Lectin binding patterns in nonsensory regions of rat cochlea during postnatal development

The distribution of glycoconjugates was examined in the nonsensory regions of the rat cochlea during postnatal development using biotin-conjugated lectins. Temporal bones of rats at postnatal d 1 and at wk 2, 4 and 6 were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde and processed for paraffin wax embedding. The dewaxed sections were incubated with 7 biotinylated lectins, followed by avidin-biotin-peroxidase complex. A different staining pattern was observed in the stria vascularis, spiral ligament and spiral limbus in the age groups examined. The staining intensity varied between lectins and the reaction product exhibited limited disparity. The staining intensity for WGA increased with age in all the 3 nonsensory regions. The staining patterns for the other lectins differed in the various nonsensory regions examined indicating tissue specificity. The limited variations in the lectin binding patterns after 2nd wk of postnatal life also indicate that the changes in the carbohydrate moieties are established during the fetal period of cochlear development and limited changes take place during postnatal maturation of the nonsensory regions.     

Mitotic activity and specification of fibrocyte subtypes in the developing rat cochlear lateral wall

Spiral ligament fibrocytes (SLFs) in the mammalian cochlear lateral wall participate in K⁺ recycling; they are classified into five subtypes based on their morphology, distribution, and function. Regeneration of SLFs is a potential therapeutic strategy for correcting several types of hearing loss, prompting us to investigate how SLF subtypes are established during development. We compared transitional SLF-type marker expression with mitotic activity to evaluate proliferation-differentiation relationships in SLFs from postnatal rat cochleae. I.p. injection of 5-bromo-2′-deoxyuridine (BrdU) demonstrated that the overall mitotic activity of SLFs decreased significantly between postnatal day 7 (P7) and P10. For all developmental periods, BrdU incorporation was weakest in the area where type I SLFs reside. The onset of expression of markers for type II/IV SLFs followed the reduced mitotic activity of the cells, whereas that of aquaporin-1, a marker for type III SLFs, was already detectable at P7, when the type III SLFs were still proliferating vigorously. Distribution of BrdU⁺ cells increased in the area of type I SLFs between P7 and P10, suggesting migration of SLFs from adjacent areas. We conclude that the time course of development of SLFs is subtype-specific.     

人内耳蜗神经管的应用解剖学

目的：为临床人工耳蜗植人术提供解剖学依据。方法：观测30例(60侧)硅胶蜗神经管的长度和直径。观测15例(30侧)CT蜗神经管的长度和直径。结果：蜗神经管为位于内耳道底至耳蜗底之间的一个粗短的圆柱型管道。硅胶蜗神经管的长度为(1．69±0．34)mm,平均直径为(2．53±0．27)mm。CT蜗神经管平均直径为(2．20±0．30)mm,直径的正常参考值为(2．08～2．23)mm(95%可信区间)。结论：首次测得了CT蜗神经管直径的正常参考值,该数值可作为人工耳蜗植入术前评估的解剖学依据。     

Multiple expression of glucose transporters in the lateral wall of the cochlear duct studied by quantitative real-time PCR assay

We have investigated the gene expression of the facilitated glucose transporter (GLUT), H⁺-coupled myo-inositol cotransporter (HMIT), and Na⁺ glucose cotransporter (SGLT) in the lateral wall of the cochlear duct by conventional RT-PCR and quantitative real-time PCR. The isoforms GLUT1, -3, -4, -5, -8, -10, -12 and HMIT were detected in both the stria vascularis and the spiral ligament, whereas no SGLT isoforms could be detected in these tissues. Quantitative real-time PCR analysis revealed significant differences in the gene expression of GLUT1, -4, -5, -10, and HMIT isoforms between the stria vascularis and the spiral ligament. This result reflects the tissue-dependent distributions of GLUT isoforms. These findings strongly suggest that a number of GLUT isoforms participate in glucose transport in the stria vascularis and the spiral ligament.     

Identification and localization of G protein subunits in human spermatozoa

Antibodies to alpha and beta subunits of guanine nucleotide regulatory proteins (G proteins) were used to identify which G proteins are present in mature human spermatozoa and to determine their subcellular localization. Immunoblots of membranes from spermatozoa demonstrate the presence of Galphai2, Galphai3, Galphaq/11 and Gbeta35 and the absence of Galphai1, Galpha0, Galphas, Galpha12, Galpha13, Galpha16, Galpha and Gbeta36. Indirect immunofluorescence demonstrates the presence of Galphaq/11 in the acrosome, with the highest proportion in the equatorial segment. Galphai2 is present in the acrosome, midpiece and tailpiece and Galphai3 in the postnuclear cap, midpiece and tailpiece. The Gbeta35 subunit is found mostly in the midpiece, with marginal labelling of the head, tailpiece and the equatorial segment of the acrosome. The distinct pattern of distribution of G proteins suggests that they may couple to receptors or effectors which also have discrete regions of localization in spermatozoa. These highly localized signal transduction pathways may regulate discrete functions, such as activation of the acrosome reaction, fusion with the oocyte and motility.     

Oxidative stress and the deleterious consequences to the rat cochlea after prenatal chronic mild exposure to carbon monoxide in air

Pregnant rats (starting on E5) were exposed chronically to carbon monoxide (CO) from gestational days 5-20. In the postnatal period, rat pups were grouped as follows: group A: prenatal exposure to CO only; group B: prenatal exposure to CO then exposed to CO from postnatal day (P) 5 to P20; group C, control (air without CO). Groups A and B showed similar deleterious effects after CO exposure. At P3, rat pup cochlea from group A showed a normal organization of the organ of Corti. There was no morphological deterioration, or loss of inner or outer hair cells. At P20, animals from group A and B showed vacuolization on the afferent terminals at the basal portion of the cochlea. We found synapsin-1 immunoreactivity (IR) to be decreased in efferent nerve terminals in CO-exposed pups at P3. From P12 to P20, synapsin-1-IR is low in efferent terminals. At P20, type I spiral ganglia neurons and afferent nerve fibers showed decreased neurofilament-IR in CO-exposed groups when compared with controls. Heme oxygenase-1 and superoxide dismutase-1-IR were elevated in the stria vascularis and blood vessels from CO-exposed rat pups at P12 and P20 in group B; in contrast group A showed a comparable expression to controls. Inducible nitric oxide synthase (iNOS) and nitrotyrosine-IR were increased in blood vessels of the cochlea in CO-exposed groups, from P3 to P20. iNOS up-regulation and the presence of nitrotyrosine in blood vessels of the cochlea indicated that CO exposure activates the production of nitric oxide via increased iNOS activity. Prenatal chronic CO exposure promotes oxidative stress in the cochlea blood vessels that in turn is reflected in damage to spiral ganglia neurons and inner hair cells, suggesting for the first time that prenatal exposure to CO at concentrations expected in poorly ventilated environments impairs the development of the inner ear.     

Endothelial Injury of Capillaries in the Stria Vascularis of Guinea Pigs Treated with Cisplatin and Gentamicin

The drugs cisplatin and gentamicin are used for treatment of various cancer patients suffering from infection. The authors report a detailed electron microscopic study of blood vessels in stria vascularis of guinea pigs after treatment with cisplatin alone and in combination with gentamicin. The most distinctive features expressing endothelial cellular injury were mitochondrial, including occasional paracrystalline inclusions; electron-lucent foci with depleted organelles; intracytoplasmic vacuole formations; lipid bodies; cytoplasmic extrusions located on the luminal surface; and severe luminal constriction of part of the vessels from animals treated with the combined drugs. The study suggests that the damage to strial capillaries due to treatment with cisplatin alone and in combination with gentamicin may contribute to the injurious effects of these drugs on the strial tissue. Furthermore, the results of this study may enlarge the awareness of the potential vascular damage and vascular complications in additional body systems after medical use of cisplatin alone or in combination with gentamicin.     

Coin Lesion of the Lung in a 19-year-old Male

The drugs cisplatin and gentamicin are used for treatment of various cancer patients suffering from infection. The authors report a detailed electron microscopic study of blood vessels in stria vascularis of guinea pigs after treatment with cisplatin alone and in combination with gentamicin. The most distinctive features expressing endothelial cellular injury were mitochondrial, including occasional paracrystalline inclusions; electron-lucent foci with depleted organelles; intracytoplasmic vacuole formations; lipid bodies; cytoplasmic extrusions located on the luminal surface; and severe luminal constriction of part of the vessels from animals treated with the combined drugs. The study suggests that the damage to strial capillaries due to treatment with cisplatin alone and in combination with gentamicin may contribute to the injurious effects of these drugs on the strial tissue. Furthermore, the results of this study may enlarge the awareness of the potential vascular damage and vascular complications in additional body systems after medical use of cisplatin alone or in combination with gentamicin.     

G protein betagamma subunits modulate the number and nature of exocytotic fusion events in adrenal chromaffin cells independent of calcium entry

G-protein-coupled receptors (GPCR) play important roles in controlling neurotransmitter and hormone release. Inhibition of voltage-gated Ca(2+) channels (Ca(2+) channels) by G protein betagamma subunits (Gbetagamma) is one prominent mechanism, but there is evidence for additional effects distinct from those on calcium entry. However, relatively few studies have investigated the Ca(2+)-channel-independent effects of Gbetagamma on transmitter release, so the impact of this mechanism remains unclear. We used carbon fiber amperometry to analyze catecholamine release from individual vesicles in bovine adrenal chromaffin cells, a widely used neurosecretory model. To bypass the effects of Gbetagamma on Ca(2+) entry, we stimulated secretion using ionomycin (a Ca(2+) ionophore) or direct intracellular application of Ca(2+) through a patch pipette. Activation of endogenous GPCR or transient transfection with exogenous Gbetagamma significantly reduced the number of amperometric spikes (the number of vesicular fusion events). The charge ("quantal size") and amplitude of the amperometric spikes were also significantly reduced by GPCR/Gbetagamma. We conclude that independent from effects on calcium entry, Gbetagamma can regulate both the number of vesicles that undergo exocytosis and the amount of catecholamine released per fusion event. We discuss possible mechanisms by which Gbetagamma might exert these novel effects including interaction with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex.     

Expression patterns of FGF receptors in the developing mammalian cochlea

Many studies have shown the importance of the fibroblast growth factor (FGF) family of factors in the development of the mammalian cochlea. There are four fibroblast growth factor receptors (FGFR1–4) and all four are expressed in the cochlea during development. While there are examples in the literature of expression patterns of some of the receptors at specific stages of cochlear development there has been no systematic study. We have assembled a full analysis of the patterns of receptor expression during cochlear development for all four Fgfrs using in situ hybridization. We have analyzed the expression patterns from embryonic day 13.5 through postnatal ages. We find that Fgfr1, 2, and 3 are expressed in the epithelium of the cochlear duct and Fgfr4 is limited in its expression to the mesenchyme surrounding the duct. We compare the receptor expression pattern to markers of the sensory domain (p27^kip1) and the early hair cells (math1). Developmental Dynamics 239:1019–1026, 2010. © 2010 Wiley‐Liss, Inc.     

A radioautographic study of [3H]L-glutamate and [3H]L-glutamine uptake in the guinea-pig cochlea

Since glutamate has been recently proposed as a possible transmitter of the sensory hair cells in the cochlea, a radioautographic study was performed to look for the in vitro uptake of [³H] l-glutamate and [³H]l-glutamine. Several experimental conditions were applied. The control experimental procedures consisted in an incubation with one of the labelled tracers (10 min), followed by a post-incubation (3 × 10 min) without tracer. In these experiments, either with [³H]l-glutamate or [³H]l-glutamine, the following structures were labelled: inner hair cells, glial cells of the osseous spiral lamina and areas of the inner spiral and tunnel spiral bundles. When these experiments were carried out in absence of Na⁺, these labellings were strongly decreased. When the incubation was not followed by a post-incubation, the results differed depending on the tracer: with [³H]l-glutamate, the glial cells and the areas of inner spiral and tunnel spiral bundles were labelled, whereas with [³H]l-glutamine, mainly the inner hair cells were labelled. An addition of l-methionine-dl-sulfoximine, a glutamine synthetase inhibitor, into the incubation and post-incubation media, produced a decrease of the labelling of the inner hair cells and of the glial cells. An addition of unlabelled glutamine to the post-incubation media decreased the inner hair cell labelling, while a similar addition of unlabelled glutamate did not. In either case, neither the outer hair cells, the second type of sensory cells, nor the spiral ganglion neurons were labelled.These results suggest that in the cochlea, glutamate and glutamine have their metabolisms linked together, as in some parts of the central nervous system. Correlated to biochemical and electro physiological data these results support the hypothesis that glutamate could be the neurotransmitter of the inner hair cells.     

Immunohistochemical localization of a new thiamine diphosphate-binding protein in the rat nervous system

The neurophysiological roles of thiamine-binding proteins have lately attracted considerable attention. We purified a new thiamine diphosphate-binding protein (ThDP-BP) prepared from rat livers and produced an antiserum against it. We examined the immunohistochemical distribution of ThDP-BP in the rat nervous system by the avidin-biotin complex technique. Immunoreactivity for ThDP-BP was found in several groups of neurons, and other neuronal tissues including glial cells, ependymal cells and Schwann cells. In neurons, the cytoplasm and processes were stained, but the nucleus was scarcely stained. In glial cells, the nucleus was stained, but the cytoplasm was not stained.     

Immunohistochemical localization of peptidylarginine deiminase in the rat brain

The enzyme peptidylarginine deiminase is responsible for the post-translational modification of certain proteins by catalysing the deimination of arginine residues to citrullines. Recently, peptidylarginine deiminase has been purified from rat skeletal muscle and its primary structure determined by molecular cloning. We have used antibodies raised against this enzyme to examine its distribution in the rat central nervous system. A few discrete neuronal cell groups in the telencephalon were selectively stained. These were the septofimbrial nucleus, the anterior commissural nucleus of the hypothalamus, and the anterodorsal thalamic nucleus. The subcommissural organ was intensely stained; but other circumventricular organs were not. In addition, peptidylarginine deiminase-immunoreactivity was found in a large population of small cells throughout the grey and white matter of the rat brain. Some of these cells contained glial fibrillary acidic protein and could thus be identified as astrocytes. However, in many regions these peptidylarginine deiminase-positive cells appeared distinct from the glial fibrillary acidic protein-positive astrocytes. Light and electron microscopic examination indicated that these cells had the morphology of microglia, however they were not stained by a lectin marker for microglia. These cells often surrounded cerebral capillaries, and sent ramifying processes into the neuropil. Peptidylarginine deiminase has been suggested to be involved in the synthesis of the endothelium-derived relaxing factor nitric oxide from arginine-containing molecules. Thus the distinct peptidylarginine deiminase-positive glial cells surrounding cerebral blood vessels may be in a position to regulate local blood flow in response to neuronal activity.     

Immunohistochemical localization of secretogranin II in the rat cerebellum

Secretogranin II (chromogranin C) is a peptide related to chromogranin A and secretogranin I (chromogranin B) which is secreted by a regulated pathway from both neurons and endocrine cells. In the present study we have determined by light microscopic immunocytochemistry its distribution in the cerebellum and in adjacent brain stem regions. Secretogranin II was found to be widely distributed throughout the gray matter of these regions. Highly immunoreactive structures in the cerebellar cortex included the majority of climbing fibers, a large number of mossy fibers, sparse varicose fibers in the molecular layer and a subpopulation of neuronal perikarya in the granule cell layer. The location and shape of these neurons are very similar to those of a novel type of cerebellar neurons which has been recently described. A moderate level of immunoreactivity was observed on fibers travelling among Purkinje cells and parallel to the pial surface in the Purkinje cell layer. A variable, but in general low, degree of immunoreactivity was also detectable in the perikarya of Purkinje cells. In the deep cerebellar nuclei a loose network of secretogranin II-positive fibers was visible. Neurons of the nuclei, however, were non-immunoreactive. A dense network of highly immunoreactive fibers was found throughout the brain stem regions adjacent to the cerebellum. Our results indicate that secretogranin II has in the cerebellum and adjacent regions a distribution more widespread than that of known regulatory peptides and suggest that the peptide-mediated signaling in the cerebellum plays a role more important that has been acknowledged so far.