非聚焦超声对豚鼠耳蜗毛细胞的影响

为探讨超声（Ultrasound，FU）对豚鼠耳蜗毛细胞的影响，分别以2．5MHz、8MHz非聚焦超声（NFU）照射豚鼠耳蜗3h、6h，于30min后行耳蜗毛细胞组织学及酶组织化学观察。发现照射6h组在基底膜第2周起始段0．87±0．20mm节段及基底周起始段0．80±0．20mm节段，耳蜗毛细胞琥珀酸脱氢酶（SDH）活性降低，乳酸脱氢酶（LDH）活性升高。提示：2．5MHz、8MHzNFU照射豚鼠耳蜗达一定剂量能引起不同部位耳蜗毛细胞有氧代谢改变。     

Immunocytochemical detection of peptides in the guinea pig cochlea

The cochleae of juvenile guinea pigs were investigated for the presence of several neuropeptides. Glucagon, insulin, CCK and beta-endorphin immunoreactive neurons and nerve fibers as well as hair cells were demonstrated by the peroxidase antiperoxidase technique. Small amounts of substance P were also found in different sites in the inner ear. In contrast, prolactin-like material could not be found at all. These findings have significance with regard to the putative role of neuropeptides in neuromodulation.     

Use of scanning electron microscopy in the cochlea of guinea pigs

IntroductionThe use of electron microscopy in the study of the inner ear has allowed us to observe minute details of the hair cells, especially in ototoxicity studies; however, the preparation of this material is a difficult and delicate task. In an attempt to simplify the handling of these materials, two agents, toluidine blue and ethylenediamine tetra-acetic acid were tested, in addition to the elimination of osmium tetroxide during the preparation of albino guinea pig cochleae. We also tested the applicability of these methodologies in an ototoxicity protocol.ObjectiveTo verify the quality of the images obtained with and without the use of ethylenediamine tetra-acetic acid, toluidine blue and osmium tetroxide in the preparation of cochleae of albino guinea pigs for the scanning electron microscopy.MethodsThree groups of cochleae were used. In Group 1, 10 cochleae were prepared with the usual methodology, dissecting the optical capsule without decalcification and using osmium tetroxide as a post-fixative agent. In Group 2, we prepared 10 cochleae decalcified with ethylenediamine tetra-acetic acid, injecting toluidine blue in the endolymphatic space to facilitate the identification of the organ of Corti. In Group 3, we used 4 cochleae of guinea pigs that received 3 doses of cisplatin (7.5 mg/kg, D1–D5–D6), two prepared according to the methodology used in Group 1 and two with that used in Group 2. Scanning electron microscopy images were obtained from the organ of Corti region of the basal turn of each cochlea.ResultsThe organ of Corti was more easily identified with the use of toluidine blue. The dissection of the cochlea was more accurate in the decalcified cochleae. The quality of the images and the preservation of the organ of Corti obtained with the two methodologies were similar.ConclusionThe proposed modifications resulted in images of similar quality as those observed using the traditional methodology.     

Immunocytochemical detection of peptides in the guinea pig cochlea

Summary The cochleae of juvenile guinea pigs were investigated for the presence of several neuropeptides. Glucagon, insulin, CCK and -endorphin immunoreactive neurons and nerve fibers as well as hair cells were demonstrated by the peroxidase antiperoxidase technique. Small amounts of substance P were also found in different sites in the inner ear. In contrast, prolactin-like material could not be found at all. These findings have significance with regard to the putative role of neuropeptides in neuromodulation.     

Immunohistochemical detection of vascular endothelial growth factor (VEGF) and VEGF receptors Flt-1 and KDR/Flk-1 in the cochlea of guinea pigs

Vascular endothelial growth factor (VEGF) is known as an endothelial cell-specific mitogen. There are no reports concerning the presence of VEGF in the inner ear. To gain information, immunohistochemical analysis using specific antibodies to VEGF and to both known VEGF receptors Flt-1 and KDR/Flk-1 was performed on paraffin-sectioned temporal bones from five guinea pigs. Immunoreactivity of VEGF, Flt-1 and KDR/Flk-1 was detectable in spiral ganglion cells. VEGF could also be found in the endothelium of blood vessels, in the spiral ligament and in the organ of Corti. Flt-1 was found in the limbus epithelium, in all supporting cells of the organ of Corti, in Claudius cells, cells of the sulcus and in the spiral ligament. Flk-1 could be detected in some supporting cells of the organ of Corti (inner pillar cells and Deiters' cells). Immunoreactivity to Flk-1 was also found in endothelium of blood vessels and in the spiral ligament. Hair cells showed VEGF immunostaining, but did not contain staining to Flt-1 nor Flk-1. In the stria vascularis any immunoreactivity to all used VEGF and VEGF receptor antibodies could not be detected. The findings were supported by Western blot analysis on inner ear tissues and ovaries from guinea pigs. We may conclude that the growth factor VEGF and both receptors participate in cochlear physiology.     

豚鼠耳蜗毛细胞暴露非聚焦超声后的酶组织化学观察

目的　探讨非聚焦超声 (non focusedultrasound ,NFU)对耳蜗毛细胞的影响。方法　以A型超声波诊断仪为超声发射器 ,分别以 2 5MHz、8MHz照射各 15耳豚鼠耳蜗 6h后 30min和 8h行耳蜗基底膜毛细胞铺片及冰冻切片观察琥珀酸脱氢酶 (succinatedehydrogenase ,SDH)组织化学变化。结果　 2 5MHz、8MHzNFU照射豚鼠耳蜗 6h后 8h能引起不同节段基底膜毛细胞SDH酶活性降低 ,其中外毛细胞较内毛细胞SDH酶活性降低更甚。照射后 8h组较照射后 30min组SDH酶活性有明显恢复。结论不同频率NFU超声照射耳蜗达一定剂量可以引起不同部位基底膜毛细胞的病理性缺氧改变 ,而这种缺氧病理改变在一定暴露剂量下是可逆或部分可逆的。提示耳蜗毛细胞可能与超声感受有关 ,其中外毛细胞可能起更为重要的作用 ,且不同部位毛细胞与超声感受的频率选择性可能有一定关系     

Localization of the NO/cGMP-pathway in the cochlea of guinea pigs.

The presence of nitric oxide synthase (NOS) in substructures of the cochlea of guinea pigs is an issue of current focus. Moreover, information concerning the localization of cells effected by the NO/cGMP-pathway are rare. Paraffin sections of guinea pig cochlea were incubated with specific antibodies to the three known NOS isoforms, soluble guanylyl cyclase (sGC) and cyclic guanosine-monophosphate (cGMP), the second messenger system of NO. While detection of inducible iNOS failed in all cochlear structures, expression of endothelial eNOS was found in the spiral ligament, in the stria vascularis, in cells of the organ of Corti, in nerve fibers and in some perikaryia of the spiral ganglion. The cochlear nerve showed an accentuated affinity for immunostaining in distal, basal segments of the cochlea. Neuronal bNOS was found predominantly in the endosteum of the modiolus and cochlea and was less intensively present in all perikaryia of the spiral ganglion and in the spiral ligament. Supporting cells of the organ of Corti and cells in the limbus spiralis displayed only modest immunostaining, while bNOS was not found in outer and inner hair cells. NOS detection was accompanied by immunoreactivity to sGC and to cGMP. The presence of NOS and its second messenger system gives evidence for a possible involvement in neurotransmission, regulation of the cochlear amplifier and in homeostasis.     

血管内皮舒张因子对豚鼠耳蜗功能的影响

目的 探讨血管内皮性一氧化氮合酶(endothelial nitric oxide synthase,eNOS)对耳蜗电位的影响.方法 健康豚鼠100只,随机等分为10组:①人工外淋巴液组;②L-精氨酸组;③Ca2+-ATP酶抑制剂组;④Ca2+-ATP酶抑制剂+L-精氨酸组;⑤Ca2+-ATP酶抑制剂+环磷酸鸟苷(c...     

豚鼠耳蜗毛细胞暴露非聚焦超声后的酶组织化学观察

目的 探讨非聚焦超声（non-focused ultrasound,NFU)对耳蜗毛细胞的影响。方法 以A型超声波诊断仪为超声发射器，分别以2.5MHz、8MHz照射各15耳豚鼠耳蜗6h后30min和8h行耳蜗基底膜毛细胞铺片及冰冻切片观察琥珀酸脱氢酶（succinate dehydrogenase,SDH)组织化学变化。结果 2.5MHz、8MHz NFU照射豚鼠耳蜗6h后8h能引起不同节段基底膜毛细胞SDH酶活性降低，其中外毛细胞较内毛细胞SDH酶活性降低更甚。照射后8h组较照射后30min组SDH酶活性有明显恢复。结论 不同频率NFU超声照射耳蜗达一定剂量可以引起不同部位基底膜毛细胞的病理性缺氧改变，而这种缺氧病理改变在一定暴露剂量下是可逆或部分可逆的。提示耳蜗毛细胞可能与超声感受有关，其中外毛细胞可能起更为重要的作用，且不同部位毛细胞与超声感受的频率选择性可能有一定关系。     

Histological and electrophysiological study of cryosurgery of the cochlea of guinea pigs

For studies on cochlear function after inner ear trauma, cryosurgery gives reproductive morphological changes that may help the understanding and the correlation of hair cell loss with cochlear physiology. In all cases of partial destruction of the organ of Corti we noticed a completely reversible limitation of hearing, whereas when injuring the organ of Corti in the whole cochlea a restriction of function persisted. We could never induce complete deafness.     

豚鼠耳蜗底回近末端局部微循环障碍的研究

目的 :探讨豚鼠耳蜗底回接近末端局部微循环障碍的听力损伤特点 ,建立以高频损伤为主的听力损伤模型。方法 :采用光化学法诱导豚鼠耳蜗底回接近末端 1/ 2段微循环障碍 ;常规火棉胶切片观察耳蜗形态学变化 ;Madsen2 2 5 0诱发电位系统记录各频率的耳蜗神经复合动作电位 (CAP)。结果 :各频率的 CAP N1 潜伏期、CAP阈移、CAP N1 振幅变化 ,提示其听力损伤以高频较重 ,组织病理学变化表明耳蜗形态学改变主要局限于耳蜗底回接近末端 1/ 2段。结论 :耳蜗底回接近末端局部微循环障碍可以导致高频范围为主的听力损伤。     

Ultrastructural changes of the cochlea in guinea pigs with acute bacterial otitis media

Acute bacterial otitis media was induced in guinea pigs with H. influenzae to investigate the ultrastructural changes of the organ of Corti. The results showed that the stereocilia in the organ of Corti adjacent to the round window was disoriented and partially disappeared, there was an increase in the number of lysosomes and marked aggregation of smooth endoplasmic reticula, the surface of the supporting cells was damaged and broken. The most prominent damages were observed on specimens 7 days following the middle ear inoculation with H. influenzae. The damages to the supporting cells were repaired 21 days after the inoculation though the damaged stereocilia did not show any sign of recovery.     

Immunohistochemical localization of unique enkephalin sequences contained in preproenkephalin A in the guinea pig cochlea

The guinea pig cochlea was studied for the presence of immunoreactivities to the four unique enkephalin sequences contained in the preproenkephalin A. The antisera to Met-enkephalin-Arg6-Phe7 and Met-enkephalin-Arg6-Gly7-Leu8 were used as highly specific markers for the preproenkephalin A. Contrary to Met-enkephalin and Leu-enkephalin, also included in the present study, these two peptide sequences are not contained in pro-opiomelanocortin or preproenkephalin B (prodynorphin). All the four different antisera showed identical localization for the four peptide sequences, suggesting their coexistence in the same nervous pathway. Specific immunofluorescence was found in intraganglionic spiral bundle, inner spiral bundle, tunnel spiral bundle and in association with inner hair cells. The nerve fibers were thin and varicose, suggesting that most, if not all, of them were unmyelinated. Their localization indicates that the unique enkephalin sequences contained in preproenkephalin A are present in the cochlear efferent pathway to the inner hair cell region.     

Acetylcholinesterase in the cochlea on normal guinea pigs by light and electron microscopy

The distribution of AchE activity in the cochleae of 42 normal guinea pigs was studied with thiocholine Cu-ferrocyanide method. Light microscopy revealed the AchE activity in the following areas: 1. the inner spiral bundle, 2. the spiral tunnel bundle, 3. the upper tunnel radial fibers, and 4. the large nerve endings at the base of the outer hair cells. Activity of AchE was most abundant in the basal turn and decreased gradually towards the apex. There was also a decreasing AchE from the first to the third row of outer hair cells. In the fourth turn, AchE activity was demonstrated only in the large nerve endings under the first row of outer hair cells. Under electron microscopy, the reaction was found to be localized to the following structures: 1. the axolemma of the small efferent fibers and their presynaptic enlargements in the inner spiral bundle, 2. the outer surface of the upper tunnel radial fibers and the spiral tunnel bundle, and 3. the large efferent nerve endings in contact with the base of the outer hair cells.     

Determination of the perilymph density in the cochlea of guinea pigs (author's transl)

The perilymph density of the sc. tympani, sc. vestibuli and the density of liquor cerebrospinalis in guinea-pigs are measured by determination from mass and volume of the fluid column in a glass capillary tube. For the density of perilymph in sc. vest. a value of (formula: see text) in sc. tymp. a value of (formula: see text), and for liquor a value of (formula: see text) was obtained.