内耳免疫反应诱导Fas和FasL表达与凋亡的关系

目的研究内耳免疫反应过程中是否存在细胞凋亡,以及细胞凋亡是否与Fas和FasL信号转导有关.方法选用雌性白色豚鼠16只,随机分为实验组和对照组各8只,以钥孔虫戚血蓝蛋白(keyhole limpet hemocyanin,KLH)全身免疫后,实验组以相同抗原进行内耳免疫,对照组内耳注射等量的磷酸盐缓冲生理盐水(phosphate buffered saline,PBS),在内耳免疫5d后处死动物,取内耳免疫侧耳蜗做石蜡切片.通过脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术(terminal-deoxynucleotidyl transferase mediated nick end labeling,TUNEL)检测内耳凋亡细胞,免疫组化检测内耳Fas和FasL的表达.结果实验组豚鼠内耳Corti器毛细胞,血管纹的缘细胞和螺旋神经节细胞存在TUNEL染色阳性细胞,而对照组动物切片仅在支持细胞、血管纹和螺旋神经节细胞中发现极少数TUNEL染色阳性细胞.免疫组化染色实验组Corti器、螺旋神经节细胞、血管纹和螺旋韧带Fas和FasL蛋白表达阳性,而对照组只有螺旋神经节细胞和血管纹有较弱的Fas蛋白表达,FasL蛋白表达阴性.结论内耳免疫反应可诱导细胞凋亡的发生,Fas-FasL途径是参与此过程重要的信号转导途径之一.     

Expression of Growth Factors and Their Receptors in the Postnatal Rat Cochlea

RT-PCR was used to assay for growth factors and receptors from seven different protein families in cochlea tissues of the juvenile rat. There was a broad representation of the growth factor families in all the cochlea tissues examined, though the organ of Corti and stria vascularis expressed a greater variety than the spiral ganglion. This broad expression suggests that a variety of known growth factors play significant roles in the development, maintenance, and repair of the inner ear. The results of this survey serve as a basis for the design of future in vitro experiments that will address the ability of growth factors to protect hair cells from damage and to evoke a repair-regeneration response by injured hair cells.     

Involvement of calpain-I and microRNA34 in kanamycin-induced apoptosis of inner ear cells

Inner ear cells, including hair cells, spiral ganglion cells, stria vascularis cells and supporting cells on the basilar membrane, play a major role in transducing hearing signals and regulating inner ear homoeostasis. However, their functions are often damaged by antibiotic-induced ototoxicity. Apoptosis is probably involved in inner ear cell injury following aminoglycoside treatment. Calpain, a calcium-dependent protease, is essential for mediating and promoting cell death. We have therefore investigated the involvement of calpain in the molecular mechanism underlying ototoxicity induced by the antibiotic kanamycin in mice. Kanamycin (750 mg/kg) mainly induced cell death of cochlear cells, including stria vascularis cells, supporting cells and spiral ganglion cells, but not hair cells within the organ of Corti. Cell death due to apoptosis occurred in a time-dependent manner with concomitant up-regulation of calpain expression. Furthermore, the expression levels of two microRNAs, mir34a and mir34c, were altered in a dose-dependent manner in cochlear cells. These novel findings demonstrated the involvement of both calpain and microRNAs in antibiotic-induced ototoxicity.     

Enzyme-histochemical localization of carbonic anhydrase in the inner ear of the guinea pig and several improvements of the technique

We have made several improvements in the method of fixation of the inner ear and the enzyme-histochemical technique for carbonic anhydrase (CA) detection. The results confirmed that CA is localized in the hair cells of the organ of Corti, Deiters' cells or nerve endings, inner pillar cells, Boettcher's cells, stria vascularis, spiral ligament, spiral limbus, and spiral ganglion cells. These results generally agree with previous histochemical observations but showed some differences. Our method preserved tissue morphology and showed more detailed localization of CA activity in the inner ear. In particular, the marginal zone of stria vascularis and the epithelial cells of spiral prominence, facing the endolymph, showed no CA activity, while the suprastrial region of the spiral ligament and the supralimbal region of the spiral limbus, juxtaposed to the perilymph, showed CA activity. In outer hair cells, the cuticular plate, which faces the endolymph showed CA activity, but the lateral membrane, which faces the perilymph showed no CA activity. In contrast, the inner hair cell cytoplasm showed diffuse CA activity. These results will be useful in considering ion exchange between endolymph and its adjacent cells, and between perilymph and its adjacent structures.     

Expression and immunohistochemical localization of TMEM16A/anoctamin 1, a calcium-activated chloride channel in the mouse cochlea

Sound transduction in the cochlea depends on the unique high concentrations of K⁺ in the endolymph. The production and maintenance of high K⁺ concentrations are accompanied by Cl^- cycling. In this study, we report on an investigation of the expression and localization of TMEM16A/anoctamin 1 (ANO1), a recently cloned Ca²⁺-activated Cl^- channel, in the mouse cochlea by Western blot and immunhistochemistry. The ANO1 protein was identified in the cochlea by Western blotting. The immunoreactivity was found in stria vascularis as a line and in the organ of Corti as three plaques. The cellular localization of ANO1 was examined by means of double-labeling experiments with anti-claudin 11, a marker for basal cells of the stria vascularis. The results demonstrated that ANO1 colocalized with claudin 11, indicating its expression in basal cells. We also examined ANO1 localization in the organ of Corti by double- and triple-labeling techniques with anti-myosin VI, a marker for hair cells, and anti-synaptophysin, a marker for olivocochlear efferent nerve endings under hair cells. The results clearly showed that ANO1 is colocalized with synaptophysin, but not with myosin VI, indicating that ANO1 is localized at medial olivocochlear efferent nerve endings under outer hair cells. These results suggest that ANO1 may be specifically involved in synaptic transmission from medial olivocochlear efferent nerve endings to outer hair cells in the organ of Corti, as well as Cl^- cycling in basal cells of the stria vascularis.     

Expression and distribution of creatine transporter and creatine kinase (brain isoform) in developing and mature rat cochlear tissues

Physiological processes in the cochlea associated with sound transduction and maintenance of the unique electrochemical environment are metabolically demanding. Creatine maintains ATP homeostasis by providing high-energy phosphates for ATP regeneration which is catalyzed by creatine kinase (CK). Cellular uptake of creatine requires a specific high affinity sodium- and chloride-dependent creatine transporter (CRT). This study postulates that this CRT is developmentally regulated in the rat cochlea. CRT expression was measured by quantitative real-time RT-PCR and immunohistochemistry in the postnatal (P0–P14) and adult (P22–P56) rat cochlea. The maximum CRT expression was reached at the onset of hearing (P12), and this level was maintained through to adulthood. CRT immunoreactivity was strongest in the sensory inner hair cells, supporting cells and the spiral ganglion neurons. Cochlear distribution of the CK brain isoform (CKB) was also assessed by immunohistochemistry and compared with the distribution of CRT in the developing and adult cochlea. CKB was immunolocalized in the organ of Corti supporting cells, and the lateral wall tissues involved in K⁺ cycling, including stria vascularis and spiral ligament fibrocytes. Similar to CRT, CKB reached peak expression after the onset of hearing. Differential spatial and temporal expression of CRT and CK in cochlear tissues during development may reflect differential requirements for creatine–phosphocreatine buffering to replenish ATP consumed during energy-dependent metabolic processes, especially around the period when the cochlea becomes responsive to airborne sound.     

Localization of β1-Adrenergic Receptors in the Cochlea and the Vestibular Labyrinth

Sympathetic activation in a “fight or flight reaction” may put the sensory systems for hearing and balance into a state of heightened alert via β₁-adrenergic receptors (β₁-AR). The aim of the present study was to localize β₁-AR in the gerbil inner ear by confocal immunocytochemistry, to characterize β₁-AR by Western immunoblots, and to identify β₁-AR pharmacologically by measurements of cAMP production. Staining for β₁-AR was found in strial marginal cells, inner and outer hair cells, outer sulcus, and spiral ganglia cells of the cochlea, as well as in dark, transitional and supporting cells of the vestibular labyrinth. Receptors were characterized in microdissected inner ear tissue fractions as 55 kDa non-glycosylated species and as 160 kDa high-mannose-glycosylated complexes. Pharmacological studies using isoproterenol, ICI-118551 and CGP-20712A demonstrated β₁-AR as the predominant adrenergic receptor in stria vascularis and organ of Corti. In conclusion, β₁-AR are present and functional in inner ear epithelial cells that are involved in K⁺ cycling and auditory transduction, as well as in neuronal cells that are involved in auditory transmission.     

Cellular distribution of myosin-V in the guinea pig cochlea

The importance of unconventional myosins to hearing has recently been revealed by the identification of myosins-VI and -VII as the defective genes in mouse mutations and in a human syndrome which lead to profound hearing loss. Another class of novel myosins (V) has been implicated in the trafficking of intracellular vesicles in neurons and other secretory cells. We used affinity-purified antibodies to determine the localization of myosin-V in the guinea pig inner ear. In the sensory epithelium of the cochlea, myosin-V epitopes were recognized in neuronal and supporting cells. Neuronal labelling was most intense in the afferent innervation of inner and outer hair cells. Supporting cells labelled were cells of Hensen and Deiters, and inner border, inner phalangeal, inner sulcus and interdental cells. In the vascular tissue of the cochlea, we observed staining of intermediate cells of the stria vascularis and of border cells between the stria and the spiral prominence. Staining of afferent chalice nerve endings was observed on type I vestibular hair cells. The results suggest that, like myosins VI and VII, myosin-V is localized in positions that may be critical to auditory function.     

Murine CMV-Induced Hearing Loss Is Associated with Inner Ear Inflammation and Loss of Spiral Ganglia Neurons

Congenital human cytomegalovirus (HCMV) occurs in 0.5–1% of live births and approximately 10% of infected infants develop hearing loss. The mechanism(s) of hearing loss remain unknown. We developed a murine model of CMV induced hearing loss in which murine cytomegalovirus (MCMV) infection of newborn mice leads to hematogenous spread of virus to the inner ear, induction of inflammatory responses, and hearing loss. Characteristics of the hearing loss described in infants with congenital HCMV infection were observed including, delayed onset, progressive hearing loss, and unilateral hearing loss in this model and, these characteristics were viral inoculum dependent. Viral antigens were present in the inner ear as were CD3+ mononuclear cells in the spiral ganglion and stria vascularis. Spiral ganglion neuron density was decreased after infection, thus providing a mechanism for hearing loss. The lack of significant inner ear histopathology and persistence of inflammation in cochlea of mice with hearing loss raised the possibility that inflammation was a major component of the mechanism(s) of hearing loss in MCMV infected mice.     

Developmental regulation of TRPC3 ion channel expression in the mouse cochlea

Canonical transient receptor potential type 3 (TRPC3) ion channels assemble from TRPC3 subunits and exhibit multiple activation mechanisms. TRPC3 has been proposed to contribute to Ca²⁺ entry supporting Ca²⁺ homeostasis in cochlear hair cells and to be activated by G protein-coupled receptor (GPCR) signaling in spiral ganglion neurons. The present study was designed to determine the spatiotemporal profile of TRPC3 expression during mouse cochlear ontogeny. TRPC3 immunofluorescence of cryosectioned cochleae was performed using E16–adult tissue. We found that prior to birth, TRPC3 expression was strongest in epithelial cells that form the cochlear partition. In the early postnatal period, to the onset of hearing (~P12), immunofluorescence was strongest in the hair cells, with increased expression in stria vascularis and Reissner’s membrane. Afferent neurite labeling in inner spiral plexus and outer spiral bundles developed transiently in the perinatal period, corresponding to the critical period of synaptic consolidation, while signal in the spiral ganglion soma increased from the perinatal period through to adulthood. Compared with the late embryonic/early postnatal levels, hair cell expression was relatively weaker from the third postnatal week, whereas spiral ganglion soma labeling was stronger. In the adult, TRPC3 expression was primarily in the soma of spiral ganglion neurons, the hair cells, and the inner and outer sulcus regions. This spatiotemporal profile of TRPC3 expression was consistent with this ion channel contributing to development of sensory, neural and epithelial cochlear tissues, as well as hair cell Ca²⁺ homeostasis and regulation of auditory neurotransmission via GPCR signaling.     

Reinnervation of hair cells by auditory neurons after selective removal of spiral ganglion neurons

Hearing loss can be caused by primary degeneration of spiral ganglion neurons or by secondary degeneration of these neurons after hair cell loss. The replacement of auditory neurons would be an important step in any attempt to restore auditory function in patients with damaged inner ear neurons or hair cells. Application of beta-bungarotoxin, a toxin derived from snake venom, to an explant of the cochlea eradicates spiral ganglion neurons while sparing the other cochlear cell types. The toxin was found to bind to the neurons and to cause apoptotic cell death without affecting hair cells or other inner ear cell types as indicated by TUNEL staining, and, thus, the toxin provides a highly specific means of deafferentation of hair cells. We therefore used the denervated organ of Corti for the study of neuronal regeneration and synaptogenesis with hair cells and found that spiral ganglion neurons obtained from the cochlea of an untreated newborn mouse reinnervated hair cells in the toxin-treated organ of Corti and expressed synaptic vesicle markers at points of contact with hair cells. These findings suggest that it may be possible to replace degenerated neurons by grafting new cells into the organ of Corti.     

NTPDase1 and NTPDase2 immunolocalization in mouse cochlea: implications for regulation of p2 receptor signaling.

Cellular, molecular, and physiological studies have demonstrated an important signaling role for ATP and related nucleotides acting via P2 receptors in the cochlea of the inner ear. Signal modulation is facilitated by ectonucleotidases, a heterologous family of surface-located enzymes involved in extracellular nucleotide hydrolysis. Our previous studies have implicated CD39/NTPDase1 and CD39L1/NTPDase2, members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, as major ATP-hydrolyzing enzymes in the tissues lining the cochlear endolymphatic and perilymphatic compartments. NTPDase1 hydrolyzes both nucleoside triphosphates and diphosphates. In contrast, NTPDase2 is a preferential nucleoside triphosphatase. This study characterizes expression of these E-NTPDases in the mouse cochlea by immunohistochemistry. NTPDase1 can be immunolocalized to the cochlear vasculature and neural tissues (primary auditory neurons in the spiral ganglion). In contrast, NTPDase2 immunolabeling was principally localized to synaptic regions of the sensory inner and outer hair cells, stereocilia and cuticular plates of the outer hair cells, supporting cells of the organ of Corti (Deiters' cells and inner border cells), efferent nerve fibers located in the intraganglionic spiral bundle, and in the outer sulcus and root region of the spiral ligament. This differential expression of NTPDase1 and 2 in the cochlea suggests spatial regulation of P2 receptor signaling, potentially involving different nucleotide species and hydrolysis kinetics.     

Protein expression of pigment-epithelium-derived factor in rat cochlea

Pigment-epithelium-derived factor (PEDF) is a 50-kDa glycoprotein with well-recognised expression in various mammalian organs showing diverse (e.g. anti-angiogenic and neuroprotective) activities. However, at present, no information is available regarding the potential function of this cytokine in the inner ear. As a first approach to investigating whether PEDF is involved in cochlear function, we have explored its protein expression in the rat cochlea by immunocytochemistry. Our results show that PEDF expression in the cochlea is most prominent in the basilar membrane below the organ of Corti, in the lateral wall (especially in the stria vascularis), in ganglion neurons, and in the endothelia of blood vessels. Our findings on its distribution in the cochlea suggest that PEDF in the basilar membrane prevents blood vessel formation that would disturb cochlear micromechanics and would interfere with the mechano-electrical transduction in the organ of Corti. In cochlear ganglion neurons, PEDF might serve a neuroprotective function possibly protecting these neurons from excessive glutamate released by the inner hair cells. Our data constitute the first report on the morphological protein distribution of this multifunctional molecule in the rat cochlea and suggest its role in important functions of the internal ear. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Purinergic signaling in cochleovestibular hair cells and afferent neurons

Purinergic signaling in the mammalian cochleovestibular hair cells and afferent neurons is reviewed. The scope includes P2 and P1 receptors in the inner hair cells (IHCs) of the cochlea, the type I spiral ganglion neurons (SGNs) that convey auditory signals from IHCs, the vestibular hair cells (VHCs) in the vestibular end organs (macula in the otolith organs and crista in the semicircular canals), and the vestibular ganglion neurons (VGNs) that transmit postural and rotatory information from VHCs. Various subtypes of P2X ionotropic receptors are expressed in IHCs as well as P2Y metabotropic receptors that mobilize intracellular calcium. Their functional roles still remain speculative, but adenosine 5′-triphosphate (ATP) could regulate the spontaneous activity of the hair cells during development and the receptor potentials of mature hair cells during sound stimulation. In SGNs, P2Y metabotropic receptors activate a nonspecific cation conductance that is permeable to large cations as NMDG⁺ and TEA⁺. Remarkably, this depolarizing nonspecific conductance in SGNs can also be activated by other metabotropic processes evoked by acetylcholine and tachykinin. The molecular nature and the role of this depolarizing channel are unknown, but its electrophysiological properties suggest that it could lie within the transient receptor potential channel family and could regulate the firing properties of the afferent neurons. Studies on the vestibular partition (VHC and VGN) are sparse but have also shown the expression of P2X and P2Y receptors. There is still little evidence of functional P1 (adenosine) receptors in the afferent system of the inner ear.     

Pax3 function is required specifically for inner ear structures with melanogenic fates

Pax3 mutations result in malformed inner ears in Splotch mutant mice and hearing loss in humans with Waardenburg’s syndrome type I. In the inner ear, Pax3 is thought to be involved mainly in the development of neural crest. However, recent studies have shown that Pax3-expressing cells contribute extensively to multiple inner ear structures, some of which were considered to be derived from the otic epithelium. To examine the specific functions of Pax3 during inner ear development, fate mapping of Pax3 lineage was performed in the presence or absence of functional Pax3 proteins using Pax3^Cre knock-in mice bred to Rosa26 reporter (R26R) line. β-gal-positive cells were widely distributed in Pax3^Cre/+; R26R inner ears at embryonic day (E) 15.5, including the endolymphatic duct, common crus, cristae, maculae, cochleovestibular ganglion, and stria vascularis. In the absence of Pax3 in Pax3^Cre/Cre; R26R inner ears, β-gal-positive cells disappeared from regions with melanocytes such as the stria vascularis of the cochlea and dark cells in the vestibule. Consistently, the expression of Dct, a melanoblast marker, was also absent in the mutant inner ears. However, when examined at E11.5, β-gal positive cells were present in Pax3^Cre/Cre mutant otocysts, whereas Dct expression was absent, suggesting that Pax3 lineage with a melanogenic fate migrated to the inner ear, yet failed to differentiate and survive without Pax3 function. Gross inner ear morphology was generally normal in Pax3^Cre/Cre mutants, unless neural tube defects extended to the cranial region. Taken together, these results suggest that despite the extensive contribution of Pax3-expressing cells to multiple inner ear tissues, Pax3 function is required specifically for inner ear components with melanogenic fates.     

Deafness and stria vascularis defects in S1P2 receptor-null mice

The S1P(2) receptor is a member of a family of G protein-coupled receptors that bind the extracellular sphingolipid metabolite sphingosine 1-phosphate with high affinity. The receptor is widely expressed and linked to multiple G protein signaling pathways, but its physiological function has remained elusive. Here we have demonstrated that S1P(2) receptor expression is essential for proper functioning of the auditory and vestibular systems. Auditory brainstem response analysis revealed that S1P(2) receptor-null mice were deaf by one month of age. These null mice exhibited multiple inner ear pathologies. However, some of the earliest cellular lesions in the cochlea were found within the stria vascularis, a barrier epithelium containing the primary vasculature of the inner ear. Between 2 and 4 weeks after birth, the basal and marginal epithelial cell barriers and the capillary bed within the stria vascularis of the S1P(2) receptor-null mice showed markedly disturbed structures. JTE013, an S1P(2) receptor-specific antagonist, blocked the S1P-induced vasoconstriction of the spiral modiolar artery, which supplies blood directly to the stria vascularis and protects its capillary bed from high perfusion pressure. Vascular disturbance within the stria vascularis is a potential mechanism that leads to deafness in the S1P(2) receptor-null mice.