Nerve fibers in human myocardial scars.

The relationships between ischemic heart disease, myocardial scars, ventricular nerve fibers, and ventricular arrhythmias have not been established despite considerable evidence suggesting important correlations. We recently described the reactions of nerve fibers in necrotic, healing, and healed rat myocardium. Prompted by these studies and by the lack of similar information for humans, we studied the structural relationships between nerve fibers and human myocardial scars. Hearts were obtained from transplant surgery and autopsy. Nerve fibers were labeled with antibody to S-100 protein. Light and electron microscopy of left ventricular scars revealed (1) fiber densities greater than those in adjacent intact myocardium, (2) fiber aggregates concentrated irregularly along the periphery of lesions, (3) fibers few in number or absent in the deeper aspects of scars, and (4) axonal enlargements containing clear and dense storage granules within the fiber aggregates. Like all other elements of the scars, the nerve fibers appeared to be oriented predominantly in the long axis of myocytes located at the edges of the lesions. Based on our experimental findings in rat hearts, these studies suggest that human myocardial nerve fibers regenerate after necrotizing injuries and that at least some of the resulting scar-associated fibers have structural features differing from those in uninjured myocardium. We suspect that these structural differences might be associated with functional alterations that could affect the triggering of ventricular arrhythmias.

The mineralization of elastic fibers and alterations of extracellular matrix in pseudoxanthoma elasticum. Ultrastructure, immunocytochemistry, and X-ray analysis.

Histologic paraffin sections of pseudoxanthoma elasticum (PXE)-involved skin of forearm and axilla were used for histochemistry and immunohistochemical and analytical electron microscopy to study the progressive mineralization in the dermis of patients with PXE. The von Kossa technique identified mineral deposits throughout the reticular PXE dermis. X-ray analysis revealed patterns of calcium and phosphorus deposition in the von Kossa-positive areas, and the immunohistochemical staining using monoclonal antibodies identified increased chondroitin-6-sulfate in these areas when compared with normal skin. Scanning transmission electron microscopy observation combined with X-ray dot mapping show calcium and phosphorus to be codistributed within the mineralized area. This study confirms by new methods the increase in chondroitin-6-sulfate, alterations in elastin and collagen, and a high calcium and phosphorus elemental distribution matching the mineralized area in the PXE dermis.

Composition of the cement line and its possible mechanical role as a local interface in human compact bone.

Human compact bone may be viewed as a fiber reinforced composite material in which the secondary osteons act as the fiber reinforcements. The cement line, which is the interface between the 'fibers' (osteons) and extraosteonal bone matrix, may impart important mechanical properties to compact bone. The nature of these properties is not known partly because the composition of the cement line is unknown. This analysis examines the constituents of the osteon cement line using scanning electron microscopy and X-ray microprobe analysis to address its biomechanical functions as a local interface. The analysis suggests that the cement line is a region of reduced mineralization which may contain sulfated mucosubstances. This composition is consistent with the hypothesis that the cement line provides a relatively ductile interface with surrounding bone matrix, and that it provides the point specific stiffness differences, poor 'fiber'-matrix bonding and energy transfer qualities required to promote crack initiation but slow crack growth in compact bone.

The subdural space interpreted as a cellular layer of meninges.

The subdural region within the cranial meninges is examined in guinea pigs by electron microscopy. The fine structures of the arachnoid membrane and dura are described separately in specimens that have been isolated from each other during removal from the cranial cavity. In addition, the fine structure of the intact dura-arachnoid is described, where the subdural space would be present in an undisrupted state. Lastly, the inner surface of the dura and the outer surface of the arachnoid membrane are examined at the point of separation between the two specimens where the dura is reflected from the arachnoid by experimental dissection. From these observations morphologic criteria are established for identifying the constituents and boundaries of the subdural space and for explaining mechanisms in the histogenetic process of "opening" or enlarging this space. The morphologic identity of the classic subdural space is reinterpreted in light of the findings. The subdural space, traditionally described as a fluid-filled potential cavity existing in an extracellular compartment, is not apparent in the guinea pig. Instead, fragile cells designated as light cells occupy the compartment between the dura and arachnoid, with very little extracellular space available. Experimental opening of the subdural space occurs, significantly, along pathways extending by fracture through the cytoplasm and intercellular separation of these light cells rather than by enlargement of a preexisting mesothelial-lined intercellular space between these cells and the true arachnoid cells. Cytoplasmic fine structure of light cells suggests a close kinship with cells in the meningeal layer of the dura. The functional significance of the light cells and their possible role in subdural hematomas is discussed.

Morphology of temperature-sensitive strains of viral-transformed 3T3 mouse fibroblasts in vitro as determined by SEM and STEM.

The morphological characteristics of BALB/c 3T3 mouse fibroblasts transformed with two temperature-sensitive strains of simian virus 40 (SV-40) were studied. When grown at the permissive temperature (32 degrees C, transformed phenotype) strains A7B4b and A209B4a both closely resemble the morphology described for wild-type SV-40 cells. Scanning electron images show numerous knobs and filopodia as characteristic surface features in both subconfluent and confluent cultures. Scanning-transmission electron images reveal that the two strains are also similar in intracellular structure. When grown at the restrictive temperature (39 degrees C, untransformed phenotype) the two strains differ from each other. The A209B4a strain is more elongated in subconfluent cultures, and shows interactions of filopodia at cell-cell interfaces when confluent similar to those described for wild-type 3T3 cells. This strain also shows intracellular accumulation of polymorphic material thought to be secondary lysosomes. The A7B4b cells are flattened but generally not elongated when subconfluent, and do not show any obvious interactions at confluency. Intracellularly, this strain also differs from A209B4a.

Morphology of differentiating epithelial cells in the respiratory tract of the postnatal opossum (Didelphis virginiana).

Correlative microscopy of the developing respiratory epithelium during mucous cell differentiation reveals a decrease in the concentration of ciliated cells concomitant with proliferation of mucous cells and formation of an inner layer of the mucous blanket. Nonciliated cells increase in concentration between 40 and 80 days postnatal and provide the major surface area of the tracheal epithelium. Nonciliated cells change from isolated small patches with flat, finely-corrugated surfaces to larger, rounded aggregates with bulging cytoplasmic contents. Individual, nonadjacent ciliated cells are distributed around the borders of aggregates. By 120 days the precise boundary of each aggregate is lost as the epithelium develops more smooth contour. In the major bronchi, ciliated cells occur primarily in the clefts between bronchial folds. Cells in these clefts also provide for the origin of goblet cells, observed by 80 days and heavily concentrated here at 120 days. Nonciliated cells with smooth surfaces (dome cells) occupy the ridges of bronchial folds and their morphology varies little up to 120 days. A tannic acid technique was used to demonstrate the development of the inner layer of the mucous blanket. The layer begins as a thin fibrillar coat on the surface of the tracheal and bronchial epithelium at 40 days. It becomes progressively thicker in the trachea but changes little in the bronchi.

Pentastomid nymph from the brain of a squirrel monkey (Saimiri sciureus). I. Morphology of the nymph.

A Pentastomid nymph of the genus Porocephalus was identified following its removal from the brain-meningeal interface of a squirrel monkey, Saimiri sciureus. It was characterized by two inner and two outer hooks adjacent to the mouth opening, the presence of accessory lobes (or spines) on the outer hooks, a vertical slit-like mouth opening surrounded by a U-shaped conformation of integument, and annulation of the body surface. Stigmata, representing the openings of integumental chloride cells, were present on the dorsum of the head and tail, on anterior aspects of annulae on the body, and on anterior and posterior edges of annulae located on the ventral surface of the tail. These openings were not present on integumental folds at the bases of the inner and outer hooks, on the dorsal aspect of the tail flap, or in the deep grooves separating annulae. Although it was not possible to determine the species, based on the number of annulae this specimen may represent P. stilesi.

Pentastomid nymph from the brain of a squirrel monkey (Saimiri sciureus). II. Morphology of the host response.

A Porocephalus nymph found in the meninges of a squirrel monkey was encapsulated by connective tissue cells and fibrils presumably derived from the pia mater. The capsule was composed of an inner layer (IL) adjacent to the nymphal integument and an outer layer (OL) adherent to the brain surface. Separating the two layers was a capsular space. The IL was lined by a granular material adjacent to the nymph surface and possessed impressions of annulae and the apical pits of chloride cells. The surface of IL facing the capsular space was characterized by a monolayer of cells possessing extensive anastomosing plasmalemmal processes. The OL was composed of several tiers of fibroblasts and associated collagen fibrils that adhered to the brain surface in the form of a thickened pia mater. It is suggested that the capsule was formed by a modification of the pia that isolated the nymph and created an "intracapsular space" with specialized lining cells to facilitate fluid exchange.

High-voltage electron microscopy of extracellular fibrillogenesis.

High-voltage electron microscopy was employed to observe developing extracellular connective tissue elements in the cervical perinotochordal and perivertebral regions in the chick embryo from 2 through 15 days' incubation. During days 2 and 3, small (10 nm) and large (18-20 nm) microfibrils surrounded the notochord, becoming evident around fibroblast-like cells in day 4. Amorphous material, globular granules and microfibrillar bundles were present at this time. Microfibrillar length increased as did the total population of microfibrils. At four days microfibrils 3-5 nm in diameter arose in all directions from globular granules. During day 9 and thereafter to day 15, microfibrillar diameters increased. This growth formed unit collagenous fibrils 30 nm in diameter or greater. Axial periodicity became evident at day 14. Small microfibrils appear to be composed largely of glycoproteins and do not contain a significant amount of collagen. The globular granules and associated filaments are probably proteoglycans. The amorphous material is believed to provide molecular collagen to developing fibrils. Large microfibrils and unit collagenous fibrils contain significant amounts of molecular collagen.

Morphology of the osteonal cement line in human bone.

While current consensus suggests the absence of collagen in osteonal cement lines, the extent of cement line mineralization and the nature of the ground substance within the cement line are unclear. Samples of human radius were examined by using scanning electron microscopy, electron microprobe, and histochemical techniques. X-ray intensities were used to compare the amount of calcium, phosphorus, and sulfur in cement lines with amounts in surrounding lamellar bone. The results indicate that cement lines contain significantly less calcium and phosphorus, but significantly more sulfur, than surrounding bone matrix. The Ca/P ratio of cement lines was significantly greater than that of lamellar bone, suggesting that the mineral in cement lines may not be in the form of mature hydroxyapatite. No selective staining of the cement lines could be demonstrated by using periodic acid-Schiff, Sudan black B, or alcian blue critical electrolyte concentration techniques.

Spatial arrangements of microfibrils in myocardial scars: application of antibody to fibrillin.

Acute myocardial infarction kills myocytes; viable and necrotic myocytes disconnect and the ends of the viable cells become anchored to collagen fibers during reparative scar tissue formation. These anchorages have not been examined in detail, although previous studies have shown that microfibrils (MFs) concentrate at the edges of scars and at the tips of normal papillary muscles. We examined the spatial arrangements of MFs at these two sites in human hearts. Light and electron microscopic observations were made on tissue samples oriented in the long axis of myocytes and stained with monoclonal antibodies to fibrillin, a glycoprotein component of human microfibrils. MFs had identical arrangements at both sites, where they formed fibrous connections between myofibers and collagen fibers. These connections were oriented in the long axis of the muscle cells. At the myocyte ends, MFs appeared to intertwine with MFs in the normal endomysium; in the main body of the connections, MFs formed compact, 200 to 500 nm thick, fibrillin-positive fibers; and at the collagen ends, MFs splayed out among collagen fibrils. These observations indicate that MFs form myofiber-collagen fiber linkages at sites where the power of myocardial contraction is being transmitted to the extracellular connective tissue framework. Formation of such linkages seems to be an important step in the successful repair of necrotic myocardial lesions.

Fate of nerve fibers in necrotic, healing, and healed rat myocardium.

Myocardial infarction, myocardial scar tissue formation, and cardiac arrhythmogenesis seem to be associated. There are electrophysiological data suggesting that myocardial nerves are involved in arrhythmia development; however, there are no morphologic studies describing the fate of these nerves following necrotizing myocardial injuries. To describe the reactions of Schwann cells and axons following such injuries, we induced lesions in rat hearts with ischemia or transdiaphragmatic freeze-thawing and examined the acutely necrotic, healing, and healed lesions with light and electron microscopy. Antibodies to Schwann cell-associated S-100 protein were used to facilitate histologic detection. Both forms of injury produced focal lesions in which Schwann cells were killed and axonal segments destroyed; however, the basal lamina sheaths of cardiac myocytes and capillaries, and probably also of nerve fibers, remained largely intact. During 4 weeks of sequential observations, Schwann cells and axons were components of a hypercellular healing front that began at the periphery and moved toward the center of each lesion. Their proliferation and growth may have occurred within the original nerve basal lamina sheaths, and reparative axonal enlargements contained an abundance of 50- to 100-nm clear and dense storage granules. Fully developed nerve fibers were not only present in newly formed scar tissue but also appeared to be present in significantly greater density than in uninjured myocardial tissue. These findings demonstrate that proliferative nerve fiber regeneration occurs from the edges of necrotizing myocardial injuries, that healing results in relatively large number of nerve fibers in newly formed scars, and that axons in these scar-associated nerve fibers contain an abundance of neurosecretory granules. The functional significance of these observations remains to be determined.

Connective tissue cells in healing rat myocardium. A study of cell reactions in rhythmically contracting environment.

To better understand the tendency of myocardium to heal by scarring rather than regeneration, the authors examined the responses of connective tissue cells (CTCs) after three types of necrotizing injuries. Derived from myocardial interstitial cells, CTCs proliferated in both the connective tissue space and the compartment of necrotic myocytes. They assumed various cell forms: fibrocytelike CTCs throughout the sites of injury deposited extracellular scar tissue elements, established CTC-myocyte contacts, and helped anchor myocytes to scar tissue with myotendonlike specializations; CTCs with more complex forms established CTC-myocyte relationships, suggesting important roles in communication and tissue remodeling. CTCs within scar tissue differentiated into myofibrocytes, chondrocytes, and possibly smooth muscle cells. Most scar tissue elements were disposed in the long axis of myocytes. These alterations in form indicate that CTCs have various roles in myocardial repair and suggest that a number of the roles are modulated by contractile forces.

Basal lamina of rat myocardium. Its fate after death of cardiac myocytes.

As part of a study of the interactions between myocardial cells and extracellular matrix during healing of necrotic lesions, we have examined the fate of myocyte basal lamina (BL) after injury with ischemia, freeze-thawing, or isoproterenol. Using light and electron microscopy, and antibodies to three BL-associated antigens, we found that the BL of necrotic myocytes remained largely intact and continued to delineate the myocyte compartment from connective tissue space. Inflammatory cells entered the myocyte compartment through holes in the acellular BL and removed cell debris. The holes may have been produced by inflammatory cells and/or by the stretching force of the beating heart. After removal of debris, some BL sheaths of necrotic myocytes collapsed, resulting in spatial approximation of vessels. Interstitial cells deposited collagen and elastic fibers in the connective tissue space and within portions of the myocyte compartment. The acellular myocyte BL, collapsed or not, retained normal antigen staining for type IV collagen, laminin, and heparan sulfate for about 10 days, then showed diminished staining in patchy areas. These areas may correspond to BL disruption and degradation in conjunction with fibrosis, although a substantial amount of acellular BL remained in situ and became embedded in scar tissue. At least two types of granulo-vesicular bodies, measuring 25 to 60 and 60 to 160 nm respectively, were associated with the acellular BL; these were of unknown origin and function. The study shows that the fate of acellular BL in injured myocardium is similar to the fate of BL in other injured tissues; however, the appearance of holes in acellular BL, within hours after injury, is unusual and may enhance scar tissue formation. Whether the acellular BL contributes to regeneration of myocardium, as do acellular BLs in other injured tissues, remains to be determined.

Myocyte reactions at the borders of injured and healing rat myocardium.

To better understand the apparent tendency of myocardium to heal by scarring rather than by restoration of normal structure, we have examined by light and electron microscopy the basic reactions of rat ventricular myocytes and their interactions with the extracellular matrix. Using three different types of necrotizing injuries, we found that necrotic myocytes separated from viable myocytes at intercalated discs leaving blunt-ended stumps at the edge of each lesion; the basal lamina of necrotic myocytes remained largely intact and spanned the gap between viable myocytes on opposite sides of each lesion. A small number of stumps were capped off by a new layer of basal lamina and showed no evidence of proliferative activity. The majority of the stumps developed cell processes that extended along the acellular myocyte basal lamina sheaths. These processes had one of two different fates. Some became apposed to similar processes, formed intercalated disc attachments, increased myofibrillar mass, and appeared to be associated with muscle reconstruction. Others developed elongate tapered ends, which terminated in myotendinous connections to scar tissue. The outcome of healing necrotic myocardium, like the healing of noncardiac necrotic tissue injuries, appears to be a function of cell growth and extracellular framework guidance; however, unlike healing of noncardiac tissues, healing of myocardium is uniquely complicated by continuing muscle contractions. We think the rhythmic pulling at the edges of necrotic lesions induces formation of myotendinous attachments, which anchor myocytes to scar tissue and probably prevent further growth.

Simplified non-enzymatic technique for isolation of epithelium and muscle from the dog prostate gland.

Epithelium and muscle from the normal dog prostate were isolated with a rapid, simple, non-surgical separation (NSS) technique, involving mechanical agitation of organ cubes in a high concentration (30 microM) of EDTA for tissue dispersion, and gentle pressure filtration for recovery of epithelium and muscle. Derivation of this NSS procedure utilized the guinea pig seminal vesicle, since it can be surgically separated (SS) into pure epithelium and muscle to serve as controls for the biochemical viability of NSS prepared tissues. The NSS procedure adopted for use yielded not only pure (greater than 95%) epithelium and muscle from intact seminal vesicle, but also activities of 5 alpha-reductase and concentrations of the cytosol estrophile in each tissue, which were not significantly different from the SS counterparts. In the dog prostate gland, the concentrations of 5 alpha-reductase and the cytosol estrophile in the NSS epithelium were 1.58 and 0.43 of the NSS muscle, respectively.

Opossum adrenal medulla: II. Differentiation of the chromaffin cell.

The ultrastructure of the opossum adrenal medulla was examined in its postnatal development. Maturation of chromaffin cells and genesis of chromaffin vesicles were of particular interest. The primitive sympathetic cell was seen to contain few organelles with no apparent polarity. Initial pheochromoblasts contained more organelles with some polarity. Endoplasmic reticulum and the Golgi complex increased as the pheochromoblasts matured, which suggested increased synthetic activity. Structures resembling Golgi/endoplasmic reticulum/lysosome (GERL) systems were seen in the pheochromoblasts. It is suggested that some of the components of the chromaffin vesicle may be processed by the GERL while others come directly through the Golgi complex. It is stressed that the developing pheochromoblast in the opossum presents an interesting model in which to study the genesis of the chromaffin vesicle.

Intracast muscle stimulation prevents bone and cartilage deterioration in cast-immobilized rabbits.

Tibial articular cartilage and the knee meniscus from cast-immobilized rabbits whose quadriceps were electrically stimulated for 17 days were compared with those from cast-immobilized rabbits without muscle stimulation. Cartilage from non-stimulated rabbits showed evidence of deep fibrillation and loss of Safranin O metachromasia. Scanning electron microscopy (SEM) showed large areas of cavitation and cartilage erosion. Cartilage from cast-immobilized muscle-stimulated rabbits demonstrated no fibrillation, pitting, or surface erosion at either the light microscopic or SEM levels. Electrical muscle stimulation prevented bone loss by significantly increasing bone turnover rate. These observations suggest that electrical muscle stimulation can prevent bone and cartilage deterioration in short-term cast-immobilized limbs.

Opossum adrenal medulla: I. Postnatal development and normal anatomy.

The anatomy and histology of the adrenal gland in the adult opossum were found to be typical for mammals. The development of the adrenal medulla was also found to follow the typical mammalian pattern. Primitive sympathetic cells were found in both intra- and extra-adrenal locations in the newborn at a time when chromaffin precursor cells were migrating to the adrenal anlage. Pheochromoblasts first appeared within the forming medulla where at a later stage chromaffin cells could be observed forming columns of cells between adjacent sinusoids. Unlike in other mammals, much of this development takes place postnatally when the neonate is in the mother's marsupium. The value of the developing opossum adrenal medulla as an experimental model is stressed, since a significant amount of development takes place in an environment that is accessible to experimental manipulation.

The characterization of normal and scleroderma skin fibroblasts cultured in a collagen gel matrix.

Fibroblasts from normal and progressive systemic sclerosis involved skin more closely resemble in vivo cells when cultured in a collagen gel matrix, than do fibroblasts cultured on plastic. Ultrastructural studies show that the cytoskeleton and secretory organelles from these cells are better developed in the presence of a collagen gel. Fibronectin, glycosaminoglycans and collagen fibrils are produced by the cells and deposited in the preformed matrix. Collagen synthesis is greater in the scleroderma derived fibroblasts for all culture conditions with increased deposition in the matrix. Total collagen production form cells associated with the gel is less than that from those cultured on plastic, suggesting an inhibition of collagen synthesis by the preformed collagen matrix.