The development of sites of metaplastic change in regenerating tendon

Summary The development of cartilage and bone in the regenerating segment of the tendon of Achilles following transection has been studied with regeneration taking place in situ, and also following transplantation to a subcutaneous site. Prior to transplantation regeneration was allowed to proceed in situ for various periods of time.It was observed that cartilage and bone develop from the cells of certain pre-cartilaginous areas which represent a metaplasia from fibroblasts. Transplantation to the subcutaneous site at a stage of regeneration when pre-cartilaginous or cartilaginous foci are present leads to the eventual development of bone in the transplant. Transplants made prior to the development of pre-cartilage or cartilage do not show bony metaplasia.It is concluded that the tension of muscle pull is a factor stimulating the metaplastic transformation of fibroblasts to chondroblasts, but once this transformation has occurred the progression to bony metaplasia continues, independently of tension.Supported by a grant from the Medical Research Council of Canada.     

Zur Frage der Exocytose im Vorderlappen der Hypophyse

Zusammenfassung Serienschnitte vom Vorderlappen der Rattenhypophyse wurden elektronenmikroskopisch untersucht, um das Verhalten der Granula bei der Hormonabgabe aufzuklären. — Granula werden einzeln oder zu mehreren durch Exocytose aus der Zelle ausgeschleust. Serienschnitte lassen erkennen, daß granulahaltige Vakuolen, die innerhalb des Zytoplasmas in einer Schnittebene zu liegen scheinen, sich bereits im Extrazellularraum befinden. Die Granula gelangen nicht als Ganzes in die Blutbahn, sondern unterliegen einem Auflösungsprozeß. Hierbei verlieren sie an Massendichte. Wir nehmen an, daß für ihre Lyse und die Freisetzung der Hormone Milieuveränderungen und Diffusionsvorgänge von Bedeutung sind. Neben der Exocytose von Granula scheinen der Zelle noch andere Wege offen zu stehen, um hormonal wirksame Stoffe freizusetzen.

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On the problem of exocytosis in the anterior pituitary

Summary The question of the granular extrusion from normal anterior pituitary cells of the rat was investigated electromicroscopically (serial sections). Granules are released from the cell either separately or several together by exocytosis. Serial sections indicate that vacuoles containing granules appear in sections taken within the cytoplasm, but are actually situated in the extracellular space. The granules are dissolved, i.e. do not enter into the blood stream. During this process they partially loose electron density. Their lysis and the release of hormones is believed to be due to a change in the milieu and to a process of diffusion. Besides exocytosis of granules possibly other mechanisms of release of hormonally active components exist.

Herrn Prof. Dr. Werner Rotter, Homburg (Saar), zur Vollendung des 75. Lebensjahres in Dankbarkeit gewidmet.     

Immunocytochemical staining of supraoptic neurons from homozygous Brattleboro rats by use of antibodies against two domains of the mutated vasopressin precursor

Summary By use of an antibody against the 14 amino acids in the mutated vasopressin precursor (CP-14) characteristic of the homozygous Brattleboro rat, an immunohisto- and-cytochemical study was performed on the supraoptic nuclei of homozygous Brattleboro rats. At the light-microscopic level, varying numbers of perikarya per section exhibited a positive reaction. The most intense staining was observed in a patchy manner on the peripheral portions of the cytoplasm, its central portion being stained less intensely. The antiserum did not react with the supraoptic perikarya of the Wistar rat. In the homozygous Brattleboro rat, antibodies against normal vasopressin only rarely resulted in a positive immunoreaction. However, when it was observed, incubation of the subsequent section with CP-14-antiserum suggested a co-localization of both peptides in the same perikaryon. At the ultrastructural level, CP-14 immunoreactivity was demonstrated on the secretory cisternae of the Golgi apparatus, on lysosome-like bodies and on parts of the rough endoplasmic reticulum. With the use of an antibody against normal vasopressin, immunoreactivity was confined to very limited areas of the rough endoplasmic reticulum. The oxytocin immunoreactivity in supraoptic perikarya of Brattleboro rats did not differ from that in the Wistar rat, either at the light- or at the electron-microscopic levels.     

Form und Verteilung subependymaler Basalmembranlabyrinthe am Ventrikelsystem der Ratte

Zusammenfassung Mit der Perjodsäure-Bisulfit-Aldehydthionin-Färbung (Specht) lassen sich am ganzen Ventrikelsystem des Rattengehirns sub- und interependymale Strukturen lichtmikroskopisch nachweisen, die elektronenmikroskopisch als Basalmembranlabyrinthe (BML) identifiziert werden können. Verschiedene Formen dieser BML lassen sich unterscheiden. Sie stehen in Verbindung mit subependymalen Kapillaren. Die Verteilung der BML wird in Ventrikelkarten dokumentiert.

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The shape and distribution of subependymal basement membrane labyrinths of the ventricular system of brain in rat

Summary Sub- und interependymal structures, being demonstrable by a period-acid-bisulfit-aldehydthionine-method (Specht), occur in the whole ventricular system by light microscopy. They can be identified electron microscopically as basement membrane labyrinths (BML). Different shapes of these BML can be distinguished. They are in communication with the basement membrane of subependymal capillaries. The distribution of BML is documented in ventricular maps.

Die Untersuchungen wurden mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft (Le 69/7) durchgeführt.     

Effects of prolonged sodium restriction on the morphology and function of rat adrenocortical autotransplants

Summary Regenerated adrenocortical nodules were obtained by implanting fragments of the capsular tissue of excised adrenal glands into the musculus gracilis of rats (Belloni et al. 1990). Five months after the operation, operated rats showed a normal basal blood level of corticosterone, but a very low concentration of circulating aldosterone associated with a slightly increased plasma renin activity (PRA). Regenerated nodules were well encapsulated and some septa extended into the parenchyma from the connective-tissue capsule. The majority of parenchymal cells were similar to those of the zonae fasciculata and reticularis of the normal adrenal gland, while zona glomerulosa-like cells were exclusively located around septa (juxta-septal zone; JZ). In vitro studies demonstrated that nodules were functioning as far as glucocorticoid production was concerned, while mineralocorticoid yield was very low. Prolonged sodium restriction significantly increased PRA and plasma aldosterone concentration, and provoked a marked hypertrophy of JZ, which was due to increases in both the number and average volume of JZ cells. Accordingly, the in vitro basal production of aldosterone and other 18-hydroxylated steroids was notably enhanced. The plasma level of corticosterone, as well as zona fasciculata/reticularis-like cells and in vitro production of glucocorticoids by regenerated nodules were not affected. These findings, indicating that autotransplanted adrenocortical nodules respond to a prolonged sodium restriction similar to the normal adrenal glands, suggest that the relative deficit in mineralocorticoid production is not due to an intrinsic defect of the zona glomerulosa-like JZ, but is probably caused by the impairment of its adequate stimulation under basal conditions. The hypothesis is advanced that the lack of splanchnic nerve supply and chromaffin medullary tissue in regenerated nodules may be the cause of such an impairment.     

Microtubular number in the tractus hypophyseus of newborn normal rats and newborn rats with congenital diabetes insipidus

Summary Evidence has already been adduced suggesting that an increase in microtubular number occurs in the tractus hypophyseus of rats stressed by the administration of hypertonic saline, and of rats with congenital diabetes insipidus (CDI). Since the tractus hypophyseus in these animals shows high secretory activity, it seems likely that the microtubular increase reflects the participation of microtubules in axoplasmic transport. To exclude, however, a congenital microtubular abnormality in CDI, affected newborn rats were examined. In these, the microtubular number was normal, thus suggesting that the increase in microtubular number seen in adult animals was not a congenital morphological abnormality. However, by 4 days of age there was a slight but statistically significant increase in microtubular number in affected rats, a change probably attributable to increased secretory activity.This project was supported by the Medical Research Council     

Microglia and reactive “M” cells of degenerating central nervous system: Does similar morphology and function imply a common origin?

Summary Penetration of the median eminence and ventrolateral walls of the III ventricle by intraventricularly injected horseradish peroxidase was studied in rats. Experimental times varied between 10 to 70 min (short-term experiments) and 12 hrs to 30 days (long-term experiments).In short-term experiments, the median eminence was found to be completely stained whereas the lateral walls of the III ventricle were penetrated only up to 1 mm in depth. Spreading of the tracer takes place predominantly through the extracellular space and cellular uptake and transport do not seem to play a role during the first 70 min following the injection.In long-term experiments, the tanycytes exhibit a variety of intracellular inclusions marked by HRP precipitate. Tanycytic perikarya contain dense bodies and lipofuscin-like aggregates. Lipoprotein granules are thought to arise from these and are interpreted as lysosomal residual bodies. In tanycytic processes and perivascular endfeet, accumulations of HRP-containing tubules and polymorphous granules are encountered suggesting a transport towards the blood vessels of the portal plexus or of the arcuate and ventromedial areas, respectively. Sporadic tanycytes which are completely and evenly stained from the ventricular surface to the end of their processes were observed in the arcuate and ventromedial area. Whether this appearance can be taken as a sign of normal cell function seems doubtful. Some possible routes of transport through the median eminence—extracellular and transcellular—are summarized in a schematic drawing, taking into account the present findings and those published elsewhere in the literature.We gratefully acknowledge an Anglo-German Collaboration Grant of the Wellcome Trust enabling stimulating discussions with other workers in the field.     

Regeneration of soleus muscles of rat autografted in toto as studied by electron microscopy

Summary Rat soleus muscles were autografted from right to left legs, and regeneration following necrosis of all original myofibres was studied after 7 to 250 days. The best regenerates were from grafts replacing all calf muscles and sutured to the tendon stumps. After 30 days the size of such regenerates was equal to those from minced gastrocnemius muscles: the cross sectional area of muscle tissue was 30% (1.7 mm²) and the number of fibres was 180% (4500) of normal soleus muscles; the fibre diameters were 10 to 40 m. To increase the number of myoblasts before grafting some muscles were injured by Ringer solution of 70° C and transplanted after 2 days. Nevertheless, this did not influence regeneration.After 7 days clusters of myotubes occurred in the periphery of the muscle. These myotubes originated from myoblasts growing like endothelial cells on the inner face of the persisting basal lamina tubes of necrotic fibres. After 30 days the muscles were vascularized. Fibres formed in a common basal lamina detached and so looked split. Satellite cells of new fibres came from undifferentiated cells associated with myotubes, i.e. from myoblasts. After 30 days and more regenerates contained three sorts of fibres. 1. Thin (5 to 20 m) fibres resembling fetal muscle fibres. They were most prominent after 30 days, and probably not yet innervated. 2. Thin (10 m) degenerating fibres as in long-time denervated muscles. 3. Thick (more than 30 m) mature looking fibres which were innervated and revealed end-plates.Half of the grafts studied after 30 and 60 days contained unmyelinated and myelinated axons which had grown along strands of surviving Schwann cells. After 250 days, only two muscles were studied which both lacked innervation. Almost all regenerates contained muscle spindles, which, however, were not innervated. Within the persisting spindle capsules new muscle fibres had been formed from satellite cells of the former intrafusal fibres.This study was supported by grants from the Danish Medical Research Council, and the National Danish Association against Rheumatic Diseases. I wish to thank Miss U. Hellhammer for valuable technical help and Dr. T. Tobias for correcting my English     

Pneumocystis carinii: Freeze-fracture study of stages of the organism

The morphology and life cycle of Pneumocystis carinii were studied in cortico-steroid-treated rats by ultrathin section and freeze-fracture electron microscopy. The following stages of P. carinii were noted: trophic, precyst, and cyst. The crescent-shaped cysts appeared to be intermediate forms between precyst and cyst. The cell wall of the trophic stage showed membrane structures suggestive of protozoan endocytosis, whereas the surface of the precyst stage was smooth. The cell wall of the cyst lacked the specialized structural differentiation of yeasts and resembled that of Plasmodium spp. We conclude that P. carinii belongs to the Protozoa, and is presumably Rhizopoda.     

Ultrastructural changes in rat epididymis after vasectomy

Summary Epididymal biopsies from rats that had undergone unilateral or bilateral vasectomies from one to eight months previously were compared with biopsies from their contralateral side or from normal controls to ascertain what ultrastructural changes had occurred. After vasectomy, spermatozoa appeared to dissolve in the lumen of the caput epididymidis and to be absorbed by the principal cells. About 5 weeks after vasectomy, numerous lamellar accumulations became apparent in the supernuclear region. Their resemblance to lysosomes or residual bodies was confirmed by an acid phosphatase reaction. After 10 weeks, similar lamellar and polymorphic accumulations on the contralateral side of animals with unilateral vasectomies indicated that resorption had also increased on the unligated side.Publication No. 627 of the Oregon Regional Primate Research Center. This study was supported by NIH Grants No. RR-00163 and HD-05969.The author wishes to thank Ms. J. Hren for her excellent technical assistance.     

Electron microscopic immunocytochemical demonstration of separate neurophysin-vasopressinergic and neurophysin-oxytocinergic nerve fibres in the neural lobe of the rat hypophysis

Summary By means of the unlabeled antibody peroxidase-antiperoxidase (PAP) technique at the electron microscopic level, it was demonstrated that the hormones of the neural lobe of the rat hypophysis are located in separate neurophysin-vasopressinergic and neurophysin-oxytocinergic nerve fibres. These observations confirm the results of our previous immunocytochemical studies at the light microscopic level.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Extracellular perivascular connective tissue space in the medial terminal nucleus of the accessory optic system in rats

Summary Two types of capillaries were found in the medial terminal nucleus of the rat accessory optic system. Type one capillaries are surrounded by glial processes and lack a perivascular space, whereas the type two capillaries and arterioles show a distinct extracellular perivascular space often filled with collagen fibrils. An internal as well as an external basal lamina lines these spaces. No fenestration of the endothelium was observed.This study was supported by N. I. H. Grant RR-00165 to Yerkes Regional Primate Research Center, and N. I. H. Grants R01-EY 00565-03 and R01-EY 00638-02 to J. Tigges. The excellent technical assistance of Mrs. G. L. Luttrell is gratefully acknowledged. We thank Dr. W. K. O'Steen for providing the rats and Mr. F. H. Kiernan for photographic help. Special thanks are due to Mrs. B. A. Olberding, in charge of the maintenance of the electron microscope.     

Fine structure of the corpus luteum of the autografted ovary in the rat

Summary The rat ovary has been transplanted successfully to subcutaneous tissue areas by several investigators. Light microscopy has revealed that corpora lutea in ovarian autografts are formed by luteinization of intact follicles and contain entrapped ova. In the present study, corpora lutea from autografted ovaries in castrate rats were obtained at metestrus and examined electron microscopically to determine whether their cellular morphology correlated with the normal progesterone levels in these animals. Cellular features usually accepted as regressive were apparent. The findings suggest either structural luteolysis is occurring before functional luteolysis or that the adrenal has increased steroidogenic activity in the castrate with ovarian autografts to account for the normal progesterone levels.Supported in part by USPHS Grant T01-DE00241-04The authors wish to thank J. Canale and Y. Tablada for technical, G. Kerimian for photographic, and M.A. Anderson for secretarial assistance     

Histological and ultrastructural alterations of rat thyroid gland after short-term treatment with high doses of thyroid hormones

The aim of the present study was to investigate histological alterations of rat thyroid gland after short-term treatment with supraphysiological doses of thyroid hormones. Rats from experimental groups were treated with triiodothyronine (T3) or thyroxine (T4) during five days. In both treated groups, thyrocyte height was reduced and follicular lumens were distended. Progressive involutive changes of the thyroid parenchyma were apparent, including follicular remodeling (fusion) and death of thyrocytes. Morphological changes confirmed by quantitative analysis were more pronounced in the T4-treated group. Our results demonstrate that thyrotoxicosis, whether induced by T3 or T4, leads to different grades of thyroid tissue injury, including some irreversible damages. These changes might be explained at least in part by lack of trophic and cytoprotective effects of the thyroid stimulating hormone. Since the period required for morphophysiological recovery may be unpredictable, findings presented here should be taken into consideration in cases where the thyroid hormones are used as a treatment for thyroid and non-thyroid related conditions.     

Über Hirnhautkörperchen des Kaninchens

Zusammenfassung Am Dach des III. Ventrikels in der Nähe des Splenium corporis callosi kommen umschriebene runde Körperchen vor. Diese Hirnhautkörperchen enthalten eine zentrale Gefäßzone, die von zwei Zellmänteln umgeben wird. In der Gefäßzone treten markscheidenfreie präsynaptische Nervenfasern unmittelbar an die Arterie und die Kapillaren heran. Die hellen Zellen des inneren Mantels sind durch hohen Glykogengehalt und durch zahlreiche Vakuolen charakterisiert. Die Interzellularspalten sind stellenweise verbreitert und angefüllt mit sauren Mukopolysacchariden. Der äußere Mantel, der unmittelbar das Hirnhautkörperchen umgibt, besteht aus dicht gepackten dunklen Zellen. Diese enthalten zahlreiche Mitochondrien und Filamente, die in eine dichte amorphe Matrix eingebettet sind und für kontraktil gehalten werden. Beide, der innere und der äußere Mantel, werden von Bündelchen kollagener Fasern durchzogen. Die enge Nachbarschaft zu Blutgefäßen und Plexus chorioideus läßt vermuten, daß die Hirnhautkörperchen mit der Kontrolle der Blutströmung zu tun haben.

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On leptomeningeal bodies in the rabbit

Summary On the roof of the 3rd ventricle of brain near the splenium corporis callosi circumscribed round bodies occur. These leptomeningeal bodies contain a central vascular zone enveloped by two sheaths of cells. In the vascular zone, some unmyelinated presynaptic nerve fibers can be seen very close to the artery and to the capillaries. The clear cells of the internal sheath are characterized by a high content of glycogen and by numerous vacuoles. The intercellular spaces are sporadically enlarged and filled with acidic nucopolysaccharides. The external sheath immediately surrounding the leptomeningeal body consists of densely packed dark cells. These cells contain numerous mitochondria and filaments embedded in a dense amorphous matrix. The filaments are interpreted as a contractile material. Both, the internal and external sheath, are trasversed by bundles of collagenous fibers. The close association with blood vessels and choroid plexus suggests that the leptomeningeal bodies are involved in blood flow control.

Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft (Le 69/7).     

Hepatocyte differentiation during early fetal development in the rat

Summary Rat hepatocyte differentiation between day 12 and 19 of fetal life was studied by electron microscopy. The cytoplasmic structures involved in synthetic and secretory function, i.e., rough endoplasmic reticulum and Golgi apparatus, appear to be the first to differentiate, and their development is probably related to the secretion of different kinds of plasma proteins. The cytoplasmic organelles involved in other hepatic functions develop later: lysosomes from day 15, peroxysomes, glycogen rosettes and smooth endoplasmic reticulum still later. However, the morphological differentiation of bile canaliculi begins from day 12.This work was supported in part by a grant from C.N.R. n. 78.02265.04