Unbiased quantification of the microdissected human Scarpa's ganglion neurons

OBJECTIVE: The objective of the present study was to obtain unbiased estimates of the total number of Scarpa's vestibular ganglion neurons in individuals with normal vestibular function. STUDY DESIGN: Application of unbiased stereology using microdissected human temporal bone specimens. METHODS: Postmortem temporal bones were obtained from five young subjects with no history of audiovestibular disease (age range 42-49 years). The vestibular nerve containing the Scarpa's ganglion was microdissected, embedded in paraffin, and cut into 40 microm serial sections. Unbiased estimates of the total number of neurons were obtained using the optical fractionator technique of stereology. RESULTS: An average of 23,599 (coefficient of variation [CV] = 0.11) vestibular ganglion neurons was obtained. There was no significant difference between the results obtained from the microdissected specimens and results that had been obtained from an age-matched group derived from a previously published report from our laboratory using archival human temporal bone specimens. CONCLUSIONS: This study represents the first report to demonstrate the combination of the microdissection technique and the unbiased stereologic technique in the human temporal bone. This study demonstrates the reliability of the microdissection technique as an alternative method of human temporal bone processing for unbiased stereology. The utility of the microdissection technique is that specimens can be used for quantification, immunohistochemistry, and other powerful applications.     

Application of unbiased stereology on archival human temporal bone

OBJECTIVE: The current report presents unbiased stereological sampling and counting strategies for estimating the total number of neurons in the spiral and vestibular ganglia of the archival human temporal bone. STUDY DESIGN: Application of unbiased stereology using archival human temporal specimens. Observational study. METHODS: The practical sampling and counting strategies for estimating the number of neurons are presented. To illustrate the method described, the total number of neurons in spiral and vestibular ganglia from archival human temporal bone was estimated. RESULTS: The study found, on average, 41,643 (coefficient of variation = 0.12) spiral ganglion neurons in the subjects with an average age of 35.6 years (age range, 22-60 y) and, on average, 27,635 (coefficient of variation = 0.10) vestibular ganglion neurons in the subjects with an average age of 28.6 years (age range, 2-58 y). CONCLUSIONS: The total number of primary auditory and vestibular ganglion neurons from the current study differs significantly from the previously published data using assumption-based methods. The unbiased stereological technique presented in the current report thus would be invaluable for future studies on such specimens, including the study of the changes of neurons during aging and various pathological conditions.     

Age-related change of the neuronal number in the human medial vestibular nucleus: a stereological investigation

An unbiased stereological method was used to assess the effect of aging on the number of neurons in the human medial vestibular nucleus. We studied 13 normal brainstem specimens (age at death from 40 to 93 years) that were part of a prior study that counted neuronal profiles and used a correction factor to estimate the number of neurons in the human vestibular nucleus. On average, we found 151.10(3) (CV = 0.15) neurons in the medial vestibular nucleus, which is 18% more than that in the prior study. Regression analysis showed a significant decrease in the number of neurons with aging. This age-related neuronal loss in the vestibular nucleus could have important functional implications regarding the well-known deterioration in balance that occurs with aging.     

Temporal bone histopathological study of Noonan syndrome.

Four temporal bone specimens, obtained from three individuals 1--6 years of age with Noonan syndrome (NS), were studied histopathologically. All four specimens were accompanied by similar inner ear abnormalities including the reduced number of spiral ganglion cells, enlarged lateral semicircular canal, and dislocated endolymphatic sac and vestibular aqueduct. The mean population of spiral ganglion cells (15,699 cells) was approximately half of those (32,978 cells) in four age-matched control cases. In addition, they had several middle ear abnormalities including the remaining mesenchyme and dehiscence of the facial canal. To our knowledge, this is the first report to describe the histopathological temporal bone findings in patients with NS. We discuss the implications of the observed abnormalities with regard to clinical issues.     

Middle ear instillation of gentamicin and streptomycin in chinchillas: morphologic appraisal of selective ototoxicity.

OBJECTIVE: To determine selective cochlear and vestibular ototoxicity of two aminoglycoside antibiotics (gentamicin and streptomycin) in the chinchilla model. Middle ear application of these agents mirrors the clinical practice of chemical vestibular ablation used in Meniere's disease. BACKGROUND: Middle ear instillation of gentamicin or streptomycin has become a popular form of vestibular ablative treatment for disabling Meniere's disease. The vestibular selectivity of these two drugs applied in this fashion has clinical support but is not fully established in humans. Our understanding in this regard has largely been limited to animal models exposed to systemic infusion of aminoglycosides. METHOD: Ten chinchillas underwent left middle ear instillation of one of three agents using variable dosing schedules: gentamicin (n = 6), streptomycin (n = 2), and saline (n = 2) as control. Animals were sacrificed for temporal bone studies using scanning electron microscopy. Morphologic changes in the cochlear and vestibular neuroepithelia were identified. RESULTS: Widespread cochlear and vestibular neuroepithelial injuries were observed with both gentamicin and streptomycin. Contralateral ototoxicity was variable and not related to the total dose of drug delivered. The effect of these two aminoglycosides on the dark cells of the vestibular system appeared negligible. CONCLUSION: We were unable to confirm the selective damage of vestibular end-organ in the chinchilla by either gentamicin or streptomycin, a phenomenon that is generally perceived to occur in humans. Chinchillas, like other small mammals, may not be an ideal model for the study of human ototoxicity.     

Susceptibility genes for gentamicin-induced vestibular dysfunction

BACKGROUND: Approximately 5% of patients administered gentamicin (GM), an aminoglycoside antibiotic, experience vestibular ototoxicity resulting in balance dysfunction. In the present study, we sought to identify susceptibility genes associated with GM-induced vestibular dysfunction using a case/control design. METHODS: White cases (n=137; 55 men, 82 women) were recruited based on physician-confirmed unilateral or bilateral vestibular dysfunction attributed to GM administration. Controls (n=126; 54 men, 72 women) were healthy, age-matched individuals without vestibular dysfunction or balance impairment. Buccal cell samples were obtained from all subjects and DNA was genotyped for 15 polymorphisms in 9 genes. Candidate genes were identified primarily for their roles in oxidative stress based on predicted mechanisms of gentamicin-induced ototoxicity. Statistical analyses included the multi-dimensionality reduction (MDR) method for identifying gene x gene interactions across multiple candidate genes. RESULTS: Both single gene and MDR analyses revealed the NOS3 (ENOS) p.Glu298Asp polymorphism as significantly associated with GM-induced vestibular dysfunction (both p 相似文献   

Aminoglycoside ototoxicity: a human temporal bone study.

OBJECTIVE: Hearing loss after aminoglycoside administration has been thought to result primarily from hair cell injury. The purpose of the study was to determine the potential for direct injury of spiral ganglion cells and hair cells in cases of documented human aminoglycoside ototoxicity. STUDY DESIGN: Retrospective case review. METHODS: The clinical course of two individuals with aminoglycoside ototoxicity are documented, including the details of administration of tobramycin and other ototoxic medication and serial audiograms. The temporal bones were processed, and the cochlear elements quantified. RESULTS: Histopathological study of the temporal bones from the individuals in the study demonstrated reduction of both ganglion cell and hair cell populations. Spiral ganglion cell loss was not necessarily subadjacent to areas of hair cell loss in cases of aminoglycoside ototoxicity. Instead, spiral ganglion cell reduction may be present in segments of the cochlea with normal-appearing hair cells. CONCLUSIONS: The study suggests that aminoglycoside antibiotics can injure spiral ganglion cells directly, as well as hair cells. Thus, the characteristic hearing loss of ototoxicity can result from degeneration of either cochlear element.     

Diameter of the cochlear nerve in deaf humans: implications for cochlear implantation.

Although the parameters that are most important for postoperative speech perception in cochlear implantation have not been identified, it is assumed that the numbers of remaining cochlear neurons and spiral ganglion cells in the implanted deaf ears are critical. In this study, we evaluated the correlation of the maximum diameter of the cochlear and vestibular nerve trunks with the number of spiral ganglion cells in horizontal sections of the temporal bone of 42 patients who were profoundly deaf during life, and in 5 patients with normal hearing. The maximum diameters of the cochlear, vestibular, and eighth cranial nerves were significantly smaller in the deaf population as compared to normal-hearing controls. In addition, the counts of the remaining spiral ganglion cells were significantly correlated with the maximum diameter of the cochlear (p = .0006), vestibular (p = .001), and eighth cranial nerves (p = .0003). The regression equation estimated that 25% of the variance of the spiral ganglion cell count was predicted by the maximum diameter of the eighth nerve. Although the results of this study suggest that preoperative radiographic imaging of the diameter of the eighth nerve may be helpful in predicting the residual spiral ganglion cell count, the wide variability of diameters of the eighth nerve in hearing and deaf subjects militates against this theoretic usefulness.     

儿童感音神经性聋77例分析

对７７例感音性神经性聋患儿的病因，前庭，听功能进行分析，中重度以上耳聋者１０９耳，平衡及半规管异常者占８８．９％。大前庭导水管综合征１９例３８耳均呈重度聋，为胚胎发育性疾病，平均年龄７．１岁，在言语形成期以后发病者有一定的言语表达能力，若早期发现进行相应的防范，可保存听力，当听力重度障碍应辅以助听器，帮助言语康复；药物中毒性聋１５例中，１３例２６耳中重工聋且呈聋哑状态，平均年龄２．８岁，此种后天获     

Development of a biophysical model for vestibular prosthesis research

Physiologic properties of primary vestibular neurons are compared and contrasted with properties of primary auditory neurons. The differences and similarities suggest possible coding strategies for a vestibular implant. The degree of spike rate variability, or coefficient of variation (CV), is a prominent physiological property of vestibular neurons with undetermined functional significance. At the very least, CV is highly correlated with threshold to electrical stimulation in the intact vestibular labyrinth. If CV is also important for vestibular coding, then electrical stimulation strategies should be designed to restore relatively physiologic patterns of CV. Simulations using a stochastic model of primary afferent vestibular neurons reveal that this should be possible using combinations of low and high-rate pulsatile stimulation. They also demonstrate that differences in the number and independence of synaptic inputs can significantly affect CV.     

Unbiased stereologic type I and type II hair cell counts in human utricular macula

OBJECTIVE: The objective of the study was to obtain unbiased estimates of the total number of type I and type II hair cells in human utricular macula from individuals with documented normal vestibular function. STUDY DESIGN: Application of unbiased stereology using microdissected human temporal bone specimens was conducted in an observational study. METHODS: Postmortem temporal bones were obtained from 10 normal patients (age range, 42-96 y; mean age, 82 y). The utricular maculae were microdissected, embedded in plastic, and cut into serial 2-microm sections. Unbiased estimates of the total number of type I and type II hair cells were obtained using the physical fractionator technique of stereology. RESULTS: The average total number of hair cells was 27,508 (CV = 11%) consisting of 17,326 (coefficient of variation [CV] = 11%) type I hair cells and 10,182 (CV = 13%) type II hair cells. The ratio of type I to type II hair cells was 1.70:1. In the age range of the study, there was no statistically significant correlation between hair cell counts and age. CONCLUSIONS: Morphometric studies of the human utricular sensory epithelium can be accomplished using unbiased stereology on microdissected specimens. There was no effect of age on total hair cell counts or on the ratio of type I to type II hair cells in the age range of the study. Further studies on younger subjects are needed to establish the effect of age. The results from the present study are closely aligned with prior studies that estimated total hair cell counts using surface mount preparations. The current data represent the first total type I and type II hair cell counts in human utricular neuroepithelium.     

Loss of vestibular neurons in clinical otosclerosis

Summary Although the principal functional deficit caused by otosclerosis is hearing loss, some patients also experience dysequilibrium and/or vestibular test abnormalities. A study was performed to determine the populations of vestibular neurons in subjects with clinical otosclerosis. Reduced neuronal counts were found when the otosclerotic lesions were large enough to involve the cribrose areas. Decreased cell counts were found principally in the superior division of the vestibular ganglion. In some cases, however, normal populations of neurons were found in spite of involvement of the cribrose areas. In some ears there were bundles of vestibular nerve fibers deviating from their normal course to pass within the focus of otosclerotic bone. It seems probable that a loss in vestibular neuronal population caused by involvement of dendritic fibers in the cribrose areas is at least partially responsible for the dysequilibrium or vestibular test abnormalities occurring in some patients with otosclerosis.Supported by a grant from the Research Fund of the American Otological Society     

Otopathology in Mohr-Tranebjaerg syndrome

BACKGROUND: Mohr-Tranebjaerg syndrome (MTS) is an X-linked, recessive, syndromic sensorineural hearing loss (HL) characterized by onset of deafness in childhood followed later in adult life by progressive neural degeneration affecting the brain and optic nerves. MTS is caused by mutations in the DDP/TIMM8A gene, which encodes for a 97 amino acid polypeptide; this polypeptide is a translocase of the inner mitochondrial membrane. OBJECTIVES: To describe the otologic presentation and temporal bone histopathology in four affected individuals with MTS. MATERIAL AND METHODS: All four subjects belonged to a large, multigenerational Norwegian family and were known to carry a frame shift mutation in the TIMM8A gene. Temporal bones were removed at autopsy and studied by light microscopy. Cytocochleograms were constructed for hair cells, stria vascularis, and cochlear neuronal cells. Vestibular neurons were also counted. RESULTS: All four subjects developed progressive HL in early childhood, becoming profoundly deaf by the age of 10 years. All four developed language, and at least one subject used amplification in early life. Audiometric evaluation in two subjects showed 80- to 100-dB HL by the age of 10 years. The subjects died between the ages of 49 and 67. The otopathology was strikingly similar in that all bones examined showed near-total loss of cochlear neuronal cells and severe loss of vestibular neurons. When compared with age-matched controls, there was 90% to 95% loss of cochlear neurons and 75% to 85% loss of vestibular neurons. CONCLUSIONS: We infer that the HL in MTS is likely to be the result of a postnatal and progressive degeneration of cochlear neurons and that MTS constitutes a true auditory neuropathy. Our findings have implications for clinical diagnosis of patients with MTS and management of the HL.     

Middle ear instillation of gentamicin and streptomycin in chinchillas: electrophysiological appraisal of selective ototoxicity

The purpose of this study was to investigate selective vestibular ototoxicity of gentamicin and streptomycin in the chinchilla model. In total, 10 chinchillas underwent left middle ear instillation of one of three agents: gentamicin, streptomycin and saline. Electrophysiological data (otoacoustic emissions (OAEs), auditory brainstem evoked response (ABRs), and ice-water electronystagmography were recorded before and after instillation. Animals were sacrificed for temporal bone studies using scanning electron microscopy. Morphological changes in the cochlear and vestibular neuroepithelia were correlated with electrophysiological changes. Widespread ipsilateral cochlear and vestibular neuroepithelial injuries were observed and correlated with loss of OAEs, ABRs and ice-water caloric response. This study provides no evidence of selective vestibular ototoxicity of gentamicin or streptomycin. Morphological damage correlates with, but precedes loss of electrophysiological parameters. Chinchillas, like other small mammals, may not be an ideal model for the study of human ototoxicity.     

Optical recording of membrane potential in dissociated mouse vestibular ganglion cells using a voltage-sensitive dye

We investigated membrane electrophysiological features of dissociated vestibular ganglion neurons, using a voltage-sensitive dye and a multiple site optical imaging system. The neuronal nature of the cultured vestibular ganglion cells was confirmed by positive staining with the anti-neurofilament 200 kDa antibody, using immunocytochemical methods. Optical absorption of the dye which binds to the external surface of neuron membranes increased while the cells were depolarized during perfusion with 150 mM potassium solution. The relative ratio (deltaI/I) of optical absorption change was 0.23 +/- 0.08% (means +/- S.D., n = 16). These optical responses were wavelength dependent, therefore, the optical response apparently originated from the voltage-sensitive dye. Under our experimental conditions, photodynamic damage and pharmacological effects of the dye were either absent or insignificant. We therefore concluded that optical recording is a new, practical and non-invasive method to simultaneously monitor changes in membrane potential from cultured vestibular ganglion cells. Optical recording is expected to provide further insight into mechanisms of information processing by vestibular ganglion neurons.     

Calpain and caspase inhibitors protect vestibular sensory cells from gentamicin ototoxicity

OBJECTIVE: Apoptosis may play an important role in the mechanism of aminoglycoside ototoxicity. Caspases and calpains are regarded to be important factors in the regulation of cell death in the inner ear. We hypothesized that caspase or calpain inhibitors would protect hair cells from aminoglycoside ototoxicity. MATERIAL AND METHODS: In order to test this hypothesis we carried out a pilot study to determine if gentamicin (GM) would induce caspase and calpain immunolabeling in guinea pig hair cells Having confirmed this we carried out the main experiment using guinea pig vestibular organ culture to determine if caspase and calpain would protect the hair cells from GM ototoxicity. RESULTS: Immunoreactivity for caspase-3 and m-calpain was detected in the vestibular sensory cells and ganglia after GM treatment. Both caspase and calpain inhibitors protected hair cells against gentamicin ototoxicity. CONCLUSION: It is suggested that inhibition of apoptosis is essential in order to block aminoglycoside ototoxicity.     

Are vestibular sensory cells preserved after destruction of Scarpa's ganglion? A study based on metastatic tumors of temporal bone.

OBJECTIVE: The contribution of nerve fibers to the maintenance of vestibular sensory cells is a controversial issue in previous studies using animals and has not yet been studied in humans. The authors investigated this issue by observing vestibular end organs in the temporal bone of three patients in whom the internal auditory canal was infiltrated with tumor cells, and Scarpa's ganglion cells showed complete degeneration. STUDY DESIGN: Retrospective case review. SETTING: University Hospital, Department of Otolaryngology. PATIENTS: Three patients with malignant metastatic temporal bone tumors. INTERVENTION: We investigated the preservative state of vestibular sensory hair cells with the Scalpa's ganglion was destructed. MAIN OUTCOME MEASURES: Maintenances of vestibular sensory hair cells. RESULTS: We found that sensory cells were intact despite the severe destruction of Scarpa's ganglion cells in two of the patients. CONCLUSION: The findings suggest that human vestibular sensory cells can be maintained for an indefinite period after denervation.     

Morphological changes of vestibular ganglion cells in human fetuses and in pediatric patients.

The temporal bone histopathology of human vestibular ganglion cells of fetuses and pediatric patients was studied. In the first study, we traced the morphological changes in vestibular ganglion cells in human fetuses ranging from 13 weeks to 39 weeks of gestational age by using 13 temporal bone serial sections. Vestibular ganglion cells had reached histological maturity by the 24th week of gestation and the volume of vestibular ganglion cell cytoplasm increased until the 39th week of gestation. In the second study, the temporal bone serial sections of seven neonates, eight infants and five children were investigated to reveal pathological changes in vestibular ganglion cells. Morphological changes in vestibular ganglion cells in human fetuses were revealed. Vestibular ganglion cells were changed pathologically by intracranial disease and variety etiology affecting the inner ear, because these are located in the internal auditory canal between the brain and labyrinth.     

Unbiased Stereological Estimation of the Spiral Ligament and Stria Vascularis Volumes in Aging and Ménière’s Disease Using Archival Human Temporal Bones

The present study applies the unbiased stereological technique—Cavalieri principle to measure the volumes of the stria vascularis (SV) and the spiral ligament (SL) using postmortem archival human temporal bones from normal young and older subjects and subjects with Ménière’s disease. Normative data was obtained from subjects without ages ranging from 15 to 84 years old who had no history of audiovestibular disease (N = 25). For comparison purposes, the normative specimens were divided into three groups: group 1 (n = 8) had ages ranging from 15 to 38 years old, average age = 23.9; group 2 (n = 8) had ages ranging from 51 to 59 years old, average age = 55.1; group 3 (n = 9) had ages ranging from 64 to 84 years old, average age = 74.3. The average SV volume of group 3 (0.479 mm³) was significantly lower than that of group 1 (0.705 mm³) (p < 0.0005) and was significantly lower than that of group 2 (0.603 mm³) (p = 0.01). The average SL volume of group 3 (8.42 mm³) was significantly lower than that of group 1 (9.54 mm³) (p<0.05), but was not significantly lower than that of group 2 (8.58 mm³). Five subjects with Ménière’s disease, confirmed by histopathological examination (ages ranging from 63 to 91 years old, average age = 73.4), were studied. The average SV volume in Ménière’s subjects (0.378 mm³) was significantly lower than age-matched controls (p<0.05). The average SL volume in Ménière’s subjects (7.01 mm³) was also significantly lower than age-matched controls (p<0.05). The SV and SL volumes were unaffected by gender. The present study demonstrates for the first time the use of the unbiased stereological technique—Cavalieri principle—as a reliable and efficient method to obtain volumetric estimates of the SV and the SL by using archival human temporal bone specimens.     

Three-dimensional anatomy of human Scarpa's ganglion.

Using a computer-aided three-dimensional reconstruction and measurement method, the authors studied the shape and dimensions of Scarpa's ganglion and interrelations of the ganglion to nearby important anatomical structures in 10 normal human temporal bones obtained from 10 individuals of different ages (12 gestational weeks to 72 years). In the postnatal cases, Scarpa's ganglion was shaped like a distorted hourglass. Although the shape of the ganglion varied somewhat among specimens, a superior division, inferior division, and isthmus ganglionaris were distinguishable in each case. Volume of the ganglion was relatively consistent after birth, so that width and height of the ganglion were inversely correlated with each other (linear regression analysis, P less than .05). There seemed to be, however, a possible postnatal increase in the distances from the ganglion to neighboring structures and in the length of the internal auditory canal; these increases were probably the result of postnatal growth of the temporal bone. Moreover, the width of the ganglion in any single specimen was found to correlate significantly positively with the distances from the ganglion to the vestibular end-organs and the length of the internal auditory canal in that specimen (linear regression analysis, P less than .01). This finding suggests that Scarpa's ganglion may be elongated secondary to the elongation of the vestibular nerve due to the growth of the temporal bone. In fetuses, each portion of the ganglion was less clearly identifiable than that portion in postnatal cases. The dimensions of Scarpa's ganglion were found to have reached maturity around the time of birth.