0000000000194728
AUTHOR
Ulf-rüdiger Heinrich
Die Renaissance der Elektronenmikroskopie. Transmissions-Elektronenmikroskop mit Energiefilter für biologische Proben
Seit Entwicklung der Transmissions-Elektronenmikroskopie durch Ernst Ruska und Max Knoll im Jahre 1931 ist das Auflosungsvermogen der Elektronenmikroskope bis zum atomaren Bereich gesteigert worden, und auch dickere Praparate oder ganze Zellen konnen jetzt durch Erhohung der Beschleunigungsspannung durchstrahlt werden (high voltage electron microscopy). Eine wesentliche Verbesserung in der Anwendung des Transmissions-Elektronenmikroskops (TEM) uber das deskriptive Niveau der Feinstruktur-Analyse hinaus wird durch die Ausstattung der Mikroskope mit analytischen Zusatzgeraten erreicht, wie beispielsweise durch die Rontgen-Mikroanalyse (energy dispersive X-ray analysis − EDX) oder die Elektron…
Possible Ca 2+ -dependent mechanism of apical outer hair cell modulation within the cochlea of the guinea pig
Calcium ions were precipitated with potassium antimonate after injection of the inorganic calcium channel blocker MnCl2 or the inorganic potassium channel blockers BaCl2 or CsCl into the perilymph of the scala vestibuli of the guinea pig. The spatial distribution of the formed histochemical reaction products within the organ of Corti was studied by energy-filtering transmission-electron microscopy. Compared with untreated control ears, the number of the formed precipitates drastically increased at the extracellular side of the lamina reticularis after application of the various inorganic channel blockers. The apical side of the outer hair cells and the intervening Deiter cells were covered …
Frequent chromosomal gains in recurrent juvenile nasopharyngeal angiofibroma.
Juvenile nasopharyngeal angiofibroma (JNA) is a rare benign tumor, mostly affecting adolescent males. Some patients develop recurrences after surgery independently of completeness of removal. Only very limited data concerning underlying chromosomal changes are available. We therefore analyzed samples of 22 JNAs, including six recurrences, with comparative genomic hybridization (CGH). Additionally, quantitative image cytometry was used for measurement of DNA aneuploidy in representative samples. Of the 13 primary JNAs without later recurrence, DNA gains were identified on autosomes in only two samples. Four patients with one or two recurrences were included in the study; for one of these, no…
Immunoelectron microscopic localization of nitric oxide synthase III in the guinea pig organ of Corti
Nitric oxide synthase III (NOS III) was identified in the guinea pig cochlea on an ultrastructural level using a post-embedding immunolabeling procedure. Ultrathin sections of London Resin (LR) White-embedded specimens were incubated with various concentrations of a commercially available antibody to NOS III and the immunoreactivity visualized by a gold-labeled secondary antibody. Analysis of ultrathin sections of the organ of Corti in the second turn of the cochlea showed that NOS III could be localized in the endothelial cells of the blood vessels under the basilar membrane, which was comparable to its location in similar cells types in various biological systems. Besides this, NOS III wa…
Morphologic Damage and Changes of Intracellular Calcium-Binding Sites after Acute Noise Trauma in the Organ of Corti of the Guinea Pig
In guinea pigs, an acute acoustic trauma was created by 6 consecutive gunshots. The sound pressure at the ear drum was 156 +/- 4 dB, the frequency maximum was between 4 and 6 kHz. Sixty hours after the noise trauma, the animals were decapitated, and the cochleae were prepared for microscopic analysis of the resulting trauma to the organ of Corti. During the process of fixation, the potassium-pyroantimonate precipitation reaction was performed to localize calcium-binding sites. The pattern of cell morphology and the distribution of calcium-binding sites was compared to that of normal control animals. Morphologic changes of the cells in the organ of Corti correlated with changes of the cellul…
Ascorbic acid reduces noise-induced nitric oxide production in the guinea pig ear.
Objectives: Noise-induced hearing loss can be caused, among other causes, by increased nitric oxide (NO) production in the inner ear leading to nitroactive stress and cell destruction. Some studies in the literature suggest that the degree of hearing loss (HL) could be reduced in an animal model through ascorbic acid supplementation. To identify the effect of ascorbic acid on tissue-dependent NO content in the inner ear of the guinea pig, we determined the local NO production in the organ of Corti and the lateral wall separately 6 hours after noise exposure. Study Design: Prospective animal study in guinea pigs. Methods: Over a period of 7 days, male guinea pigs were supplied with minimum (…
Localization of the two constitutively expressed nitric oxide synthase isoforms (nNOS and eNOS) in the same cell types in the saccule maculae of the frog Rana pipiens by immunoelectron microscopy: evidence for a back-up system?
There is growing evidence for a nitric oxide/cyclic GMP pathway of signal transduction in the vestibular system. Recently, two isoforms of nitric oxide (NO) synthase (nNOS and eNOS) and NO itself have been identified at the light microscopic level in the vestibulocochlear system of mice using specific antibodies and a new fluorescence indicator. In order to acquire more information about signal transduction and tissue modulation in this neuroepithelium at the cellular and subcellular levels, ultrathin sections of London Resin White-embedded saccule maculae of the frog Rana pipiens were incubated with various concentrations of commercially available antibodies to nNOS and eNOS. The immunorea…
Localization of Ca(2+)-stores and tissue compartments with a Ca(2+)-binding capacity in the organ of Corti of the guinea-pig by electron energy-loss spectroscopy.
SUMMARY The addition of 10 mM CaCl2 to glutaraldehyde fixative leads to the formation of small electron-dense deposits in the organ of Corti of the guinea-pig. These precipitates are mainly attached to cell membranes in contact with different extracellular lymphatic fluids. A higher number of precipitates is localized in the acellular parts of tectorial and basilar membrane. Electron energy-loss spectroscopy (EELS) was used to determine the elemental composition of the deposits formed. The spectra showed a prominent signal at the Ca2+ L2,3 ionization edge. Oxygen could also be detected in all the precipitates analysed. EELS analysis of mitochondria of the inner and outer hair cells after co…
Gentamicin alters Akt-expression and its activation in the guinea pig cochlea
Gentamicin treatment induces hair cell death or survival in the inner ear. Besides the well-known toxic effects, the phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway was found to be involved in cell protection. After gentamicin application, the spatiotemporal expression patterns of Akt and its activated form (p-Akt) were determined in male guinea pigs. A single dose of 0.1 mL gentamicin (4 mg/ear/animal) was intratympanically injected. The auditory brainstem responses (ABRs) were recorded prior to application and 1, 2 and 7 days afterward. At these three time points the cochleae (n=10 in each case) were removed, transferred to fixative and embedded in paraffin. Seven ears were used as u…
Dexamethasone prevents hearing loss by restoring glucocorticoid receptor expression in the guinea pig cochlea
Objectives/Hypothesis Dexamethasone is widely used in the treatment of various inner ear diseases. However, knowledge about its direct impact on glucocorticoid receptor (GR) expression is still limited. Study Design Prospective animal study in male guinea pigs. Methods A therapeutic concentration of dexamethasone (8mg/mL) or a physiological concentration of NaCl (0.9% solution) were intratympanically injected into the ears of guinea pigs (n = 10 in each case) 14 hours prior to 90 dB noise exposure (1 hour). Eighteen ears were exposed to noise only. Seven untreated ears were used as controls. Auditory brainstem responses were recorded prior to noise exposure or treatment and 2 hours thereaft…
Progress in electron microscopic diagnostics: semi-quantitative determination of precipitable calcium in different cell types of the organ of Corti in the guinea-pig
Potassium antimonate was used to precipitate calcium in the cochlea of the guinea-pig. The distribution of the calcium antimonate precipitates was analysed by electron microscopy. The precipitate density was determined in different cell types in the organ of Corti by counting the number of calcium binding sites in a 10-micron 2 area. The size of the precipitates varied considerably, and thus the relative amount of the precipitable calcium was estimated only semi-quantitatively. As the prominent carbon signal is superimposed over the nearby small Ca(2+)-edge signals, the combined signal of the antimony M4,5-edge and the oxygen K-edge of the calcium antimonate salt formed was chosen for the s…
Immunoelectron-microscopic localization of synaptophysin in the organ of Corti of the guinea pig.
Synaptophysin has been localized previously in the mammalian cochlea at the light-microscopic level and in few reports by electron microscopy using either a preincubation procedure or the avidin-biotin reaction. Here we present results of the electron-microscopic analysis for postembedding immunoreactivity of synaptophysin in the cochlea of the guinea pig of LR-White-embedded samples. Strong synaptophysin immunoreactivity is located in the cytoplasm of the efferent nerve endings at the base of inner and outer hair cells. Besides this, some antibodies to synaptophysin were also identified in the cytoplasm of outer hair cells. To get more information about the cellular content of synaptophysi…
Ultrastructural localization of Ca2(+)-binding sites in the middle ear mucosa of the rat.
Cations were precipitated with potassium-pyroantimonate in the middle ear mucosa of the rat and the distribution of the formed precipitates was studied by electron microscopy. The precipitate density in various cells of the different epithelia of the middle ear mucosa was determined on electron micrographs by counting the number of precipitates per unit area. Electron spectroscopic imaging was also performed to obtain information about the spatial distribution of the precipitates and their elemental composition.
Histamine Upregulates Gene Expression of Endothelial Nitric Oxide Synthase in Human Vascular Endothelial Cells
Background— Histamine has a short-term, transient, stimulating effect on endothelial nitric oxide synthase (eNOS) activity; however, long-term effects on eNOS have not been described yet. In addition, the vascular effect of histamine seems to depend critically on eNOS functionality. Therefore, we studied the effects of histamine on eNOS gene expression and function. Methods and Results— In human umbilical vein endothelial cells (HUVECs) and HUVEC-derived EA.hy 926 cells, histamine upregulated eNOS mRNA (RNase protection assay) and protein (electron microscopic immunocytochemistry) expression. The upregulation of eNOS could be prevented by mepyramine, a selective antagonist at the H 1 recep…
Immuno-electron microscopic localization of the alpha(1) and beta(1)-subunits of soluble guanylyl cyclase in the guinea pig organ of corti.
Guanylyl cyclases (GC) catalyze the formation of the intracellular signal molecule cyclic GMP from GTP. For some years it has been known that the heme-containing soluble guanylyl cyclase (sGC) is stimulated by NO and NO-containing compounds. The sGC enzyme consists of two subunits (alpha(1) and beta(1)). In the present study, the alpha(1) and beta(1)-subunits were identified in the guinea pig cochlea at the electron microscopic level using a post-embedding immuno-labeling procedure. Ultrathin sections of LR White embedded specimens were incubated with various concentrations of two rabbit polyclonal antibodies to the alpha(1)- and beta(1)-subunit, respectively. The immunoreactivity was visua…
Gentamicin alters nitric oxide production in semicircular canals and otolith organs.
Objectives/Hypothesis: Intratympanic application of gentamicin is an important therapeutic option to control vertigo spells in Meniere's disease. Low doses eliminate the function of semicircular canal ampullae (SCCA) and saccule in most patients, although utricular function is maintained in many cases. Local alteration in free radical production might be responsible for these differences. Therefore, the gentamicin-induced nitric oxide (NO) production was determined in an animal model using separate organ cultures of SCCA, saccule, and utricle. Study Design: Prospective pilot study in male guinea pigs. Methods: SCCA, saccule, and utricle of 28 guinea pigs were isolated and incubated separate…
Electron microscopic localization of nitric oxide I synthase in the organ of Corti of the guinea pig
Nitric oxide synthase (NOS) activity has been detected previously in the mammalian cochlea at a light microscopic level. Here we present results of electron microscopic analysis for post-embedding immunoreactivity of neural-type NOS I in the cochlea of the guinea pig. Strong enzyme immunoreactivity was identified in the cytoplasm of inner and outer hair cells. Gold-labeled NOS I antibodies were mainly located in electron-dense areas of the cytoplasm, whereas electron-lucent regions of the receptor cells were nearly free from any immunoreactivity. In both types of hair cells anti-NOS I antibodies were also visible in the cuticular plates, hair bundles and nuclei. Further ultrastructural anal…
The VEGF/VEGF-R Axis in Sporadic Vestibular Schwannomas Correlates with Irradiation and Disease Recurrence
<b><i>Background/Aims:</i></b> The molecular mechanisms downstream of mutated neurofibromatosis type 2 (NF2) gene resulting in the growth and development of vestibular schwannoma (VS) are controversial. Several lines of evidence suggest the involvement of the vascular endothelial growth factor (VEGF) pathway in VS development. Given that recent studies of VEGF blockade in patients with NF2-associated VS showed positive effects on VS growth control, we initiated this comprehensive study of the VEGF pathway in sporadic VS. <b><i>Methods:</i></b> A tissue microarray analysis of 182 sporadic VS was conducted. The expression of VEGF and its recepto…
Immunohistochemical localization of cyclooxygenase isoforms in the organ of Corti and the spiral ganglion cells of guinea pig cochlea.
Prostaglandins have been used in experimental models and clinical studies for the therapy of sudden hearing loss and tinnitus with conflicting results. However, little is known about the rate-limiting enzymes of prostaglandin synthesis in the inner ear, the generally constitutively expressed cyclooxygenase 1 (COX-1) and the distress-inducible cycloxygenase 2 (COX-2). To extend our knowledge concerning the physiological expression and localization of these two enzymes, immunohistochemical stainings of the guinea pig cochlea were performed. Light microscopical analysis revealed a homogenous distribution of COX-1 within nearly all cell types of the organ of Corti, but no COX-1 expression in th…
17β-Estradiol Reduces Nitric Oxide Production in the Guinea Pig Cochlea
Intense noise exposure and the application of ototoxic substances result in increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as nitric oxide (NO). In order to reduce the free NO concentration in the inner ear under pathological conditions, the use of natural cytoprotective substances such as 17β-estradiol is a promising therapeutic concept. In male guinea pigs the organ of Corti and the lateral wall were isolated from the cochlea and afterwards incubated for 6 h in cell-culture medium. 17β-Estradiol was adjusted in 2 concentrations to organ cultures of the right ears (12 animals per concentration). The left ears were used as controls. The NO produc…
Endothelial nitric oxide synthase upregulation in the guinea pig organ of Corti after acute noise trauma.
Endothelial nitric oxide synthase (eNOS) upregulation was identified 60 h after acute noise trauma in morphologically intact cells of the reticular lamina in the organ of Corti of the guinea pig in the second turn of the cochlea. Using gold-coupled anti-eNOS antibodies and electron microscopy, it was shown that eNOS expression was upregulated in all cell areas and cell types except inner hair cells. Furthermore, eNOS was found in the organelle-free cytoplasm and in mitochondria of various cell types. The density of eNOS in mitochondria was considerably higher compared with the surrounding cytoplasm. Since eNOS activity is regulated by calcium, the eNOS detection was combined with calcium pr…
Cell-specific accumulation patterns of gentamicin in the guinea pig cochlea.
Intratympanic gentamicin therapy has become a popular treatment modality for Meniere's disease (MD) through controlled elimination of vertigo spells caused by the balance organ. However, the known ototoxic properties of aminoglycosides lead to cochlear damage. In order to gain more information about cellular preferences for aminoglycoside accumulation within the cochlea, gentamicin was immuno histochemically localized by light microscopy in male guinea pigs 1 and 7 days after intratympanic application (n = 8 ears/incubation time). Differences in the gentamicin-specific cellular storage capacities were quantified by determination of the local immuno staining intensities. Gentamicin was detec…
Calcium-binding sites in the inner ear after pure-tone stimulation
Five guinea pigs were exposed to an interrupted 90 dB SPL pure tone of 3.2 kHz for a total application time of 5 min. Following sound application all animals were decapitated and the cochleae were removed. After that, calcium-binding sites were located by the potassium pyroantimonate precipitation method. Another three animals served as control animals and did not receive the sound treatment. Findings confirmed previous studies showing the spatial arrangements of precipitate rich regions in the inner ear's two acellular structures (the basilar membrane and tectorial membrane) and the two cellular structures (the inner hair cells and Huschke's teeth). By using semiquantitative evaluation we …
Nitric oxide--a versatile key player in cochlear function and hearing disorders.
Nitric oxide (NO) is a signaling molecule which can generally be formed by three nitric oxide synthases (NOS). Two of them, the endothelial nitric oxide synthase (eNOS) and the neural nitric oxide synthase (nNOS), are calcium/calmodulin-dependent and constitutively expressed in many cell types. Both isoforms are found in the vertebrate cochlea. The inducible nitric oxide synthase (iNOS) is independent of calcium and normally not detectable in the un-stimulated cochlea. In the inner ear, as in other tissues, NO was identified as a multitask molecule involved in various processes such as neurotransmission and neuromodulation. In addition, increasing evidence demonstrates that the NO-dependent…
Doxycycline reduces nitric oxide production in guinea pig inner ears.
Abstract Objective Gentamicin application is an important therapeutic option to control vertigo spells in Meniere's disease. However, even in the case of low-dose intratympanic application, gentamicin might contribute to a pathological NO-increase leading to cochlear damage and hearing impairment. The study was performed to evaluate the nitric oxide (NO) reducing capacity of doxycycline in the inner ear after NO-induction by gentamicin. Methods In a prospective animal study, a single dose of gentamicin (10 mg/kg body weight) was injected intratympanically into male guinea pigs (n = 48). The auditory brainstem responses (ABRs) were recorded prior to application and 3, 5 and 7 days afterwards…
Alteration of loosely bound calcium in the guinea pig organ of Corti after treatment with diltiazem as calcium channel blocker
After oral administration of the organic calcium channel blocker diltiazem to guinea pigs for 7 days, calcium ions were precipitated with potassium antimonate in the cochleae. The spatial distribution of the precipitates was studied by energy-filtering transmission electron microscopy and the amount of the ultrastructural reaction products formed was determined semiquantitatively by an image processing system. Compared with untreated control ears, the number of the formed precipitates was reduced drastically in the inner hair cells after diltiazem treatment. In addition, electron microscopic analysis revealed that the number of calcium precipitates attached at the basolateral membrane of th…
Use of electron spectroscopic imaging to determine element composition of the melanin granules in the stria vascularis of the guinea pig.
Electron spectroscopic imaging (ESI) was used to analyze the element content of melanin granules in the stria vascularis seen in ultrathin sections of Spurr-embedded cochleae of the guinea pig. To determine element composition, ESI images were taken at different ionization edges, and non-specific background signals were subtracted digitally by an image processing system. The presence of calcium and nitrogen in the melanin granules could be demonstrated clearly. The calcium identified in the melanin granules was then compared with the spatial distributions of calcium binding sites after the application of an antimonate precipitation method, which was used to localize loosely bound calcium. D…
Ultrastructural evidence for protection of the outer hair cells of the inner ear during intense noise exposure by application of the organic calcium channel blocker diltiazem.
Outer hair cells could be protected during intense noise exposure by the application of the calcium channel blocker diltiazem given before and after noise treatment. After various experimental approaches, the ultrastructural morphology was analysed for the different animal populations in the basal part of the second turn of the cochlea, which was the most destroyed area after an acute noise trauma caused by a gun shot (sound pressure at the ear drum 156 dB, frequency maximum between 4 and 6 kHz). Compared to untreated control specimens (experimental animal group I), the outer hair cells in the basal part of the second turn of the cochlea were mostly destroyed without any diltiazem applicati…
Image cytometric DNA analysis of mucosal biopsies in patients with primary achalasia.
(P < 0.0001), and 9c- (P = 0.0001) exceeding rate with progredient DNA alterations in the respective order. CONCLUSION: The finding that DNA aneuploidy was identifi ed by image cytometry in esophageal specimens of patients with achalasia, which may be due to specifi c chromosomal alterations presenting as precancerous lesions in 27% of patients, leads us to conclude that image cytometry represents a valuable screening tool. Abstract AIM: To determine DNA aneuploidy in mucosal biopsies of achalasia patients for subsequent rapid diagnosis. METHODS: Biopsies from the middle third of the esophagus were obtained in 15 patients with achalasia. Immunohistochemical staining was carried out with mon…
Gentamicin increases nitric oxide production and induces hearing loss in guinea pigs.
Objectives/Hypothesis: Gentamicin application is an important therapeutic option for Meniere's disease. However, even if given at intervals, a destruction of the cochlea was often observed in various animal models together with an increased content of nitric oxide (NO) and reactive oxygen species. The present study was undertaken to identify the correlation between hearing threshold alteration and the NO production in the lateral wall and organ of Corti of the guinea pig in response to gentamicin application. Study Design: Prospective animal study in guinea pigs. Methods: A single dose of gentamicin (10 mg/kg body weight) was injected intratympanally into male guinea pigs and the auditory b…
Mechanisms underlying noise-induced hearing loss
Noise-induced hearing loss (NIHL) is the worldwide leading occupational disease and presents an important socio-economic factor. Despite numerous identified details about its etiology, the underlying mechanisms, which induce NIHL, have been only partially identified. In the present article, we shall discuss possible mechanisms focusing on failure in cellular calcium regulation, leading to a rise in mitochondrial NO production and reactive oxygen species formation. In cases where radical production is significantly elevated, pathological concentrations lead to alterations of cell physiological conditions and finally contribute to NIHL. A more detailed knowledge about the induction of free ra…