Wilfried Naumann

The secretion of the subcommissural organ

SummaryThe secretion of the subcommissural organ (SCO) has been studied immunocytochemically by use of the following antisera: (1) antiserum against an aqueous extract of bovine Reissners fiber (RF), (anti-RF-DC antiserum); (2) antiserum against the protein fraction F1 obtained by gel electrophoresis of the aqueous RF-extract (RF-DF1-antiserum); (3) antiserum against the protein fraction F2 prepared in the same manner (RF-DF2-antiserum). As shown by immunological and/or immunocytochemical experiments in bovines and rats, the three antisera are of high specificity, i.e., react exclusively with the secretion of the SCO, which appears to be a unique product of the vertebrate organism. Concerning the distribution of the reaction products within the SCO-cells, no differences were found light microscopically after use of the RF-DC-antiserum, the RF-DF1-antiserum, or the RF-DF2-antiserum.Comparative studies were carried out with the RF-DC-antiserum only. A positive immunocytochemical reaction of the SCO-secretion was obtained in many vertebrate species (mammals, birds, reptiles, amphibians, bony fishes, sharks, and cyclostomes). RF gives a positive reaction in mammals only; to date RF of non-mammalian vertebrates did not react immunocytochemically with the present antiserum. Comparative immunocytological studies have shown that (1) the SCO-cells of the ependymal layer as well as the SCO-cells of the hypendymal layer contain immunoreactive material, (2) in the majority of vertebrates hypendymal structures are more common than has been previously supposed, and (3) RF or constituents of this structure are produced by the SCO. The immunocytochemical studies have led to the impression that the SCO-secretion is not only discharged into the cerebrospinal fluid, but also in hypendymal vessels and/or leptomeningeal spaces, as has been postulated previously by Oksche and others (for review, see Oksche 1969).

Immunocytochemical investigation of the subcommissural organ in the rat

SummaryThe results of a preliminary immunocytochemical investigation on the subcommissural organ (SCO) in rats show that (1) Reissners fiber (RF) or essential compounds of the RF are produced by the SCO, (2) the immunoreactive material is produced in the epithelial cells of the SCO as well as in the hypendymal cells, and (3) the immunoreactive material of the SCO belongs to a category of endogenous peptides to date not demonstrable immunocytochemically in other brain structures.

Histochemische Untersuchungen am Subcommissuralorgan und am Reissnerschen Faden von Lampetra planeri (Bloch)

Summary1.The Subcommissural organ, Reissners fibre and ventriculus terminalis of Lampetra planeri (Bloch) has been studied histochemically.2.The secretion product of the subcommissural organ represents a neutral polysaccharide protein-complex containing an increased concentration of cystin, tyrosin and tryptophan.3.Differences between subcommissural cistern-secretion, Reissners fibre and massa caudalis cannot be revealed by methods of histochemistry.4.It is supposed that in the subcommissural organ the apical secretion into the ventricle takes place with the aid of hydrolytic enzymes. There are no symptoms of a basal secretion.5.The constant occurrence of a series of enzymes refers to a continuous secretion process of the subcommissural organ.6.Monoaminooxydase activity on the site of the subcommissural ependymal processes may be considered to be an adrenergic transmission mechanism.7.According to the investigations of enzyme histochemistry energy production in the subcommissural organ of Lampetra planeri (Bloch) largely takes place in anaerobic metabolism.8.The degradation of fibre-secretion takes place on the site of the ventriculus terminalis, where an increased activity of hydrolytic and glycolytic enzymes is demonstrable.Zusammenfassung1.Das Subcommissuralorgan, der Reissnersche Faden und die Endampulle von Lampetra planeri (Bloch) wurden mit baustein- und enzymhistochemischen Methoden untersucht.2.Das Sekret des Subcomissuralorgans ist ein neutraler Polysaccharid-Protein-Komplex, der Cystin, Tyrosin und Tryptophan in erhöhter Konzentration enthält.3.Unterschiede zwischen dem subcommissuralen Zisternensekret, dem Reissnerschen Faden und der Massa caudalis sind mit histochemischen Methoden nicht nachweisbar.4.Im Subcommissuralorgan wird für die apikale Sekretabgabe in den Ventrikel eine Beteiligung hydrolytischer Enzyme angenommen. Hinweise für eine basale Sekretion wurden nicht gefunden.5.Das konstante Auftreten der nachgewiesenen Enzyme spricht für einen kontinuier-lichen Sekretionsprozeß des Subcommissuralorgans.6.Die Aktivität der Monoaminooxidase im Bereich der subcommissuralen Ependymfortsätze kann im Sinne eines adrenergen Überträgermechanismus gewertet werden.7.Die Energiegewinnung erfolgt nach fermentativen Untersuchungen im Subcommissuralorgan von Lampetra planeri (Bloch) vorwiegend im anaeroben Stoffwechsel.8.Der Abbau des Fadensekrets findet im Bereich der Endampulle statt, wo eine vermehrte Aktivität hydrolytischer und glykolytischer Enzyme nachweisbar ist.

Evidence for neuroprotective properties of human umbilical cord blood cells after neuronal hypoxia in vitro

BackgroundOne of the most promising options for treatment of stroke using adult stem cells are human umbilical cord blood (HUCB) cells that were already approved for therapeutic efficacy in vivo. However, complexity of animal models has thus far limited the understanding of beneficial cellular mechanisms. To address the influence of HUCB cells on neuronal tissue after stroke we established and employed a human in vitro model of neuronal hypoxia using fully differentiated vulnerable SH-SY5Y cells. These cells were incubated under an oxygen-reduced atmosphere (O2< 1%) for 48 hours. Subsequently, HUCB mononuclear cells (MNC) were added to post-hypoxic neuronal cultures. These cultures were characterized regarding to the development of apoptosis and necrosis over three days. Based on this we investigated the therapeutic influence of HUCB MNC on the progression of apoptotic cell death. The impact of HUCB cells and hypoxia on secretion of neuroprotective and inflammatory cytokines, chemokines and expression of adhesion molecules was proved.ResultsHypoxic cultivation of neurons initially induced a rate of 26% ± 13% of apoptosis. Hypoxia also caused an enhanced expression of Caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP). Necrosis was only detected in low amounts. Within the next three days rate of apoptosis in untreated hypoxic cultures cumulated to 85% ± 11% (p ≤ 0.001). Specific cytokine (VEGF) patterns also suggest anti-apoptotic strategies of neuronal cells. Remarkably, the administration of MNC showed a noticeable reduction of apoptosis rates to levels of normoxic control cultures (7% ± 3%; p ≤ 0.001). In parallel, clustering of administered MNC next to axons and somata of neuronal cells was observed. Furthermore, MNC caused a pronounced increase of chemokines (CCL5; CCL3 and CXCL10).ConclusionWe established an in vitro model of neuronal hypoxia that affords the possibility to investigate both, apoptotic neuronal cell death and neuroprotective therapies. Here we employed the therapeutic model to study neuroprotective properties of HUCB cells.We hypothesize that the neuroprotective effect of MNC was due to anti-apoptotic mechanisms related to direct cell-cell contacts with injured neuronal cells and distinct changes in neuroprotective, inflammatory cytokines as well as to the upregulation of chemokines within the co-cultures.

Elektronenmikroskopische Untersuchungen ber den Reissnerschen Faden und die Ependymzellen im Rckenmark von Lampetra planeri (Bloch)

1. Der Reissnersche Faden von Lampetra planeri (Bloch) last sich nicht nur lichtmikroskopisch, sondern auch elektronenmikroskopisch als kompakter Faden darstellen. Er besteht aus feingranuliertem Material, das keine Zellorganellen einschliest und an der Oberflache durch keine Membran begrenzt wird. In seiner ganzen Ausdehnung vom Subkommissuralorgan uber den gesamten Zentralkanal bis zur Endampulle bleibt die Feinstruktur unverandert. In der Endampulle selbst lockert sich der Faden auf, und das feingranulierte Sekretmaterial tritt durch Interzellularspalten in das umgebende Gewebe aus. 2. Die den Zentralkanal auskleidenden Ependymzellen bilden pseudopodienartige Vorstulpungen, die feingranuliertes Material und Blaschenstrukturen enthalten. 3. Die Vorstulpungen treten bis an den Reissnerschen Faden heran. Zwischen Zellmembran und Faden kommt es zu einer engen, sehr charakteristischen Verzahnung. Der vorubergehende Kontakt wird als morphologischer Ausdruck eines Stoffaustausches angesehen.Summary1.The electron microscope as well as the lightmicroscope reveals that the Reissner fibre of Lampetra planeri (Bloch) is of a compact structure. It consists of a finely granulated material which does not include cell organelles and is not enveloped by a membrane. In its entire length, from the subcommissural organ over the entire central canal to the terminal ventricle, its fine structure remains unchanged. In the terminal ventricle itself the fibre loosens up and the finely granulated secretory substance enters into the surrounding tissue through intercellular gaps.2.The ependyma cells lining the central canal form pseudopodial processes which contain finely granulated substance and vesicular structures.3.The processes of the cells abut upon the Reissner fibre. Cellular membrane and fibre are very closely interdigitated. The temporary contact is regarded as a morphological equivalent for the exchange of substances.Zusammenfassung1.Der Reissnersche Faden von Lampetra planeri (Bloch) läßt sich nicht nur lichtmikroskopisch, sondern auch elektronenmikroskopisch als kompakter Faden darstellen. Er besteht aus feingranuliertem Material, das keine Zellorganellen einschließt und an der Oberfläche durch keine Membran begrenzt wird. In seiner ganzen Ausdehnung vom Subkommissuralorgan über den gesamten Zentralkanal bis zur Endampulle bleibt die Feinstruktur unverändert. In der Endampulle selbst lockert sich der Faden auf, und das feingranulierte Sekretmaterial tritt durch Interzellularspalten in das umgebende Gewebe aus.2.Die den Zentralkanal auskleidenden Ependymzellen bilden pseudopodienartige Vorstülpungen, die feingranuliertes Material und Bläschenstrukturen enthalten.3.Die Vorstülpungen treten bis an den Reissnerschen Faden heran. Zwischen Zellmembran und Faden kommt es zu einer engen, sehr charakteristischen Verzahnung. Der vorübergehende Kontakt wird als morphologischer Ausdruck eines Stoffaustausches angesehen.

Immuno-electron-microscopic analysis of the basal route of secretion in the subcommissural organ of the rabbit

SummaryLow-temperature-embedded tissue of the subcommissural organ (SCO) of the rabbit was analyzed for the basal route of secretory product by means of indirect immuno-metal cytochemistry (protein A-gold technique) at the electron-microscopic level. By use of (1) an antiserum against bovine Reissners fibre (see Sterba et al. 1981) and, thereafter, (2) particulate gold-marker solution, immunoreactive sites could be clearly visualized within the extracellular matrix of both (a) the basal part of the ependymal cell layer, and (b) the hypendyma proper. Abundant secretory material was identified within (i) dilated intercellular spaces (a + b) as well as (ii) branching basal lamina labyrinths and distinct perivascular spaces (b). All these compartments are thought to belong to a system of extracellular channels, which may function in secretion directed toward hypendymal blood vessels.

Exohypothalamic Axons of the Classic Neurosecretory System and their Synapses

Publisher Summary The classic neurosecretory system of the vertebrates is considerably more differentiated than has hitherto been supposed. Unlike the fibers of the descending part, the ascending fibers (exohypothalamic fibers) are not connected to capillaries, but rather make true synapses with other neurons. The most interesting feature of these peptidergic synapses is the presence of large vesicles containing neurophysins and nonapeptides. The existence of an ascending part makes it clear that the classic neurosecretory system cannot be viewed simply as a system involving signal transfer by neurohormone release and transfer of the mediators to the targets via the blood stream. Instead, the view that the classic neurosecretory system also affects subsystems of the brain by direct neuronal intervention have to be accepted, and thus directly takes part in a variety of cerebral functions. The chapter discusses the possibility of the system being involved in such disparate processes as memory and the regulation of blood pressure.

The central nervous system of sea cucumbers (Echinodermata: Holothuroidea) shows positive immunostaining for a chordate glial secretion

BackgroundEchinoderms and chordates belong to the same monophyletic taxon, the Deuterostomia. In spite of significant differences in body plan organization, the two phyla may share more common traits than was thought previously. Of particular interest are the common features in the organization of the central nervous system. The present study employs two polyclonal antisera raised against bovine Reissners substance (RS), a secretory product produced by glial cells of the subcomissural organ, to study RS-like immunoreactivity in the central nervous system of sea cucumbers.ResultsIn the ectoneural division of the nervous system, both antisera recognize the content of secretory vacuoles in the apical cytoplasm of the radial glia-like cells of the neuroepithelium and in the flattened glial cells of the non-neural epineural roof epithelium. The secreted immunopositive material seems to form a thin layer covering the cell apices. There is no accumulation of the immunoreactive material on the apical surface of the hyponeural neuroepithelium or the hyponeural roof epithelium. Besides labelling the supporting cells and flattened glial cells of the epineural roof epithelium, both anti-RS antisera reveal a previously unknown putative glial cell type within the neural parenchyma of the holothurian nervous system.ConclusionOur results show that: a) the glial cells of the holothurian tubular nervous system produce a material similar to Reissners substance known to be synthesized by secretory glial cells in all chordates studied so far; b) the nervous system of sea cucumbers shows a previously unrealized complexity of glial organization. Our findings also provide significant clues for interpretation of the evolution of the nervous system in the Deuterostomia. It is suggested that echinoderms and chordates might have inherited the RS-producing radial glial cell type from the central nervous system of their common ancestor, i.e., the last common ancestor of all the Deuterostomia.

Preparation and discharge of secretion in the subcommissural organ of the rat

SummaryThe secretion of the subcommissural organ (SCO) of the rat was studied by means of immunocytochemistry at the electron-microscopic level with the use of (1) the polar embedding medium Lowicryl K4M at -30° C, (2) the protein A-gold technique, and (3) a rabbit antiserum against bovine Reissners fiber (see Sterba et al. 1981).Two different substructures of the ependymal and the hypendymal SCO-cells display a positive immunocytochemical reaction: (1) sacs containing flocculent secretion, which originate from the granular endoplasmic reticulum, and (2) vacuoles filled with fine granular secretion, which are pinched off from the Golgi apparatus. The secretory material of the sacs and the vacuoles is discharged both (i) apically into the cerebrospinal fluid and (ii) basally into intercellular spaces of the SCO-hypendyma. The apically released secretion is condensed to a lamina-like formation, which more caudally assumes the form of Reissners fiber. The route of the basally released secretion remains, however, vague. The “periodically striated bodies”, which were thought to be morphological mediators of the discharge of the secretion into the capillaries, are never labeled by gold particles.

Reissner’s substance expressed as a transient pattern in vertebrate floor plate

 The function of the floor plate in dorso-ventral patterning of the developing nervous system and in the guidance of commissural axons is well established. However, several morphological aspects concerning the exact localization of its rostral and caudal end and the regional and temporal specialization are still controversial. We present new insights revealed by the expression of Reissner’s substance in the floor plate during early neurogenesis of zebrafish, Xenopus, chick and rat. We used a polyclonal antiserum raised against Reissner’s substance, which is a secretory product of radial glia in the roof plate of the adult vertebrate brain. In early embryonic stages the rostral boundary of floor plate immunoreaction vary in the different vertebrates. Immunoreactive cells are not only present in the epichordal region (rat) but also in prechordal areas of the midbrain (chick) and forebrain (zebrafish and Xenopus). During further development, Reissner’s substance expression disappears first in the most rostral areas and later also in the spinal cord. However, immunopositive labelling in the isthmus region at the mes-metencephalic boundary, described originally as the flexural organ, is most extensive and detectable during a long period of embryonic development. It is proposed that the gradual restriction of Reissner’s substance expression to the isthmus reflects the complex differentiation processes in this region also in later embryonic development. Furthermore, the expression pattern in zebrafish indicates that Reissner’s substance could play a role in axonal decussation.