by PGE2.