Symposium: new data for noise standards. 3. Organ culture of the mammalian inner ear: a tool to study inner ear deafness

Twelfth gestation day kreisler otocysts were explanted into an organ culture system and allowed to develop for nine days. The homozygotic (kr/kr) kreisler otocysts showed significant developmental differences when compared to the development that occurred in the organ culture specimens of the otocysts of its heterozygotic (+ /kr) litter mates. The differences in development observed in vitro were the same major developmental differences that had been observed in vivo. The phenotypic expression of the kreisler genome has expressed itself in vitro in the homozygotic kreisler otocyst.     

Biochemical studies on the embryonic development of the mammalian inner ear in organ culture

Summary Adenylate cyclase activity and phospholipid labeling were compared during embryonic development of the mouse inner ear in vivo and in vitro. Inner ears were explanted on the 16th gestational day and cultured in vitro for 3–12 days. The gestation time in vivo is 21 days.During the 1st week in vitro there is very little growth of the inner ear with regard to total protein content. In contrast, the labyrinth increases its protein content threefold during the corresponding period of time in vivo.The activity of adenylate cyclase develops parallel in vivo and in vitro until the 19th gestational day whereafter the specific activity of the enzyme in vitro surpasses that of the enzyme in vivo three- to fivefold suggesting a lack of control mechanisms in organ culture. Phospholipids are labeled by ³²P in an essentially similar quantitative relationship in vivo and in vitro, while some quantitative differences exist.According to the present study the usefulness of the organ culture for the investigation of inner ear development appears limited to a culture period corresponding to an age prior to birth.Presented at the 17th Workshop on Inner Ear Biology in Stockholm, June 23–25, 1980     

Organ culture system for inner ears

This paper reports an organ culture system using 2-hydroxyethylmethacrylate (HEMA) hydrogel as a substrate to study the development of otocysts and statoacoustic ganglions of the mouse embryos in vitro. Twelfth and thirteenth gestation-day otocysts and statoacoustic ganglions with otic sensory epithelium and/or rhombencephalon developed well on the HEMA hydrogel. Normal morphogenesis of the inner ears and cytodifferentiation of their sensory epithelia were noted. Neurons of the statoacoustic ganglions differentiated well with outgrowth of nerve fibers. The new organ culture system can be used for cultivating otocysts extracorporeally, facilitating investigation of the effects of various biologically active extracellular proteins, glycoproteins and glycosaminoglycans on statoacoustic ganglion-target tissue explants.     

Development of endolymph during maturation of the mammalian inner ear

Summary The development of the elemental composition in the endolymphatic space was investigated during embryologic and early post natal maturation of the CBA/CBA mouse. At birth the elemental distribution was similar in the endo- and perilymphatic spaces. Mature composition of endolymph was reached 6–8 days post partum. The maturation of endolymph corresponded well in time with the morphological maturation of the stria vascularis.Supported by grants from the Swedish Medical Research Council (12X-720), The Swedisch Society of Medical Sciences and funds from Tysta Skolan     

Surgery of the inner ear with hearing preservation: serial histological changes

OBJECTIVES/HYPOTHESIS: Surgery of the inner ear can result in hearing preservation under certain conditions, but the mechanisms responsible for hearing preservation or loss are not well understood. The specific aim of the study is to examine histological sections obtained at different time intervals after varying degrees of surgical entry into the inner ear, to understand how the cochlea is protected. The hypothesis is that internal partitioning occurs. STUDY DESIGN: Histologic examination of guinea pig inner ears by light microscopy. METHODS: Guinea pigs underwent lateral semicircular canal transection and plugging, ampullectomy, or vestibulotomy, and tone-burst auditory brainstem response thresholds at 2, 8, and 24 kHz were measured at intervals before and after surgery. Animals were killed after 1, 3, 7, or 21 or more days, and temporal bones were examined histologically. RESULTS: The histological response to surgical trauma consists of fibrosis and varying amounts of inflammation near the site of surgical entry. Cochlear hair cells are nearly always preserved, even when hearing loss occurs. Extension of the inflammatory response to the cochlea is associated with greater degrees of hearing loss. CONCLUSION: The guinea pig inner ear is capable of withstanding surgical trauma to the semicircular canals and vestibule without complete loss of cochlear function. Fibrosis creates an effective partition between the site of surgical entry and the rest of the inner ear. Cochlear preservation might be enhanced if the inflammatory response can be contained.     

Gene transfer into the mammalian inner ear using HSV-1 and vaccinia virus vectors.

The introduction of foreign genes into cells has become an effective means of achieving intracellular expression of foreign proteins, both for therapeutic purposes and for experimental manipulation. Gene delivery to the nervous system has been extensively studied, primarily using viral vectors. However, to date less work has focused on gene delivery to the inner ear, and existing studies have primarily used adenovirus and adeno-associated virus. Using two recombinant viral vectors, herpes simplex type 1 (HSV-1), and vaccinia virus, bearing the Escherichia coli lacZ gene, we tested gene delivery to the guinea pig cochlea in vivo with beta-galactosidase staining as an assay. The HSV-1 and vaccinia virus vectors were both found to infect and elicit transgene expression successfully in many cells in the guinea pig cochlea, including cells in the organ of Corti. These data demonstrate the feasibility of gene delivery to the inner ear using these two viral vectors. Such techniques may facilitate study of the auditory systems, and might be used to develop gene therapy strategies for some forms of hearing loss.     

RNA干扰载体活体内耳转染的可行性

目的：初步探讨RNAi技术应用于活体动物内耳的可行性。方法：实验分为2组，左耳为手术耳，右耳为内部对照耳。手术中将阳离子聚合物混合的RNAi质粒载体通过手术于圆窗注入，测量手术前及术后1、3、7d载体表达及畸变产物耳声发射和听性脑干反应以判别载体的表达特点及对听力的影响。结果：载体在内耳术后存在表达，对听力的影响为一过性。结论：RNAi质粒载体在内耳可以无害表达，将来可以应用于负性调节基因治疗。     

Normal anatomy of the inner ear.

The three parts of the inner ear have been reviewed: the membranous (endolymph containing) labyrinth surrounded by the osseous (perilymph containing) labyrinth, and the otic capsule of bone that encases the osseous labyrinth. This is a brief survey of the normal anatomy, but one must always remember that the hallmark of the temporal bone is variation.     

Immune-mediated inner ear disease.

Recent clinical studies, experimental research, and various testing techniques in otoimmunology have resulted in presentation of the synonymous terms autoimmune sensorineural hearing loss, autoimmune inner ear disease, immune-mediated sensorineural hearing loss, and immune-mediated inner ear disease or disorder. The development of this terminology and the clinical presentation of this disease as well as whether it is really a distinct clinical entity are discussed. Laboratory tests for immune-mediated inner ear disease are presented along with a discussion of progress being made in the growing field of otoimmunology.     

Type 1 vanilloid receptor expression by mammalian inner ear ganglion cells

The type 1 vanilloid receptor (VR1) is a non-specific cation channel activated by capsaicin, lipoxygenase (LOX) products, heat and acid. This study demonstrates VR1 and 5-LOX expression by inner ear ganglion cells. A PCR product (210 bp) was amplified from both oligo(dT)- and random primer-generated cDNAs of rat spiral ganglion cells using VR1 gene-specific primers constructed from the 3' non-homologous region. This PCR product shared 100% sequence homology to a rat VR1 cDNA (GenBank accession no. AF029310) and a rat vanilloid receptor splice variant mRNA (GenBank accession no. AF158248). Frozen sections of PLP-fixed, decalcified Long-Evans rat temporal bones were stained immunohistochemically for VR1. Neurons and satellite cells in both the vestibular and spiral ganglia were VR1-immunopositive. Neurons and supporting cells in adjacent sections of these ganglia were immunopositive for 5-LOX. These findings raise the hypothesis that activation of VR1 by endogenous ligands may contribute to hypersensitivity of the eighth nerve to hair cell inputs in a variety of pathologic conditions, such as tinnitus, Meniere's disease and migraine. In particular, these data suggest that LOX activation during inflammatory processes or during cyclo-oxygenase inhibition (e.g. by aspirin) is a potential intrinsic source of VR1 activation in inner ear ganglia.     

Homing of lymphocytes to the inner ear.

The migration of lymphocytes to the inner ear was studied during an immune response in the cochlea. Sensitized lymphocytes from peripheral blood, neck lymph nodes and spleen from strain 13 inbred guinea pigs were labelled with 51Cr and injected intravenously into strain 13 recipients undergoing an inner ear immune response. Eighteen hours later the temporal bones and immune organs of the recipients were assayed for radioactivity to detect the infiltration of labelled cells. In addition autoradiography was performed to localize labelled cells in the inner ear. More lymphocytes from the peripheral blood entered the inner ear during the immune response than spleen or lymph node cells. This indicates that the inner ear comes under the immuno-surveillance of the peripheral circulation in response to antigenic stimulation. Most labelled lymphocytes were observed in the basal turn of the scala tympani and in and around the spiral modiolar vein of the challenged cochlea. A few cells were seen also in the control cochleas but almost all where inside the blood vessels. This pattern suggests that the blood vessels of the spiral modiolar vein are the initial site through which lymphocytes entered the inner ear.     

In vitro isolation and cell culture of vestibular inner ear melanocytes

INTRODUCTION: Melanocytes of the membranous labyrinth of the inner ear have been described morphologically in various contexts. Nature and functions of these cells are as yet not completely clear, even though several hypotheses exist regarding the same. The limited knowledge is due in part to a lack of methods regarding in vitro cell culture. The aim of this study was to describe conditions for the successful cell culture of vestibular inner ear melanocytes (VIEM), to compare their growth properties with those of epidermal melanocytes, and to characterize them immunohistochemically. MATERIALS AND METHODS: Membranous labyrinth cells from freshly slaughtered sheep were isolated, and melanocytes and fibroblasts subsequently cultured. In addition, melanocytes from the skin of the same sheep were cultured. Antibodies specific to tyrosinase, tyrosinase-related protein 1 (TRP-1/Mel-5), and melanoma-specific antigen A (Melan A) were used to analyze the cultured cells. RESULTS: The proliferation of VIEM was retarded in comparison to epidermal melanocytes. After 14 days, VIEM began to proliferate for the first time, whereas epidermal melanocytes proliferated already after 7 days. In contrast to epidermal melanocytes, the culturing process of VIEM seemed to be dependent on the presence of fibroblasts, and VIEM often accumulated in the vicinity of fibroblasts forming three-dimensional clusters. Moreover, VIEM showed a higher ratio of highly pigmented cells with a round cell shape and small dendrites in comparison to epidermal melanocytes. Immunohistochemical techniques proved the VIEM to be positive for Melan A, TRP-1 and, in the majority of cases, also for tyrosinase. CONCLUSION: We successfully cultured melanocytes of the inner ear vestibular labyrinth for the first time and demonstrated melanocytic characteristics of these cells. This accomplishment will provide the opportunity to investigate VIEM in more detail in future experiments.     

Control of the microcirculation of the inner ear.

In general, many similarities are seen between cerebral blood flow and cochlear blood flow in response to vasodilating drugs. Cochlear vessels appear to be weakly controlled by the adrenergic nervous system. Cholinergic agents dilate cochlear vessels, although the vasodilation in the cochlea does not necessarily cause an increase in cochlear blood flow because of the associated hypotension. The effect on the systemic circulation is too profound to make these agents clinically useful. Papaverine, dipyridamole, amyl nitrite, and the plasma kinins produce transient increases in cochlear blood flow. Ten per cent carbon dioxide and 90 per cent oxygen product progressive vasodilation as long as the mixture is inhaled. Hydralazine produces too profound an effect on systemic blood pressure. Histamine and betahistine increase cochlear blood flow but only in doses that produce bronchiospasm in the guinea pig. Nicotinic acid and nicotinyl tartrate have no effect on cochlear blood flow. If one considers safety and efficacy as well as patient tolerance, papaverine appears to be the drug of choice for increasing cochlear blood flow clinically. Histamine and betahistine appear to be acceptable alternatives. Betahistine is no longer available for clinical use because of the failure to demonstrate clinical efficacy of therapy.     

Hearing preservation after inner ear gene therapy: the effect of vector and surgical approach

Over seventy studies have examined the potential of gene therapy in the inner ear. For the most part, they have focused on adenoviral vectors and delivery into the cochlea. Most studies have emphasized looking at the expression of marker genes driven by a CMV promoter and have used first-generation adenoviral constructs. E1/E3/E4 deleted adenoviral vectors carrying the green fluorescent protein (GFP) gene were injected into the round window, the basal turn of the cochlea (via a cochleostomy) or into the superior semicircular canal. Hearing was then tested 24 h after viral gene transfer. Large vector titers in small volumes of fluid were well tolerated with the round window approach resulting in complete hearing preservation with transfer of GFP to hair cells and spiral ganglion cells. Injection of comparable doses of vector into a basal turn cochleostomy resulted in high-frequency hearing loss. Addition of a pancaspase inhibitor protected hearing when larger volumes of fluid were administered to the inner ear.     

Recurrent diving-related inner ear barotrauma.

OBJECTIVE: To present two cases of recurrent diving-related inner ear barotrauma (IEB) and to discuss the possible cause and pathogenesis of the increased inner ear vulnerability. STUDY DESIGN: Case series. SETTING: Tertiary referral center. PATIENTS: Two scuba divers suffering from repeated cochleovestibular barotrauma. INTERVENTIONS: Neurotological evaluation, perilymphatic fistulae repair, and conservative treatment. MAIN OUTCOME MEASURE: The increasing popularity of scuba diving expose the individuals involved in this sport to unique pathologies that are not common under terrestrial conditions. The otolaryngologist who is involved in the care of these patients is required to diagnose and treat diving-related ear injuries and to consider the risk for recurrent inner ear injury when diving is resumed. CONCLUSION: IEB carries a risk for permanent hearing loss and chronic vestibulopathy. We recommend complete neurotological evaluation including high-resolution CT of the temporal bones as a routine workup for IEB. The presence of a significant residual sensorineural hearing loss, evidence for noncompensated vestibular damage, and CT findings of possible enhanced cerebrospinal fluid-perilymph connection should be considered when a return to diving activity is considered.