Effects of fixation and decalcification on the immunohistochemical localization of bone matrix proteins in fresh-frozen bone sections

To examine the stability of bone matrix proteins for crystal dislocation, the immunolocalization of type I collagen, bone sialoprotein, and osteopontin was investigated during different stages of fixation and decalcification. Four-week-old rat femurs were rapidly frozen, and were sectioned without fixation or decalcification. Thereafter, following or bypassing fixation in 4% paraformaldehyde, these sections were decalcified in 5% EDTA for 0-5 min. Before decalcification, marked radiopacity of bone matrix was observed in contact microradiography (CMR) images, and electron probe microanalysis (EPMA) demonstrated intense localization for phosphorus and calcium. In fixed and unfixed sections without decalcification, immunolocalization of bone matrix proteins were almost restricted to osteoid. After 1 min of decalcification, reduced radiopacity was apparent in the CMR images, and less phosphorus and calcium was observed by EPMA, which completely disappeared by 5 min decalcification. After 3-5 min of decalcification, unfixed sections showed that these proteins were immunolocalized in bone matrix, but were not detectable in osteoid. However, fixed sections demonstrated that these were found in both bone matrix and osteoid. The present findings suggest that bone matrix proteins are embedded in calcified matrix which is separated from the aqueous environment and that they hardly move, probably due to firm bonding with each other. In contrast, matrix proteins in osteoid are subject to loss after decalcification because they may be bound to scattered apatite crystals, not to each other.     

Effects of various decalcification protocols on detection of DNA strand breaks by terminal dUTP nick end labelling

To analyse DNA strand breaks by terminal deoxy(d)-UTP nick-end labelling (TUNEL) in calcified tissues including bones and teeth, it is important to decalcify the tissues first. However, the effects of decalcifying reagents on the integrity of DNA are largely unknown. In the present study, we evaluated the usefulness of various decalcifying reagents including 10% EDTA (pH 7.4), 5% trichloroacetic acid (TCA), 5% formic acid, 5% HCl, 10% nitric acid, Plank–Rychlo's solution, Morse's solution and K-CX solution in TUNEL staining. Mouse maxilla was selected as the experimental system. Apoptotic cells naturally occurring in the epithelium were analysed. Tissues were assessed by soft X-ray imaging to confirm complete decalcification. The time required for decalcification of the tissue was 7 days with 10% EDTA and 2 days with other decalcifiers. Decalcified tissues were stained with Methyl/Green–Pyronine Y or 4, 6-diamidino-2-phenylindole for assessment of DNA integrity. Nuclei of epithelial cells were strongly positive for both dyes after decalcification with 10% EDTA, 5% TCA, Morse's solution and 5% formic acid. The other reagents failed to retain DNA. Our results demonstrated good TUNEL staining of the maxilla treated with 10% EDTA or 5% TCA . Based on the required time for processing and the signal-noise ratio, we recommend 5% TCA as the decalcifying reagent to analyse for DNA strand breaks.     

The membraneous nitrite reductase involved in the electron transport of Wolinella succinogenes

Wolinella succinogenes grown with nitrate as terminal electron acceptor contains two nitrite reductases as measured with the donor viologen radical, one in the cytoplasm and the other integrated in the cytoplasmic membrane. The fumarate-grown bacteria contain only the membraneous species.The isolated membraneous enzyme consists of a single polypeptide chain (M _r 63,000) carrying 4 hemeC groups and probably an iron-sulphur cluster as prosthetic groups. The enzyme amounts to about 1% of the total membrane protein.The isolated enzyme catalyses the reduction of nitrite to ammonium without accumulation of significant amounts of intermediates or alternative products. The Michaelis constant for nitrite was 0.1 mM and the turnover number of the hemeC 1.5 · 10⁵ electrons per min at 37°C.The viologen-reactive site of the enzyme in the membrane is oriented towards the cytoplasm. When the isolated enzyme is incorporated into liposomes, the viologen-as well as the nitrite-reactive site is exposed to thooutside.The cytoplasmic membrane contains a second hemeC protein (M _r 22,000) which may represent a cytochrome c.Abbreviations NQNO 2-(n-nonyl)-4-hydroxyquinoline-N-oxide - MES 2-(N-morpholino)ethanesulfonate - MOPS 3-(N-morpholino)propanesulfonate - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonate - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - MK menaquinone     

Submicroscopic structure of the membranous labyrinth

Summary Investigations were performed by light and electron microscope on the submicroscopic structure of the epithelium of Corti's organ in the white rat.Morphological and structural differences between the inner hair cells and the outer hair cells are revealed.The inner hair cells are closely inter-related with the inner supporting cells and have a polyhedral shape, whereas the outer hair cells look like cylinders and are surrounded by an intraepithelial fluid.The structural peculiarities consist of differences in the dimensions of the hairs, in the arrangement of cytoplasmic organoids and in the aspect of the receptoneural junction. In both sensory hair cells 4 zones of different structure can be distinguished from the surface inwards: apical zone, intermediate zone, perinuclear zone and receptoneuronal junction. The functional value of these different zones is discussed and compared with what has been demonstrated in other receptors.The pillar cells and the Deiters' cells are supporting cells which have a filamentous skeleton, composed of submicroscopic individual filaments. These filaments have a diameter of about 215 Å and present some analogies with the tonofilaments of the stratified squamous epithelium. The filaments are arranged differently in the pillar cells and in the Deiters' cells. Possible functional differences between these patterns are discussed.The reticular membrane is not an extracellular cuticle. It consists of intracellular cementitious material (like the terminal bars of the epithelial cells).The Hensen's and Claudius cells, the Böttcher's cells, the inner supporting cells, the inner and outer spiral sulcus cells are regular prismatic cells with few endoplasmic organoids and without filaments.This work is dedicated to the memory of the late Prof. L. Pietrantoni.     

Submicroscopic structure of the membranous labyrinth

Zusammenfassung Der Verfasser hat eine Methode entwickelt, die es gestattet, die einzelnen Teile der Schnecke (Membrana tectoria, Basilarmembran, Ligamentum spirale, Limbus spiralis, Reissnersche Membran, Cortisches Organ) in ausreichendem Reinheitsgrad und in solchen Mengen zu isolieren, daß mikroskopische Untersuchungen mit polarisiertem Licht sowie mit dem Elektronenmikroskop, diffraktographische sowie chemische Analysen durchgeführt werden können.Chemische und diffraktographische Untersuchungen haben ergeben, daß die Membrana tectoria hauptsächlich aus Proteinen bestellt. Das Vorhandensein von Kollagenprotein ist auszuschließen. Das Protein dürfte zur Gruppe der weichen Kératine mit geringem Cystingehalt gehören. Auf Grund der ausgezeichneten Übereinstimmung der Befunde am Phasenkontrastmikroskop, mit polarisiertem Licht (bei Vorhandensein Eigen- und Form-Doppelbrechung) und am Elektronenmikroskop ergibt sich, daß das in Frage stehende Protein aus Protofäden von etwa 90 Å Durchmesser besteht. Die Protofäden verlaufen leicht wellenförmig radiär, doch wurden (entlang dem exzentrischen Membranrande) auch Bereiche mit longitudinalem Faserverlauf beobachtet. Im ganzen sind sie mit einer gewissen Gleichartigkeit angeordnet, obwohl Bereiche mit dichterer — Longitudinalfasern — oder lockerer Anordnung — dem Limbus spiralis eingefügter Teil — vorhanden sind. Die Membrana tectoria ist somit epithelialer Herkunft mit augenscheinlich fadenförmig ausgerichteter Struktur.Der Verfasser nimmt an, daß die Ausrichtung der Fasern mit dem Spannungszustand der Membran in Zusammenhang steht, die sich zwischen Limbus spiralis und Hensenschen Zellen bildet. Diese entfernen sich ihrerseits während der Bildung des Cortischen Organs voneinander.

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Research financed by C.N.R. grant.

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Acknowledgements. The author expresses his thanks to Dr. S. De Petris of the INAIL Laboratory of Electron Microscopy of the Clinic for Occupational Diseases of the University of Milan for the help and technical assistance given in obtaining X-ray diagrams and electron photograms with the Siemens Elmiskop I. Grateful acknowledgements are also made to Dr. L. Amante for the —SH and —S—S— groups determinations.     

Submicroscopic structure of the membranous labyrinth

Summary Investigations were performed by light and electron microscope on the basilar membrane, limbus spiralis and spiral ligament.These different parts continue one into the other and make up a single morphological and functional structure which may be called the supporting structure of Corti's organ (s.s.C.o.).It is formed by a tissue the components of which are the cells and an intercellular substance in which are arranged the capillary vessels.The cells can be classed in two groups, the first consisting of the cells proper (basilar membrane, limbus spiralis and spiral ligament cells) which present structural changes parallel with the growth mechanism of the intercellular substance; the second of the cochlear duct covering cells (Corti's organ cells, inner and outer spiral sulcus cells, interdental cells, stria vascularis cells).The intercellular substance is organised in laminae, fibrolaminae, bundles and microscopic fibers composed of filaments with an intervening ground substance.The filaments have a diameter ranging from 85 to 105 Å. Topochemical tests with polarised light microscope, enzymatic tests, diffractographic and chemical analyses suggest that these filaments unquestionably consist of protein material which have nothing to do with collagen or elastic fibers. Perhaps it may be classed in the K.E.M.F. group.The ground substance generally appear anhistous and transparent but in some parts of the basilar membrane it presents a cottony appearance.The possible different hypotheses about the classification of the s.s.C.o. tissue are discussed.The quantity and architecture of the cells and the intercellular substance vary appreciably in the basilar membrane, limbus spiralis and spiral ligament, which are examined in detail one by one.The demonstration that the s.s.C.o. is formed of a tissue possessing an intercellular substance containing filamentous scleroproteins clearly corroborates the theory that is performs supporting activity in respect of Corti's organ. The term supporting structure of Corti's organ is based on this interpretation.Research financed by C.N.R. grant.     

Effects of passive flexion of the forepaw on the response gain of limb extensors to sinusoidal stimulation of labyrinth receptors

The main aim of the present study was to find out whether the dynamic characteristics of responses of limb extensor muscles to labyrinth stimulation were modified by the proprioceptive input elicited by appropriate displacements of the corresponding limb extremity. In cats decerebrated at precollicular or intercollicular level, the multiunit EMG activity of the medial head of the triceps brachii was recorded during roll tilt of the animal at the frequency of 0.15 Hz, +/- 10 degrees leading to selective stimulation of labyrinth receptors. This stimulation was then tested several times at regular intervals of 2 to 6 min for several hours while maintaining the ipsilateral forelimb in the horizontal extended position, i.e. with the plantar surface of the foot lying on the tilting table, or during passive flexion of the forepaw in plantar or dorsal direction. In all the experiments in which the forelimb was in the control position, the multiunit EMG responses of the triceps brachii were characterized by an increased activity during side-down tilt of the animal and a decreased activity during side up tilt. These responses were related to animal position and not to the velocity of animal displacement, thus being attributed to stimulation of macular, utricular receptors. Static displacement of limb extremities following plantar flexion of the forepaw greatly decreased the amplitude of the EMG modulation and thus the gain of the first harmonic component of the multiunit EMG responses of the ipsilateral triceps brachii to animal tilt. This reduced gain was due not only to a reduced number of motor units recruited during labyrinth stimulation, but also to a reduced modulation of firing rate of the active motor units, as shown by recording the activity of individual motor units. On the other hand, displacement of the same extremity in the opposite direction, i.e. following dorsiflexion of the forepaw, enhanced the amplitude of the EMG modulation and thus the gain of the multiunit EMG responses of the ipsilateral triceps brachii to animal tilt. This finding was mainly due to an increased recruitment of motor units during side-down tilt, although an increased modulation of the firing rate of individual motor units could not be excluded. In both instances, no changes in the phase angle to the responses were observed. The changes in response gain described above depended on the amount of passive displacement of the forepaw and persisted unmodified throughout the new maintained position.(ABSTRACT TRUNCATED AT 400 WORDS)     

Infra-red spectroscopic study for decalcification in the dentine

Summary A study was made of spectra of decalcified human dentine being treated with five decalcifying agents to investigate the differences of infrared spectra of decalcified dentine.Solutions of 20% 4-Na-EDTA, 5% HNO₃, 10% sulfosalicylic acid, 10% CCl₃COOH and 10% lithium lactate were used as decalcifying agents. Infrared absorption spectra between 1800 and 650 cm^–1 were obtained for the investigation of the absorption strength at 1033,1095; 1412–1414 to 1450–1455; 875–870; 1649 and 1550 cm^–1 before and after decalcification.The specimens following decalcification with EDTA and HNO₃ showed almost no influences to the absorption strength at 1649 and 1550 cm^–1 of amides. Such absorption spectra were relatively slight in CCl₃COOH and sulfosalicylic acid. The absorption strength at 1033 and 1095 cm^–1 bands of PO ₄ ^––– may indicate a standard of decalcification. Quick and complete decalcification of dentine was performed by HNO₃, CCl₃COOH and sulfosalicylic acid but slower and more incomplete decalcification resulted with EDTA and lithium lactate.