Werner Giebel

Distribution of marked perilymph to the subarachnoidal space

ZusammenfassungBei Meerschweinchen wurde die tympanale Perilymphe mit dem fluoreszierenden Farbstoff Rhodamin markiert, bei der ersten Versuchsreihe durch Applikation eines Tropfens der 2%igen Lösung auf das runde Fenster. Da hierbei der Perilymphraum nicht eröffnet wurde, blieb der physiologische Zustand der kommunizierenden Flüssigkeiten Perilymphe und Liquor cerebrospinalis erhalten. Der Farbstoff erreichte innerhalb von 3–5 min durch den Aquaeductus cochleae den Subarachnoidalraum. Beim toten Tier allerdings war die Ausbreitungsgeschwindigkeit signifikant geringer. Dies spricht für eine physiologische Strömung der Perilymphe durch den Aquädukt zum Subarachnoidalraum beim lebenden Tier. Bei der zweiten Versuchsreihe wurden 2 ng gefriergetrocknetes Rhodamin direkt in den Perilymphraum appliziert. Obwohl dabei nahezu kein zusätzliches Volumen eingefüllt wurde, war die Farbstoffausbreitung deutlich weitläufiger. Die geringfügige Volumenschwankung bei der Markierung hatte das empfindliche Flüssigkeitssystem erheblich gestört.SummaryIn guinea pigs the tympanic perilymph of the basal turn was marked with fluorescing rhodamine. In the first series one drop of a 2% solution of the dye was applied onto the round window membrane. Since the perilymphatic space was not opened, the physiologic state of the communicating fluids perilymph and CSF was maintained. The dye appeared within 3–5 min via cochlear aqueduct in the subarachnoidal space. In the dead animal the speed of distribution was significantly less. This most probably indicates a weak physiologic flow of the perilymph through the aqueduct to the subarachnoidal space in living animals.In the second series, 2 ng of lyophilized rhodamine was instilled directly into the tympanic perilymph near the round window. Though hardly any additional volume was brought into the perilymph, the distribution of the dye was distinctly greater as a sign of the considerable sensitivity of the fluid system, even by the slightest manipulation.

Die Ausbreitung von Tetracyclin im Innenohr nach unterschiedlicher Applikation im Mittelohr

SummaryIn 31 young healthy guinea pigs a 2% solution of tetracycline was introduced into the middle ear under urethane anesthesia. In one group tetracycline was filled in the tympanic cavity. In the other it was applied only to the round and oval windows. The solution was removed from the specimens after various periods of contact and the cochlea immediately frozen with liquid air. The head was then frozen, severed and mounted on the cryotome. The specimens were illuminated with ultraviolet light under the operating microscope. The distribution pattern of the tetracycline was determined in the following manner: Every 5 μm parallel to the modiolus a section of the labyrinth was cut away and the fluorescent face of the remaining specimen was examined and photographed. The results of each animal were recorded in diagram-form (Fig. 1). In this way, the liquid-spaces of the labyrinth and the brain remain closed throughout the application and evaluation phases of the experiment, and changes of pressure or direction of flow are excluded.In both test series the perilymph of the tympanic scale of the first coil showed a distinct fluorescence after every period of application.In the first series, in which the whole bulla was filled, tetracycline was detected after 10 min: (1) in all the scales of the first turn, (2) in the vestibule, (3) in the utricule and (4) in the two upper coils (Fig. 2a).After 30 min of application, fluorescence appeared in the whole cochlea and in the perilymphatic duct up to the subarachnoidal space (Fig. 2b). After 60 min the substance had diffused throughout all the lymphatic spaces of the inner ear, including the endolymphatic duct and sac (Fig. 2c). In addition, all the experiments of this series revealed the presence of tetracycline in the bony structure of the cochlea (Fig. 4a). The tangential cut through this structure showed a high concentration of tetracycline in its blood vessels (Fig. 5).In the second series, where tetracycline was applied only to the niches, a more sharply differentiated picture appeared. After 10 min fluorescence was observed in the first turn of the scala tympani and in the vestibule (Fig. 3a). After 1 h the substance had only penetrated the first quarter of the first coil, whereas it had already reached the subarachnoidal space via the perilymphatic duct (Fig. 3c). After 2 h the fluorescence appeared in addition in the endolymphatic duct and sac, in the utricule, and it had diffused both longitudinally and transversally throughout the first coil, but not beyond the beginning of the second coil (Fig. 3d). In contrast to the first series, no significant penetration of the bony capsule could be observed (Fig. 4b).These findings lead to the following conclusions:1.Low molecular substances reach the inner ear fluids very quickly through the round window membrane as well as through the stapes footplate and the cochlear bone.2.The transport of low molecular substances to the inner ear is facilitated by blood vessels in its bony capsule.3.The different invasion of the cochlea and the perilymphatic duct in the second series of experiments speaks for a drainage of the perilymph from the scala tympani into the subarachnoidal space.ZusammenfassungAn 31 ohrgesunden Meerschweinchen wurde Tetracyclin in gelöster Form in das Mittelohr appliziert. Dabei wurden entweder nur die beiden Fensternischen oder die gesamte Paukenhöhle gefüllt. Die gefrorenen Köpfe wurden im Kryotom geschnitten und im UV-Licht betrachtet. Auf diese Weise konnte das Tetracyclin im Innenohr nachgewiesen werden. Durch das Einfrieren des Kopfes wurden die Flüssigkeitsräume von Innenohr und Gehirn weder bei der Applikation noch bei der Auswertung eröffnet.Das Tetracyclin gelangt vor allem über das runde Fenster ins Innenohr. Außerdem kann es auch über das ovale Fenster in das Vestibulum eindringen. Als weitere Zugangswege kommen die Diffusion durch die knöcherne Labyrinthkapsel sowie der Transport über labyrinthdurchquerende Gefäße in Betracht. Von der ersten Windung der Scala tympani gelangt das Tetracyclin relativ schnell über den Aquaeductus cochleae in den Subarachnoidalraum.

Immunohistochemical findings in the vestibular ganglion from a patient with Menière's disease

SummaryImmunofluorescent staining techniques were performed on a series of cryostat sections of the vestibular ganglion taken during ganglionectomy from a patient with Menières disease. The direct technique using FITC-labelled antiserum proved the presence of immunoglobulins in the patients blood vessels and endoneural connective tissue. Preincubation with unlabelled antiserum blocked this reaction. An additional positive reaction of ganglion cells was demonstrated by incubating tissue with the patients serum. These findings prove antibodies in the patients serum against the autologous ganglion cells. The vestibular ganglion from a patient with dizziness following a skull base fracture served as a control specimen. No autoimmune reaction was found in the ganglion cells when incubation was carried out with the patients serum. A positive reaction in the blood vessels and connective tissue was less pronounced. The findings of the present study underline the occurrence of immunological reactions in the vestibular ganglion of patients with Menières disease. A subsequent degeneration of ganglion cells could provoke clinical Menières attacks.

A new immunohistochemical method for the detection of gentamicin in inner ear fluid compartments

A new method was developed for frozen section detection of antigens that natively occur in the cochlear peri- and endolymph. A combination of immunohistochemistry and immunoblot assay enabled topological and quantitative detection of small and hydrophilic molecules (such as the aminoglycoside antibiotics) in frozen sections of the inner ear compartments (scala tympani, scala vestibuli and cochlear duct). A selective localization is possible in the peri- and endolymphatic region of each coil of the cochlea. During sectioning of the cochlea, a small piece of a nitrocellulose membrane is placed to the surface of the intersection and briefly warmed. The sections are cut, simultaneously attached to a nitrocellulose membrane on which the aminoglycoside antibiotics remain adsorbed without any fixation procedure. Using this method, immunoincubation to detect gentamicin was performed in a way usually done in western blot analysis. Results with two different enzyme reactions with the enzyme conjugated to a second antibody (i.e., dye as substrate and the chemiluminescence detection system) are presented and compared. This histoimmunoblot assay provides a general non-radioactive and sensitive immunohistochemical tool for the localization of compounds occurring in extracellular body fluid compartments. For inner ear research this method now enables the investigation of the penetration and distribution of therapeutics in peri-and endolymphatic sites and can even be applied to separately quantifying concentrations of a substance in different coils of the same cochlear section.

Quantitative Bestimmung von Protein, Albumin und Antibiotika im Nasensekret gesunder Probanden

This study on the pharmacokinetics of antibiotics in nasal secretions was carried out with two orally applicable penicillin derivatives which show different resorption patterns. Each of the antibiotics (Ampicillin and Bacampicillin) was given in equimolar doses to 20 healthy young volunteers, with normal mucosa, in a double blind cross over fashion. Nasal secretions were collected 1, 2, 3, 4, 6, and 8 h after the application of a single dose to the overnight fasted persons. In 10 of them blood was taken at 0.5, 1, 1.5, 2, 3, and 4 h after the administration. For the sampling of the nasal secretions cotton wool was weighed together with an airtight vial containing 300 microliter of phosphate buffered saline (PBS). The dry cotton wool stayed in the nasal cavity for 20 min, was then put into the PBS and weighed again. The difference determines the amount of secretions collected. After 30 min the soaked cotton wool was pressed out into a vial with a sterile syringe. One hundred microliters of this solution was taken to determine the antibiotic concentration by a micromodification of the agar diffusion technique. In the remaining fluid total protein and albumin were quantitatively determined. The amount of nasal secretions which have been collected are, on average, independent of the time (Fig. 1). With rising secretion the protein content decreases (Fig. 5) as is the case with the albumin concentration. Regarding all persons, the protein content and albumin (Fig. 4) remain constant during the experiment from 8 a.m. to 4 p.m. The differences between the values shown in the figures are not significant. Comparing the mean concentration for the antibiotic at different times after the application, it is obvious that the agents show different curves (Fig. 6). With ampicillin the maximum of 0.13 microgram/ml is reached at 2 h after the administration whereas with becampicillin the maximum of 0.84 microgram/ml is reached after 1 h. The concentrations in the nasal secretions are clearly dependent of the serum values. In the serum the maximum of the mean values plotted against the time of 2.7 microgram/ml is to be found at 2 h, if ampicillin is given, whereas the maximum of 9.3 microgram/ml is reached at 1 h after bacampicillin administration. In both cases in serum and nasal secretions the mean concentration maximum is about three times higher after bacampicillin as compared with ampicillin. As a reference for the concentration of the antibiotic the total protein content of the sample is more suitable as compared with sample volume and albumin because of its easy and exact determination. The results show that the nasal secretions can be used as a model to evaluate the pharmacokinetics in the mucosa of the upper respiratory tract if an adequate number of test persons is used.SummaryThis study on the pharmacokinetics of antibiotics in nasal secretions was carried out with two orally applicable penicillin derivatives which show different resorption patterns. Each of the antibiotics (Ampicillin and Bacampicillin) was given in equimolar doses to 20 healthy young volunteers, with normal mucosa, in a double blind cross over fashion. Nasal secretions were collected 1, 2, 3, 4, 6, and 8 h after the application of a single dose to the overnight fasted persons. In 10 of them blood was taken at 0.5, 1, 1.5, 2, 3, and 4 h after the administration.For the sampling of the nasal secretions cotton wool was weighed together with an airtight vial containing 300 μl of phosphate buffered saline (PBS). The dry cotton wool stayed in the nasal cavity for 20 min, was then put into the PBS and weighed again. The difference determines the amount of secretions collected. After 30 min the soaked cotton wool was pressed out into a vial with a sterile syringe. One hundred microliters of this solution was taken to determine the antibiotic concentration by a micromodification of the agar diffusion technique. In the remaining fluid total protein and albumin were quantitatively determined. The amount of nasal secretions which have been collected are, on average, independent of the time (Fig. 1). With rising secretion the protein content decreases (Fig. 5) as is the case with the albumin concentration. Regarding all persons, the protein content and albumin (Fig. 4) remain constant during the experiment from 8 a.m. to 4 p.m. The differences between the values shown in the figures are not significant.Comparing the mean concentration for the antibiotic at different times after the application, it is obvious that the agents show different curves (Fig. 6). With ampicillin the maximum of 0.13 μg/ml is reached at 2 h after the administration whereas with becampicillin the maximum of 0.84 μg/ml is reached after 1 h. The concentrations in the nasal secretions are clearly dependent of the serum values. In the serum the maximum of the mean values plotted against the time of 2.7 μg/ml is to be found at 2 h, if ampicillin is given, whereas the maximum of 9.3 μg/ml is reached at 1 h after bacampicillin administration. In both cases in serum and nasal secretions the mean concentration maximum is about three times higher after bacampicillin as compared with ampicillin. As a reference for the concentration of the antibiotic the total protein content of the sample is more suitable as compared with sample volume and albumin because of its easy and exact determination. The results show that the nasal secretions can be used as a model to evaluate the pharmacokinetics in the mucosa of the upper respiratory tract if an adequate number of test persons is used.ZusammenfassungBei 20 gesunden Testpersonen wurden in einer Doppelblindstudie Ampicillin und Bacampicillin in einmaliger äquimolarer Dosis oral appliziert. Bei allen Versuchsteilnehmern wurde zu Versuchsbeginn sowie nach 1, 2, 3, 4, 6 und 8 h Nasensekret entnommen, dessen Menge durch Wiegen bestimmt wurde. In diesen Proben wurde der Albumin-und Gesamtproteingehalt sowie die Konzentration des Antibiotikums bestimmt. Bei 10 dieser Personen wurde zu Versuchsbeginn und nach 0,5, 1, 1,5, 2, 3 und 4 h Blut entnommen und im Serum der Antibiotikumspiegel ermittelt.Die Menge des Sekrets sowie die Konzentrationen von Albumin und Gesamtprotein weisen starke Schwankungen auf, bleiben im Mittelwert aber über die Versuchsdauer konstant.Im Nasensekret tritt das Maximum der mittleren Antibiotikakonzentration bei Ampicillin (0,13 μg/ml) nach 2 h auf, während es bei Bacampicillin (0,84 μg/ml) bereits nach 1 h vorliegt. Dieser Zeitverlauf und die Konzentrationsunterschiede sind vom Serumspiegel abhängig. Im Serum zeigen sich die Maxima der mittleren Antibiotikakonzentration bei Ampicillin (2,7 μg/ml) nach 2 h und bei Bacampicillin (9,3 μg/ml) nach 1 h. Sowohl die Penetration der Substanz über die Nasenschleimhaut in das Sekret, als auch die Elimination folgen dem Serumspiegel nur mit einer geringen zeitlichen Verzögerung.Als Bezugsgröße für die Antibiotikakonzentration im Nasensekret erscheint die Gesamtproteinmenge am besten geeignet, da die Bestimmung einfach und wenig störanfällig ist.

Zur Ausbreitung löslicher Substanzen nach Applikation in Perilymphe und Liquor

SummaryNew labelling methods using fluorochromes reduce the amount of substances to 1 μg. The volume which is temporarily taken out and then reinjected into the fluid spaces is 0.1 μl. At different times after the application, the heads of the guinea pigs are frozen by liquid air, and cut on a cryotome. The mounted object is then fotographed with an operating microscope in UV-light.Substances applicated into the perilymph of the scala tympani reach the subarachnoid space via the cochlear aqueduct in about 10 min. After filling the whole bulla tympanica, the dyes diffude into the cochlea mainly through the membrane of the round window. They are reaching the inner ear also through the oval window and by labyrinth crossing vessels. Substances applied to the cerebello-medular cistern could not be detected in the cochlea. After intraperitoneal administration the fluorochromes can be found in the cochlear nerve, in the spiral ligament, in perilymph and endolymph. Substances which have reached the cochlear endolymph can be found in the endolymphatic sac after 20 min.This leads to the conclusion that perilymph and endolymph are produced in the cochlea. The blood vessels are the origin of the substances in the inner ear fluids, but a blood-inner ear barrier seems to be existent.

Inner ear fine structures of the hamster in frozen sections used for immunohistochemical assays for inner ear diseases.

SummaryFrozen sections of the inner ear of the hamster enable detailed investigations of the fine structures in immunofluorescence assays. At high magnification single mitochondria can be identified by their reactions with an antiserum containing antibodies against mitochondria. In the positive reaction with an antiserum against nuclei, the typical green fluorescence is restricted to the nuclei, which are mostly separated by the surrounding cytoplasm. The method of immunohistochemical assay using frozen sections from the non-decalcified inner ear is very time-consuming and cannot be recommended for the routine diagnosis of inner ear diseases, although it may be useful in research and for studying critical clinical cases.

Histochemical studies of cholesteatoma

SummaryFrozen sections of cholesteatomas were compared with postauricular and auditory canal skin and studied using histochemical methods. Several dehydrogenases, lysosomal enzymes and proteolytic activity were studied. Lactate and malate dehydrogenase activity was very strong in the epithelium and subepithelial tissue of cholesteatomas; no succinate dehydrogenase activity was observed. Esterase and acid phosphatase activity was prominent in cells of granulation tissue. Considerable proteolytic activity was observed in these cells and one specimen showed possible extracellular activity. Strong evidence of fat was found in the granulation tissue, partiuclarly at the interface of granulation tissue and bone. In postauricular and auditory canal skin, enzyme activity was generally weaker; no evidence of fat was found. The findings are discussed in the light of other investigations on the importance of enzyme activity in bone destruction associated with cholesteatoma.

Enzymhistochemische Untersuchungen am Innenohr des Frosches (Rana temporaria)

SummaryThe histochemical demonstration of different enzymes was carried out on undecalcified and unfixed frozen sections of the inner ear of the frog. Thus it was possible to reduce incubation time to less than 1 h.Beside a number of dehydrogenases, the presence of unspecific esterase, creatine phosphokinase and endopeptidase was investigated.For different parts of the endolymphatic wall characteristic enzyme patterns have been shown:In the ampullae on both sides of the sensory epithelium a transitorial and a dark cell zone have been observed (Fig. 1). In the utricle the sensory epithelium, perimacular zone and dark cell zone have been identified (Fig. 2). The perimacular zone surrounding the sensory epithelium of the macula sacculi shows a slight different enzyme activity (Fig. 3). On the dorso-lateral wall of the sacculus the tegmentum vasculosum-equivalent was found, which is discussed to be the phylogenetic precursor of the stria vascularis and Reissners membrane, in regard to its function (Fig. 4). A part of the tegmentum vasculosum-equivalent is in contact with the recessus basilaris, out of which the mammalian cochlea is developing during evolution (Fig. 6b).The dark cell zones and the tegmentum vasculosum-equivalent show activities of all enzymes tested. In the sensory epithelia only low activities of the dehydrogenases compatible with aerobic glycolysis have been detected.In the apical portion of the sensory epithelia of the semicircular ducts and the statolithic apparatus most enzymes show higher activity than basal. The apical portion is mainly built up by hair-cells and the high enzyme content seems to be correlated to these (Fig. 1). In some tests the macula sacculi reacts different, which may be due to active secretion of the supporting-cells in this structure (Fig. 3). The presence of enzymes of protein- and aminoacid metabolism as well as the activity of unspecific esterase (Fig. 5b) and endopeptidases in the basal portion of the sensory epithelia of the acoustic receptors enables the conclusion on a secretory activity of these supporting-cells.ZusammenfassungDie enzymhistochemischen Untersuchungen am Innenohr des Frosches wurden an unfixierten und unentkalkten Gefrierschnitten durchgeführt.In den Ampullen und im Utriculus lassen sich neben den Sinnesepithelien der Cristae ampullares und der Macula utriculi Dunkelzellenzonen und Übergangszonen bzw. perimaculäre Randzonen unterscheiden. Ebenso wird das Sinnesepithel der Macula sacculi von einer spezifisch reagierenden Randzone flankiert.Im Bereich der Sacculushinter- und -seitenwand ist das Tegmentum vasculosum-Äquivalent gefunden worden, welchem funktionelle und entwicklungsgeschichtliche Bedeutung zukommt als Vorläufer der Stria vascularis und der Reissnermembran der Säugetiercochlea. Auf Grund seiner anatomischen Ausdehnung besteht eine direkte Beziehung zum Recessus basilaris der Papilla basilaris, aus welchem sich in der Phylogenese die Cochlea entwickelt.Die reichlichste Enzymausstattung besitzen die Dunkelzellenzonen und das Tegmentum vasculosum-Äquivalent. Charakteristische Enzyme der aeroben Glycolyse kommen in allen Sinnesepithelien nur in schwacher Konzentration vor.In den Sinnesepithelien des Bogengang- und Statolithenapparats sind deutliche enzymatische Aktivitäten bei allen Nachweisen vor allem in den apikalen Zellanteilen zu beobachten und damit hauptsächlich den Haarzellen zuzuordnen mit Ausnahme der Macula sacculi, bei der eine Modifizierung der Verteilungsmuster der Reaktionsprodukte durch aktive Sekretion der Stützzellen diskutiert werden muß.Hohe Aktivitäten zahlreicher Enzyme des Protein- und Aminosäurestoffwechsels sowie an unspezifischer Esterase und an Endopeptidasen in den basalen Anteilen des Sinnesepithels der akustischen Papillen stärken den Verdacht auf eine sekretorische Aktivität der Stützzellen.

Zur Ausbreitung von Fluorescein im Innenohr nach Applikation in das Mittelohr

SummaryA solution of 2% fluorescein in buffered physiological saline was applied into the middle ear of young healthy pigmented guinea pigs. In one series of experiments the whole middle ear was filled. In a second series the round and oval windows were left uncovered. At different time intervals after application the inner ear was frozen by liquid air and the specimen cut in a cryotom.Fluorescein quickly reached the subarachnoidal space from the first coil of the scala tympani (about 10 min). From the lower cochlear duct the substance was carried to the endolymphatic sac in about 30 min. Fluorescein penetrated the cochlear bone in all turns and was found in endolymph and perilymph. From the upper coils the substance spread slowly basalwards in all three scales. In all cases fluorescein was seen in the modiolus.