Frank Wohlrab

Morphometric parameters of Müller (glial) cells dependent on their topographic localization in the nonmyelinated part of the rabbit retina. A consideration of functional aspects of radial glia

SummaryMorphometric parameters of Müller cells were evaluated by light microscopy both in whole retinae and in enzymatically isolated cells from adult pigmented rabbits. In spite of the marked decrease in cell densities from visual streak to far periphery, a constant glia-neuron ratio of about 1∶15 was found in all regions. The volume of individual Müller cells was found to increase strongly when the cells become shorter, i.e. when the retinal centre was compared to the retinal periphery. The contribution of Müller cell volume to the total retinal volume, however, was shown to be constant at about 6%. Long Müller cells have a thin vitreal process and a small vitreal endfoot surface. The consequences of this rule for the proposed function of Müller cells in retinal K+ clearance are discussed with respect to general features of radial glia. It is suggested that foetal radial glial cells too long to perform sufficient K+ clearance are destined to be transformed into ‘adult’ multipolar glia by mitotic cell division.

Intraportal Transplantation of Pancreatic Islets Into Livers of Diabetic Rats: Reinnervation of Islets and Regulation of Insulin Secretion by the Hepatic Sympathetic Nerves

Two weeks after intraportal transplantation of 2,000 neonatal pancreatic islets, recipient rats completely recovered from streptozotocin-induced diabetes. The reversal of diabetes could be documented by the normalization of blood glucose levels, by a restored weight gain, by normal glucagon and insulin levels in blood, and by a disappearance of polyuria and polydipsia. The reversal remained stable for at least 9 months. This study determined whether intraportally transplanted pancreatic islets were reinnervated after transplantation and whether the secretion of insulin and glucagon from pancreatic islets might be modulated by the vegetative innervation of recipient livers. Predominantly catecholaminergic but also cholinergic nerve fibers were detected not only within the portal tracts around hepatic arteries, portal veins, and bile ducts, but also at the borderline of hepatocytes and β-cells and in islet cell complexes between β-cells. Corresponding electron micrographs showed β-cells in close contact with axons of nonmyelinated nerve fibers. Isolated livers were single pass perfused via both the hepatic artery and the portal vein. An increase in glucose level from 5 to 14 mmoW enhanced hepatic glucose uptake and increased insulin secretion from transplanted islets with a biphasic secretion profile but had no effect on glucagon output. Stimulation of the nerve plexus around the hepatic artery and the portal vein (7.5 Hz, 2 min), which activates primarily the sympathetic system, not only reduced glucose uptake and perfusion flow but also completely reversed the glucose-stimulated increase in insulin secretion. Nerve stimulation did not influence glucagon secretion. The α1-aradrenergic antagonist prazosin and the β-blocker propranolol completely blocked the nerve stimulation-dependent alterations in glucose balance and in perfusion flow but did not affect the reversal of the glucose-stimulated increase in insulin release caused by nerve stimulation. This indicates indirectly that the inhibition by sympathetic liver nerves of insulin secretion was mediated via α2-receptors as in normal pancreatic islets. Experiments with the α2-adrenergic antagonist yohimbine were in line with this conclusion.

Über die histochemische Erfassbarkeit der Aminosäure-Dehydrogenasen in Säugetierorganen

SummaryA study was conducted in order to contribute to the methodology and topochemical localization of amino acid dehydrogenases in mammals; Nitro-BT, Tetra-Nitro-BT and phenazine methosulphate were used in the tests. Under anaerobic incubation conditions varying amounts of some α-amino acids (dextrorotators) are oxidized in the mammalian kidney. The positive test result is regarded as the manifestation of a d-amino acid dehydrogenase activity. The investigation reveals that the kidney of carnivores (cat, dog, mink) shows the highest d-AAD-activity. However, this activity is histochemically not detectable in the kidney of genuine herbivores (guinea pig, rabbit, cattle). Rat, mouse, pig, and sheep take an intermediate position. In most of the species investigated the oxidative deamination of the d-amino acids takes place in the pars recta (renal cortex). In humans and sheep, however, the entire renal cortex gives positive results. The reaction is negative in the liver of most of the species tested.Out of the l-amino acids, tested in the course of the investigation, it is only l-proline that gives a positive result on kidney sections. In contrast to the findings concerning d-AAD activity, the formazan deposits are seen in the pars convoluta. In all probability this activity can bei regarded as a specific proline-dehydrogenase.ZusammenfassungEs wurde der Versuch unternommen, zur Methodik sowie zur topochemischen Lokalisation der Aminosäure-Dehydrogenasen beim Säugetier unter Verwendung von Nitro-BT bzw. Tetra-Nitro-BT und Phenazinmethosulfat beizutragen. Unter anaeroben Inkubationsbedingungen werden dabei einige α-Aminosäuren der d-Reihe in unterschiedlicher Rate in der Säugerniere oxydiert und der positive Reaktionsausfall als Ausdruck einer d-Aminosäure-Dehydrogenaseaktivität angesehen. Wie die Untersuchungen ergaben, zeigt die Niere von Carnivoren (Katze, Hund, Nerz) die stärkste d-ASD-Aktivität, während die Aktivität in der Niere reiner Herbivoren (Meerschweinchen, Kaninchen, Rind) histochemisch nicht faßbar ist. Ratte, Maus, Schwein und Schaf nehmen eine Zwischenstellung ein. In der Nierenrinde der meisten untersuchten Tierarten findet die oxydative Desaminierung der d-Aminosäuren vorwiegend in der Pars recta der Hauptstücke statt; bei Schaf und Mensch zeigt die gesamte Nierenrinde einen positiven Reaktionsausfall. An der Leber der meisten untersuchten Arten war die Reaktion negativ.Von den getesteten l-Aminosäuren konnte nur mit l-Prolin ein positiver Befund an Nierenschnitten erhoben werden. Im Gegensatz zur d-ASD finden sich die Formazanablagerungen bei den meisten untersuchten Spezies in der Pars convoluta der Hauptstücke. Auf Grund der Untersuchungen wird angenommen, daß es sich hierbei um eine spezifische Prolin-Dehydrogenase handelt.

Der histochemische Nachweis der Monoaminoxydase mit thiazolyl-substituierten Tetrazoliumsalzen in Gegenwart von Metallionen

ZusammenfassungEs wird eine Methode zum histochemischen Nachweis des Monoaminoxydase-systems beschrieben. Unter Verwendung thiazolyl-substituierter Tetrazoliumsalze erfolgt in Gegenwart von Metallionen und Tryptaminhydrochlorid als Substrat die Bildung eines feingranulären Metallformazankomplexes. Die Größe und Beschaffenheit des Komplexes spricht für eine intramitochondriale Lokalisation der MAO. Die Lokalisation der MAO in einigen Organen und Geweben wird beschrieben.SummaryA method is described for histochemical demonstration of monoamine oxidase system. Using tryptamine hydrochloride as substrate with thiazolyl-substituted tetrazolium salts in presence of metal ions there is formed a fine granular metalformazan complex. The size and quality of the complex suggest an intramito-chondrial localization of MAO. The distribution of MAO in some organs and tissues is described.

Nichtenzymatische TNBT-Färbung von Gewebsstrukturen

SummaryIncubating native or fixed cryostat sections in a TNBT-containing medium and reducing TNBT to formazan, produces a non-enzymatic staining of tissue structures. This phenomenon is investigated light and electron microscopically; the emphasis is put on the intracellular localization of TNBT-formazan. In the cell electron-dense TNBT-formazan is found in the immediate vicinity of the external membranes of mitochondria, membranes of the endoplasmic reticulum, and in regions, which are free from organelles. The reaction becomes weaker if it is preceded by an extraction of lipids. It is therefore concluded that TNBT is absorbed non-specifically onto the lipoproteins of the cell and that the localization of TNBT-formazan is influenced by diffusion phenomenons. It seems possible that TNBT is reduced by free diffusible reducing tissue components. If TNBT is used as an indicator for reduction in light or submicroscopical investigations of oxidative enzymes a non-specific TNBT-formazan localization in tissue structures might be encountered. However, more work along these lines is needed to finally confirm this theory.ZusammenfassungEine Inkubation nativer oder fixierter Kryostatschnitte in einem TNBT-haltigen Inkubationsmedium und eine anschließende Reduktion des TNBT zum Formazan führt zu einer nichtenzymatischen Anfärbung der Gewebsstrukturen. Diese Beobachtung wird licht- und elektronenmikroskopisch im Hinblick auf eine intrazelluläre Lokalisation des TNBT-Formazans näher analysiert. Elektronendichtes TNBT-Formazan konnte in der Zelle in unmittelbarer Nachbarschaft von Mitochondrienaußenmembranen, Membranen des endoplasmatischen Retikulums und in organellenfreien Bezirken nachgewiesen werden. Eine vorherige Lipidextraktion bewirkt eine Abschwächung der Reaktion. Es wird daraus der Schluß gezogen, daß TNBT unspezifisch an Lipoproteinstrukturen der Zelle adsorbiert wird und daß die beobachtete Lage des TNBT-Formazans durch Diffusionsphänomene beeinflußt wird. Möglicherweise kann das TNBT durch frei diffusible reduzierende Gewebskomponenten reduziert werden. Bei Verwendung von TNBT als Reduktionsindikator zum licht- und submikroskopischen Nachweis oxydativer Enzyme ist mit einer unspezifischen TNBT-Formazan-Lokalisation in Gewebsstrukturen zu rechnen. Die endgültige Bestätigung dieser Auffassung erfordert weitere Modellversuche.

Effects of α -aminoadipic acid on the glutamate-isolated P III of the rabbit electroretinogram

Abstractα-aminoadipic acid was intravitreally applied to adult rabbits. After 5 h, the retinae of these animals were examined by electroretinography and histochemistry. The retinal Müller cells were extremely swollen, and the electroretinographic slow P III was extinguished. The mass receptor potential was somewhat diminished. The results are consistent with the opinion that the slow P III is the reaction of the Müller cells to the changed external potassium ion concentration caused by the activity of the photoreceptors.

Die Leber der Fledermaus in Hibernation Licht- und elektronenmikroskopische Untersuchungen

ZusammenfassungDie Leber von Fledermäusen in Hibernation wurde licht- und elektronenmikroskopisch untersucht. Im Elektronenmikroskop zeigt die Fledermausleber die typische Ultrastruktur der Säugetierleber. Sie besitzt jedoch Besonderheiten, die mit dem Stoffwechsel im Winterschlaf im Zusammenhang stehen. Die Leberzelle ist besonders reich an Glykogen, Eisen sowie Ribonukleoproteidgranula und besitzt auffallend viele große Mitochondrion mit zahlreichen Cristae und osmiophilen Körpern. Es wird versucht, histochemische und biochemische Befunde anderer Autoren mit den Ergebnissen unserer Untersuchungen zu korrelieren. Unsere Untersuchungsergebnisse sprechen nicht für das Vorliegen einer reinen Speicherleber, sondern für eine stärkere Stoffwechselaktivität der Leber im Winterschlaf.

Glucose transporter isoform (GLUT) 2 expression in beta-cells of long-term syngeneic islet grafts

Abstract Syngeneic islets were transplanted into the liver of streptozotocin (STZ)-induced diabetic LEW.1W rats, and the expression of the glucose transporter isoform GLUT 2, an essential component of the glucose-sensing mechanism of the pancreatic beta-cell, was determined in the grafted islet tissue. Graft-bearing liver was obtained 12, 36, and 60 weeks after transplantation, and tissue sections were immunoperoxidase stained for GLUT 2 and major islet peptides. Islet cell aggregates of different sizes were found in the portal tract and in juxtaposition to the hepatocytes. At all time points, beta-cells in the grafts displayed GLUT 2 expression comparable to that of islets in nondiabetic rats. Islet cells containing immunoreactive insulin and islet amyloid polypetide were plentiful, while those staining positive for glucagon and somatostatin were scarce in these grafts. The results show that beta-cells in islets engrafted in the liver, although initially exposed to chronic hyperglycemia, have the capability of stably expressing GLUT 2 over long-term periods.

Syngeneic transplantation of rat pancreatic islets into the spleen. Light and electron microscopical findings

SummaryAn islet transplantation model was established with the two congeneic Lewis rat strains LEW.1W and LEW.1A, which were made diabetic by a single i.p. injection of 50 mg/kg b.w. streptozotocin. Isolated neonatal islets of the two strains served as a graft. Syngeneic transplantation into the spleen resulted in permanent graft survival and in normoglycemia of streptozotocin diabetic rats of the two strains. 200 days after transplantation functionally intact islets were demonstrable in the spleen by histological, histochemical and electron microscopical investigations. Histochemical findings indicate an activation of interdigitating reticular cells in the white pulp and of macrophages in the red pulp of the spleen as consequences of the islet transplantation. In addition a few electron microscopical findings concerning possible interactions between host plasma cells, lymphocytes and transplanted beta cells of islets are described and discussed.

Histochemische Untersuchungen zum Nachweis der Cholindehydrogenase beim Säugetier

ZusammenfassungEs werden die Tetrazoliumverbindungen MTT und Nitro-BT zum histochemischen Nachweis der Cholindehydrogenase sowohl unter aeroben als auch unter anaeroben Inkubationsbedingungen vergleichend getestet. Der histochemische Nachweis der Cholindehydrogenase gelingt unter anaeroben Bedingungen unter Einschaltung von Phenazinmethosulfat als intermediärem Elektronenüberträger mit beiden Tetrazoliumverbindungen bereits nach kurzen Inkubationszeiten. Die Cholindehydrogenase wird gehemmt durch p-Chlormercuribenzoat sowie geringer durch Atebrin. Mehrmaliges Frieren und Tauen des Gewebes führt zu einer Aktivitätsminderung des Fermentes, die nach Zusatz von ATP zum Inkubationsmedium wieder ausgeglichen wird.In Übereinstimmung mit den Ergebnissen biochemischer Untersuchungen findet sich Cholindehydrogenase nur in Niere und Leber der untersuchten Ratten.