A synthesis of interstitial fluid regulation and lymph formation.

The interstitial fluid spaces are filled with a mat of collagen fibers, and the interstices of this mat contain a mucopolysaccharide gel ground substance. Both the collagen fibers and the gel are elastic structures that can be expanded or compacted. In the expanded state the collagen fibers are pushed far apart and pockets of free fluid develop witin the gel. In the compacted state the elastic recoil of the compressed collagen fibers and gel reticular fibrillae seems to cause suction on the fluid within the tissue spaces, thus creating a subatmospheric pressure. Measurements of interstitial fluid pressure using a perforated capsule method indicate that this is normally slightly negative (subatmospheric) in most soft tissues. However, even very slight extra filtration of fluid into the tissue spaces increases the interstitial fluid pressure toward more positive values, which in turn increases lymph flow. The increased lymph flow then decreases the interstitial fluid volume and pressure back toward normal because of two mechanism, 1) direct removal of fluid from the tissue spaces in the lymph, and 2) removal of protein from the interstitial fluid in the lymph, thus decreasing the interstitial fluid colloid osmotic pressure and allowing more effective osmosis of fluid directly from the interstitial spaces back into the capillaries.     

Immunofluorescent and ultrastructural studies of “sarcomeric” units in stress fibers of cultured non-muscle cells

Immunofluorescence and electron microscopy were used to study the organization of actin, myosin and α-actinin in the “sarcomeric” units of the stress fibers of a selected non-muscle cell type. The results of indirect immunofluorescence confirm that myosin and α-actinin are periodically distributed in discrete units along stress fibers and demonstrate that they are alternately spaced. This relationship is required by a sarcomere model of stress fiber construction. A comparison between immunofluorescent and EM images of stress fibers confirms that α-actinin is confined to Z line-like dense bodies, myosin to the spaces in between. The most intriguing result is that by immunofluorescence a periodic distribution of actin can be detected in some fibers. This may indicate that even actin is periodically distributed in non-muscle “sarcomeres”.     

The red pulp of the human spleen. Structural basis of blood filtration

The paper summarizes the authors' findings indicative of an open blood circulation in the human spleen, particularly those obtained by immune and enzyme histo- and cyto-chemical methods, and by electron microscopy. In the red pulp the blood gets into the extravascular spaces of the pulp cords, where the individual blood components have to pass between numerous macrophages to reach the sinuses. The sinus wall is composed of elongated endothelial cells surrounded by waved annular or ring fibers of the basement membrane. In some areas annular fibers are joined by longitudinal fibers, giving rise to the filtration lattice, the fenestrated basement membrane. The sinus wall represents the last filter barrier which decises whether the blood elements get back into the blood or not. Extravasation of blood secures that all foreign as well as the altered own components, particularly cellular and particle ones naturally along with the normal constituents get from the circulating blood into extravascular spaces. In the next phase, however, the normal ones return into the circulation, whereas the abnormal components are removed from the extravascular tissue by means of the macrophagic and immune system of the spleen.     

Reticular meshwork of the spleen in rats studied by electron microscopy

The reticular meshwork of the rat spleen, which consists of both fibrous and cellular reticula, was investigated by transmission electron microscopy. The fibrous reticulum of the splenic pulp is composed of reticular fibers and basement membranes of the sinuses. These reticular fibers and basement membranes are continuous with each other. The reticular fibers are enfolded by reticular cells and are composed of two basic elements: 1) peripheral basal laminae of the reticular cells, and 2) central connective tissue spaces in which microfibrils, collagenous fibrils, elastic fibers, and unmyelinated adrenergic nerve fibers are present. The basement membranes of the sinuses are sandwiched between reticular cells and sinus endothelial cells and are composed of lamina-densalike material, microfibrils, collagenous fibrils, and elastic fibers. The presence of these connective tissue fibrous components indicates that there are connective tissue spaces in these basement membranes. The basement membrane is divided into three parts: the basal lamina of the reticular cell, the connective tissue space, and the basal lamina of the sinus endothelial cell. When the connective tissue space is very small or absent, the two basal laminae may fuse to form a single, thick basement membrane of the splenic sinus wall. The fibrous reticulum having these structures is responsible for support (collagenous fibrils) and rebounding (elastic fibers). The cells of the cellular reticulum--reticular cells and their cytoplasmic processes, which possess abundant contractile microfilaments, dense bodies, hemidesmosomes, basal laminae, and a well-developed, rough-surfaced endoplasmic reticulum, and Golgi complexes, which are characteristic of both fibroblasts and smooth muscle cells--are considered to be myofibroblasts. They may play roles in splenic contraction and in fibrogenesis of the fibrous reticulum. The contractile ability may be influenced by the unmyelinated adrenergic nerve fibers that pass through the reticular fibers. The three-dimensional reticular meshwork of the spleen consists of sustentacular fibrous reticulum and contractile myofibroblastic cellular reticulum. This meshwork not only supports the organ but also contributes to a contractile mechanism in circulation regulation, in collaboration with major contractile elements in the capsulo-trabecular system.     

Histochemistry of the lung of the Australian snake-necked tortoise chelodina longicollis

The innervation and structure of the lung of the Australian snake-necked tortoise, Chelodina longicollis, was examined by using light microscopy including fluorescence histochemical techniques. The anterior lung was divided into a number of compartments with numerous alveolar spaces. The posterior lung was simpler and saclike in structure and alveolar spaces were absent. Smooth muscle fibers occurred in discrete muscle bands and in the walls of the septal bands. Ganglion cells occurred along nerve trunks throughout the lung but were more numerous in the posterior lung. Smooth muscle bands, the extrinsic pulmonary artery, and the arteries within the lung were sparsely innervated by adrenergic fibers. Substance P-containing sensory fibers were not demonstrated. The innervation and structure of the lung are compared to published work on other reptiles.     

A unique connective tissue structure in the aortic media of the chicken

Summary The interstitium between smooth muscle cells in the media of the abdominal aorta of the chicken contains basement membranes, glycosaminoglycan, stout elastic fibers, extensive bundles of collagenous fibers, and a unique striated structure. In cross section, this striated, hexagonal structure resembles a honeycomb, each hexagon consisting of 6 isosceles triangles. Microtubule-like structures are present at each corner and center of a hexagon, and 3 delicate filaments are located equidistantly between putative microtubules. The periodicity evident in longitudinal section is the result of a constant repetition of microtubule-like elements. From staining with phosphotungstic acid it appears that the striated connective tissue structures are proteinacous and might serve as a reinforcing structure where smooth muscle cells are separated by dilated extracellular spaces.     

Immunofluorescent localization of extracellular matrix components during muscle morphogenesis. II. In chick embryos with hereditary muscular dysgenesis (cn/cn)

The immunofluorescent distribution of types I and III collagen, fibronectin, and laminin during muscle morphogenesis of the crooked neck dwarf mutant chick embryo differs from that of the normal chick. The drastic difference is related to the inability of the mutant embryo to maintain a harmonious muscle pattern. The first sign of the defect is the disaggregation of type I collagen fibers of the tendons and the disorganization of the intermuscular spaces. The organization of the connective tissue never proceeds beyond the appearance of an epimysial envelope, rich in types I and III collagen, which becomes disorganized shortly after. No perimysial envelopes displaying types I and III collagen fibers and fibronectin, nor endomysial sheaths develop. Only large spaces filled with types I and III collagen fibers subdivide groups of muscle cells irregularly. On the whole, type III collagen is less abundant than type I collagen. Fibronectin disappears from the periphery of the muscle cell. Laminin is more thickly deposited in the basal lamina around irregularly sized muscle cells than around the normal muscle cell. The results are discussed in terms of morphogenetic interactions between connective tissue cells and muscle cells, and in terms of fibrosis, which characterizes some muscle diseases.     

Innervation of the mink pineal gland with neuropeptide Y (NPY)-containing nerve fibers

Summary An immunohistochemical investigation of the mink pineal gland was performed by use of antibodies raised in rabbits against neuropeptide Y (NPY) and Cys-NPY (32–36)-amide recognizing neuropeptide Y with an amidation at position 36 (NPYamide). NPY-immunoreactive nerve fibers were located predominantly in the rostral part of the pineal gland and in the pineal stalk. Immunoreactive nerve fibers were found throughout the pineal gland, but the number of fibers in the caudal part of the gland was low. The fibers were present both in the perivascular spaces and between the pinealocytes. Many NPY-immunoreactive fibers were also located in the posterior and habenular commissures; some of these fibers were connected with the fibers in the rostral part of the mink pineal gland, indicating that at least some of the NPY-immunoreactive nerve fibers are of central origin. The nerve fibers immunoreactive to amidated NPY were distributed in a similar manner. However, the number of fibers immunoreactive to NPYamide was lower than the number of fibers immunoreactive to NPY itself. After removal of the superior cervical ganglia bilaterally 22 days or 12 months before sacrifice, NPY-immunoreactive nerve fibers remained in the gland. This immunohistochemical study of the mink pineal gland therefore shows that the NPY/NPYamide-immunoreactive nerve fibers innervating the pineal gland in this spegcies are a component of the central innervation or originnate from extracerebral parasympathetic ganglia.     

Lens metabolic cooperation: a study of mouse lens transport and permeability visualized with freeze-substitution autoradiography and electron microscopy

Transport of metabolites is demonstrated between compartments of the adult mouse lens by freeze-substitution autoradiography. In vivo patterns of lysine incorporation are compared with in vitro patterns of lysine, glucose, uridine, and deoxyglucose incorporation. Intracellular and extracellular distributions of tritiated metabolites are determined by comparison of transported substrates with the nontransported molecules of similar molecular size: mannitol and sucrose. The permeability of the lens intercellular spaces is probed with Procion Yellow at the level of fluorescence microscopy, and with horseradish peroxidase at the electron microscope level. Freeze-fracture electron microscopy reveals gap junctions between epithelial cells, between lens fibers, and between epithelial cells and lens fibers. Zonulae occludentes (tight junctions) are not routinely observed between epithelial cells in the mouse. This latter result is subject to species variation, however, since zonulae occludentes are abundant between chicken epithelial cells. The permeability results suggest that the lens cells are capable of metabolic cooperation, mediated by an extensive gap junction network.     

Distribution of muscle fiber types and EMG activity in cat intercostal muscles

The electromyogram (EMG) activity and histochemical properties of intercostal muscles in the anesthetized cat were studied. The parasternal muscles were consistently active during inspiration. The external intercostals in the rostral spaces and the ventral portions of the midthoracic spaces were also recruited during inspiration. The remaining external intercostals were typically silent, regardless of the level of respiratory drive. The internal intercostal muscles located in the caudal spaces were occasionally recruited during expiration. There was a clear correlation between recruitment patterns of the intercostals and the histochemically defined fiber type properties of the muscles. Intercostal muscles that were routinely recruited during inspiration had a significantly higher proportion of slow-oxidative muscle fibers.     

Temporary normalization of blood sugar, in a totally pancreatectomized dog, after placing an artificial insulin distributor]

Islets of Langerhans were stablished in the spaces between 9 synthetic hollowfibers in a "bio-artificial insulin distributor" which was implanted in the carotid artery of totally pancreatectomized dogs. Normo-glycemia was restored after 4 hours. The major problem encountered was coagulation in the fibers. Presently, this limits the prolonged utilization of this system.     

Über intraepitheliale nervöse Endformationen in mehrschichtigem Plattenepithel

Zusammenfassung Im Anschluß an frühere lichtmikroskopisch erhobene Befunde (Silberimprägnation; 1924, 1928) weist der Verfasser durch elektronenmikroskopische Untersuchungen nach, daß durch die Interzellularspalten des Epithels (Schnauze der weißen Ratte) Nervenfasern hindurchziehen. Diese Nervenfäserchen, ihre Varikositäten und ihre Endknöpfchen liegen in den Spalten zwischen den Epithelzellen. Die Ultrastruktur der nackten, d.h. von Schwannschen Zellen nicht umhüllten terminalen Nervenfasern und ihrer Endigungen im Epithel wird beschrieben.

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On intraepithelial nerve endings in a stratified squamous epithelium

Summary In addition to former light microscopical observations the author demonstrates electronmicroscopically that nerve fibers pass through the intercellular spaces of the epithelium. The nerve fibers, their varicosities and terminal bulbs lie within the spaces between the epithelial cells. The ultrastructure of the terminal nerve fibers in the epithelium is described.

     

NUCLEOSIDE PHOSPHATASE AND CHOLINESTERASE ACTIVITIES IN DORSAL ROOT GANGLIA AND PERIPHERAL NERVE

In dorsal root ganglia and peripheral nerve of the rat and other species, nucleoside phosphatase and unspecific cholinesterase reaction products are found in the plasma membranes and spaces between them at two sites: (1) Schwann cell-axon interfaces and mesaxons of unmyelinated fibers, and (2) sheath cell-perikaryon interfaces and interfaces between adjacent sheath cells. Acetylcholinesterase reaction product is found in the perikaryon (within the endoplasmic reticulum) and the axon (axoplasmic surface). Nucleoside phosphatase reaction product is also found in the numerous vacuoles at the surface of perineurium cells, ganglion sheath cells, and cells surrounding some ganglion blood vessels. Nucleoside phosphatase activities in the sections fail to respond, in the manner described for "transport ATPase," to diisopropylphosphofluoridate, sodium and potassium ions, and ouabain. Nucleoside diphosphates are hydrolyzed more slowly than triphosphates in unmyelinated fibers, and are not hydrolyzed at the perikaryon surface. Nucleoside monophosphates are either not hydrolyzed or hydrolyzed very slowly. In contrast to these localizations, which are believed to demonstrate sites of enzyme activity, it is considered likely that diffusion artifacts account for the nucleoside phosphatase reaction product frequently found along the outer surfaces of myelinated fibers and within vacuoles at the Schwann cell surfaces of these fibers. The diffuse reaction product seen in basement membranes of ganglion and nerve may also be artifact.     

On the intrinsic innervation of normal rat liver

Summary With the Bodian method stained fibers were observed in the lobules of the rat liver and with the modified Karnovsky and Roots thiocholine method cholinesterase (presumably acetylcholinesterase (AChE))-positive nerve fibers were found in a pattern similar to that of the Bodian-stained fibers. The AChE positive nerve fibers form a network in the liver lobules in close relation to hepatocytes and sinusoids. Fluorescent varicose nerve fibers demonstrated by the glyoxylic acid and Falck-Hillarp fluorescence methods were found only in the interlobular spaces associated with vessels. As no overlapping of distribution patterns of AChE-positive nerve fibers and fluorescent nerve fibers occurs, the AChE activity of the nerves of the liver lobules probably reflects the associated presence of acetylcholine in the nerve fibers. In consequence we suggest that nerves of the liver lobules belong to the autonomic parasympathetic nervous system.SEM of liver tissue revealed light cords apparently situated in smooth surfaced channels between adjacent hepatocytes and in the space of Disse, where fibers also cross sinusoids. We tentatively suggest that the cords of the SEM represent the AChE-positive nerve fibers of our LM observations'.The skilled assistance of Dr. Esther Hage, Department of Pathology, Odense Sygehus, Denmark, in the Falck-Hillarp fluorescence work is gratefully acknowledged     

Nascent muscle fiber appearance in overloaded chicken slow-tonic muscle

The application of a weight overload to the humerus of chickens induces a hypertrophy of anterior latissimus dorsi (ALD) muscle fibers. This growth is accompanied by a rapid and almost complete replacement of one slow-tonic myosin isoform, SM-1, by another slow-tonic isoform, SM-2. In addition, a population of small fibers appears mainly in extrafascicular spaces and, concurrently, three additional myosin bands are detected by gel electrophoresis. Five antibodies against myosin heavy chain (MHC) isoforms were selected as immunocytochemical probes to determine the cellular location and nature of these myosins. The antibodies react with ventricular, fast skeletal muscle and either SM-1 or SM-2, or both the slow-tonic MHCs. The antifast and antiventricular antibodies react with myosin present in the 10-day embryonic ALD muscle but do not react with myosin in posthatch ALD muscle. The small fibers in overloaded muscle contain a myosin isoform characteristically expressed during the embryonic stage of ALD muscle development and therefore are named nascent myofibers. Some of the nascent myofibers do not react with the antibody to both slow-tonic MHCs, indicating the lack of the normal adult slow-tonic myosins which are expressed in 10-day embryos. In order to explore the origin of the nascent fibers, an electron microscopic study was performed. Stereological analysis of the existing fibers shows a stimulation of numbers and sizes of satellite cells. In addition, the volume occupied by nonmuscle and undifferentiated cells increases dramatically. Myotube formation with incipient myofibrils is seen in extrafascicular spaces. These data suggest that new muscle fiber formation accompanies hypertrophy in overloaded chicken ALD muscle and the process may involve satellite cell migration.     

THE ULTRASTRUCTURE AND DISPOSITION OF VESICULATED NERVE PROCESSES IN SMOOTH MUSCLE

The walls of the gastrointestinal tract and urinary bladder of rats were fixed in osmium tetroxide, embedded in methacrylate, and sectioned for electron microscopy. The examination of sections of smooth muscle tissue with the electron microscope reveals the presence of bundles of unmyelinated nerve fibers within the intercellular spaces. In addition, vesiculated nerve processes, bounded on their outer surfaces by delicate plasma membranes and typically containing varying quantities of synaptic vesicles and mitochondria, make intimate contact with the surface of smooth muscle cells. These nerve processes are similar in structure and disposition to nerve endings previously described in skeletal muscle, in the central nervous system, in peripheral ganglia, in receptors, and in glands. It is concluded that the relationships existing between vesiculated nerve processes and the surface of smooth muscle cells constitute neuromuscular junctions. Profiles of protrusions of smooth muscle cells are often seen protruding into the intercellular spaces. Here they occur singly or in groups, originating from one or more cells. Because of the plane of section the protrusions may sometimes appear as individual entities between the muscle cells. In such cases care must be exercised in their identification because they have characteristics similar to sectioned nerve processes which also occur in the intercellular spaces.     

Histochemical and biochemical studies of cholinesterases in the carotid labyrinth of amphibians

Acetylcholinesterase activity was detected in the carotid labyrinth of amphibians by both biochemical and histochemical methods. The histochemical tests showed enzyme activity in surfaces of afferent and efferent nerve terminals in contact with type I cells, but none within the type I cells. Acetylcholinesterase activity occurred on some, though not all, nerve fibers in the extra-cellular spaces. These fibers might be parasympathetic cholinergic fibers innervating blood vessels.     

The coelomic envelope of Xenopus laevis eggs: a quick-freeze, deep-etch analysis

The extracellular matrix of Xenopus laevis oocytes was analyzed before and after meiotic maturation using quick-freeze, deep-etch, rotary-shadow electron microscopy. The perivitelline space (PS) of the meiotically immature oocyte contains a filamentous network which connects microvilli (MV) and follicle cell macrovilli to the folded oocyte surface below. The envelope overlying the PS is composed of bundles of large fibers which course between the tips of the MV. Spaces between these bundles contain smaller fibrils which secure the egg envelope to the microvillar tips. Meiotic maturation is accompanied by flattening of the oocyte plasma membrane, formation of an orderly array of MV, and elevation of the egg envelope. In the coelomic eggs, the reorganized envelope is composed of loosely bundled large fibers which course above the microvillar tips rather than between them. The spaces between these bundles contain small fibers similar to those seen in the meiotically immature oocyte. This reorganized envelope, however, will not bind sperm; further modifications must transpire during passage through the oviduct to render it sperm receptive.     

New fiber formation in the interstitial spaces of rat skeletal muscle during postnatal growth.

The purpose of this study was to determine whether fiber hyperplasia occurs in the rat plantaris muscle during postnatal weeks 3-20. Total muscle fiber number, obtained via the nitric acid digestion method, increased by 28% during the early postnatal rapid growth phase (3-10 weeks), whereas the number of branched fibers was consistently low. Whole-muscle mitotic activity and amino acid uptake levels showed an inverse relationship to the increase in total fiber number. The expression of MyoD mRNA (RT-PCR) levels decreased from 3 to 20 weeks of age, as did the detection of anti-BrdU- and MyoD-positive cells in histological sections. Immunohistochemical staining patterns for MyoD, myogenin, or developmental myosin heavy chain on sections stained for laminin (identification of the basal lamina) and electron micrographs clearly indicate that de novo fiber formation occurred in the interstitial spaces. Myogenic cells in the interstitial spaces were negative for the reliable specific satellite cell marker M-cadherin. In contrast, CD34 (an established marker for hematopoietic stem cells)-positive cells were located only in the interstitial spaces, and their frequency and location were similar to those of MyoD- and/or myogenin-positive cells. These findings are consistent with fiber hyperplasia occurring in the interstitial spaces of the rat plantaris muscle during the rapid postnatal growth phase. Furthermore, these data suggest that the new fibers may be formed from myogenic cells in the interstitial spaces of skeletal muscle and may express CD34 that is distinct from satellite cells.     

Tyrosine hydroxylase- and neuropeptide Y-immunoreactive nerve fibers in the pineal complex of untreated rats and rats following removal of the superior cervical ganglia

Summary The distribution of tyrosine hydroxylase (TH)- and neuropeptide Y (NPY)-immunoreactive(IR) nerve fibers in the pineal complex was investigated in untreated rats and rats following bilateral removal of the superior cervical ganglia. In normal animals, a large number of TH- and NPY-IR nerve fibers were present in the pineal capsule, the perivascular spaces, and intraparenchymally between the pinealocytes throughout the superficial pineal and deep pineal gland. A small number of TH-IR and NPY-IR nerve fibers were found in the posterior and habenular commissures, a few fibers penetrating from the commissures into the deep pineal gland. To elucidate the origin of these fibers, the superior cervical ganglion was removed bilaterally in 10 animals, and the pineal complex was examined immunohistochemically. Two weeks after the ganglionectomy, the TH-IR and NPY-IR nerve fibers in the superficial pineal gland had almost completely disappeared. On the other hand, in the deep pineal and the pineal stalk, the TH-IR and NPY-IR fibers were still present after ganglionectomy. These data show that the deep pineal gland and the pineal stalk possess an extrasympathetic innervation by TH-IR and NPY-IR fibers. It is suggested that the extrasympathetic TH-IR and NPY-IR nerve fibers innervating the deep pineal and the pineal stalk originate from the brain.