In vitro long-term development of cultured inner ear stem cells of newborn rat.

The adult mammalian auditory receptor lacks any ability to repair and/or regenerate after injury. However, the late developing cochlea still contains some stem-cell-like elements that might be used to regenerate damaged neurons and/or cells of the organ of Corti. Before their use in any application, stem cell numbers need to be amplified because they are usually rare in late developing and adult tissues. The numerous re-explant cultures required for the progressive amplification process can result in a spontaneous differentiation process. This aspect has been implicated in the tumorigenicity of stem cells when transplanted into a tissue. The aim of this study has been to determine whether cochlear stem cells can proliferate and differentiate spontaneously in long-term cultures without the addition of any factor that might influence these processes. Cochlear stem cells, which express nestin protein, were cultured in monolayers and fed with DMEM containing 5% FBS. They quickly organized themselves into typical spheres exhibiting a high proliferation rate, self-renewal property, and differentiation ability. Secondary cultures of these stem cell spheres spontaneously differentiated into neuroectodermal-like cells. The expression of nestin, glial-fibrillary-acidic protein, vimentin, and neurofilaments was evaluated to identify early differentiation. Nestin expression appeared in primary and secondary cultures. Other markers were also identified in differentiating cells. Further research might demonstrate the spontaneous differentiation of cochlear stem cells and their teratogenic probability when they are used for transplantation.

Immunocytochemical detection of synaptophysin in C57BL/6 mice cochlea during aging process.

Aged mammals frequently exhibit a bilateral, progressive, and symmetric deafness related to the degeneration of auditory receptor. However, little is still known about aging effects on synapses in this receptor. Synaptophysin (Syp) is a 38 kDa Ca2+ binding glycoprotein widely found in presynaptic membrane and vesicles. The Syp has been found in presynaptic buttons of efferent auditory fibers, within the developing and adult auditory receptor. The detection of Syp in aged cochleae could provide relevant information about synaptic changes and receptor degeneration process observed in old animals. This paper focuses on aging linked changes related to the presence of Syp in cochleae of C57BL/6J mice (from 1 to 24 months old). Results showed that during the first months of age, no significant changes were observed in the Syp distribution under the basal pole of inner (IHCs) neither the outer (OHCs) hair cells. At six months of age, a significant decrease of Syp immunocytochemical detection appeared in fibers under the most external row of OHCs, but restricted to the cochlear basal coil. Only a very scarce reduction of Syp was noted under the IHC and the other OHC rows, also at the basal coil. From mice 9 months old on, a progressive decrease of the presence of Syp was found under IHC and all OHC rows starting at the basal coil and reaching the apical coil in the oldest mice. All these data could indicate that the cochlea aging process early affects to presynaptic membrane proteins of efferent endings fibers. This early alteration of cochleae efferent synapses could be involved in the whole degeneration of the Corti's organ.

Magnetoencephalography for research of auditory cortex.

CONCLUSION: The results could indicate that, during phylogeny and human ontogeny, the central nervous system has enhanced the speech activity from any other activity even though other frequencies could be relevant for survival. OBJECTIVE: People of all ages can experience alterations of auditory perception that progressively increase with aging. The whole approach to these alterations needs not only peripheral (cochlear) or brainstem studies but also an analysis of the auditory cortex. In fact, auditory evoked fields (AEF) may contribute to the understanding of the neural correlate of sound awareness. SUBJECTS AND METHODS: The M100 response after pure tone stimulation (five frequencies ranging from 500 to 8000 Hz) was analyzed in a group of nine adult subjects with normal hearing, older than 25 years of age and under 40 years old. Average M100 field intensity was calculated for all magnetoencephalography (MEG) channels in a 60 ms window around the M100 waveform. RESULTS: The results indicate a more intense cortical response to main speech frequencies (0.5 to 2 kHz) as compared with other frequencies not involved in human conversation.

[General indications of middle-ear implants]. / Indicaciones generales de los implantes de oído medio.

Middle ear implants are indicated in bilateral neurosensorial hearing loss in the following situations: bilateral neurosensorial hearing loss with maximal thresholds of air conduction or air conduction within an established range. The differential threshold in frequencies of 500, 1,000, 2,000 and 4,000 Hz should not be above 10 dB in two or more of these frequencies. Minimal speech recognition must be 50% or more. Middle ear function and anatomy must be normal and there must be no evidence that hearing loss is of retrocochlear origin. Use of a hearing aid for at least 6 months in the ear receiving the implant must be proven to be unsatisfactory by a hearing test. Expectations must be realistic and vestibular alterations must be excluded.

Serotonergic innervation of the inner ear: is it involved in the general physiological control of the auditory receptor?

The auditory pathway of mammals is composed of two complementary ascending afferent and descending efferent independent systems. The brainstem nuclei and cochlear projections for these systems are now well-known. In addition, a highly conspicuous distribution for serotonergic fibers was recently reported. This study focused on these serotonergic fibers and their neurons of origin. We identified several different types of serotonergic brainstem neurons surrounding the superior olivary complex and around the periolivary nuclei. Even though the 5-hydroxytryptamine (5-HT) efferent cochlear innervation originates in the periolivary area of the superior olivary complex system projecting to the cochlea, it is not involved in the transduction of pure tones during auditory processing. However, recent findings, after cochlear blockade of serotonin transporters, strongly suggested that this neuroactive substance has an important turnover within the auditory receptor. The presence of a conspicuous peripheral nerve distribution together with a particular brainstem origin could define a complex role for this innervation. Therefore, 5-HT fibers projecting to the cochlea might be involved, as in other parts of the auditory pathway, in alertness, attention, control of sleep or wakefulness cycles, and state of urgency prior to the transduction processing at the auditory receptor. A lack, or reduction, of the function of these fibers could result in pathological alterations.

Simultaneous HPLC quantification of monoamines and metabolites in the blood-free rat cochlea.

Monoamine quantification in peripheral sensory receptors, such as the cochlea, is of major interest since monoamines could play a role in neurotransmission. A three-step biochemical protocol was developed to analyze monoamine content within the cochlea. Removal of the blood by aortic perfusion was carried out with an anticoagulant solution prior to the dissection of the cochlea from the temporal bone. The cochlear monoamines and some of their metabolites were then quantified, from homogenated cochlear tissue, by a new application of high performance liquid chromatography coupled to electrochemical detection. This method demonstrated enough sensitivity to detect norepinephrine (NE), dopamine (DA), serotonin (5-HT) and some of their metabolites (3,4-dihydroxyphenylacetic acid, DOPAC; homovanillic acid, HVA; and 5-hydroxyindole-3-acetic acid, 5-HIAA). Furthermore, it enabled the demonstration of noise-induced changes in the cochlear concentrations of NE, DA, DOPAC and HVA. In addition, the aortic perfusion allowed removal of the blood-borne 5-HT from the cochlea without inducing systemic alterations or monoamine degradation, as shown by the absence of effects on NE, DA, DOPAC, HVA or 5-HIAA concentrations. The present methodology may constitute a useful strategy to analyze monoamine turnover in the cochlea and other peripheral sensory receptors.

Age- and gender-related changes in the cochlear sympathetic system of the rat.

The sympathetic innervation projecting to the cochlea plays an important role in the auditory function, there is, however, no information about whether it is altered with advancing age. High performance liquid chromatography coupled to electrochemical detection was used to quantify both basal and noise-induced concentrations of norepinephrine (NE) in the rat cochlea. The cochlear concentration of NE was found to be independent of age in adult (3-12 months old) and aged (19 and 24 months old) males and the adult females. However, the concentrations of NE increased in aged females with respect to the younger ones, which suggests an increase in NE synthesis and a reduced NE release. Thus, a prominent gender effect emerged from this study, since the NE cochlear concentration was lower in adult females than in males, but tended to be the same level in aged animals. These modifications could be related to dramatic hormonal changes occurring in females with advancing age.

Biochemical evidence for the presence of serotonin transporters in the rat cochlea.

Cochlear serotonergic innervation is constituted by efferent fibers projecting both to the area below the inner and the outer hair cells. Previous detection of serotonin (5-HT) metabolites and 5-HT receptor mRNAs suggests the existence of serotonergic synaptic activity in the cochlea. The present study explores this possibility through the effect of 6-nitroquipazine (6-NQ), a 5-HT selective reuptake inhibitor, on the basal turnover of 5-HT. The concentrations of 5-HT and its metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) were quantified by high performance liquid chromatography with electrochemical detection in blood-free cochleae of rats treated with 6-NQ or saline and kept under silent conditions. Treatment with 6-NQ induced a significant increase of the cochlear concentration of 5-HT and a significant reduction of 5-HIAA concentration with respect to saline treatment. These findings could indicate that 6-NQ induced the blockade of the 5-HT selective reuptake to the cochlear serotonergic fibers. This suggests that plasma membrane 5-HT transporters are present in cochlear serotonergic fibers. Even though the role of serotonergic innervation on cochlear physiology remains unknown, the existence of cochlear serotonergic synaptic activity is strongly supported by present contributions.

[Deafness and aging: studies in experimental models]. / Sordera y envejecimiento: estudios en modelos experimentales.

Since 1970 a progressive aging of the world population, mainly in the most developed countries, has been observed. Spain could have, around 2050, the most aged human population of the world. Therefore, scientist show an increasing interest on the study of the aging-related pathologies (i.e. deafness linked to aging process: presbycusis). The deep analysis of the presbycusis physiopathology will be based on the study of patients, but also on animal models. This report summarizes our results obtained on the analysis of the deafness linked to aging on the C57/BL/6 mice.

Electrophysiological monitoring of hearing function during cochlear perilymphatic perfusions.

CONCLUSION: The cochlear perilymphatic perfusion produces, by itself, significant effects in the cochlear physiology that could be associated with the surgical procedure. These effects need to be well characterized to allow a reliable quantification of the effects of the experimental agent being tested. OBJECTIVES: The study focused on the accurate description of the electrophysiological effects on the cochlear potential recordings of perilymphatic perfusions. METHODS: Two successive cochlear perilymphatic perfusions were carried out. The first used artificial perilymph. The second used artificial perilymph alone or a kainic acid (KA) solution in artificial perilymph. The compound action potential of the auditory nerve (CAP-AN) was recorded: (1) before the first perfusion, (2) after the first perfusion and (3) after the second perfusion, and compared between groups. RESULTS: The first intracochlear perfusion with artificial perilymph produced significant effects in the CAP-AN that could be related to the surgical procedure. These effects were analysed separately from the effects produced by the KA. In particular, the KA administered intracochlearly produced a significant increase in the latency and a decrease in the amplitude of the CAP-AN N1 wave compared with the controls that were perfused twice with artificial perilymph.

The impact of comorbidity on the survival of patients with laryngeal squamous cell carcinoma.

UNLABELLED: Abstract Conclusions: In survival analysis, the combined Charlson comorbidity index (CCI) can be considered as a prognostic factor independent of the tumor node metastasis (TNM) classification, tumor stage, and tumor location. Severe comorbidity was the factor that had the greatest impact on prognosis in cases of initial tumor. OBJECTIVE: To study the influence of comorbidity on the survival of patients undergoing surgery for larynx cancer. METHODS: This was a retrospective study of the survival of 231 patients with laryngeal cancer who underwent surgery between 1995 and 2002. The CCI was used to assess comorbidity, the Kaplan-Meier method was used for survival analysis, and the Cox proportional risk regression model was used to identify independent prognostic factors. RESULTS: The multivariate analysis of specific mortality showed that patients classified as having severe comorbidity (CCI) were more likely to die (adjusted hazard ratio (adjHR) 1.85, 95% confidence interval (CI) 1.07-3.17). This difference was more important in patients with early tumor stages than in those with advanced stages.

Pharyngo-cutaneous fistula: an old problem revisited.

CONCLUSION: Pharyngo-cutaneous fistula is the most common complication after total laryngectomy (TL), with many factors linked to its emergence. However, it has rarely been associated with the type of pharyngeal suture. We conclude that the technique of surgical closure of the pharynx and care in the tightness of the suture seem to be fundamental factors for pharyngo-cutaneous fistula development. OBJECTIVE: The aim of present work was to determine whether the type of pharyngeal suture can be considered as a major risk factor for developing a pharyngo-cutaneous fistula following TL. METHODS: We carried out a series of 157 consecutive TLs. In the first 90 procedures, we performed a pharyngeal closure technique with T-shaped interrupted stitches reinforced with constrictors. In the other 67 cases, a doubled continuous suture technique with reinforcement with the cutaneous flap was developed. RESULTS: In all, 25.5% of the cases sutured with the interrupted stitches developed a fistula while only 2.9% of the patients that underwent continuous suture developed a fistula.

Cellular and molecular bases of neuroplasticity: brainstem effects after cochlear damage.

After a cochlear lesion or auditory nerve damage, afferent connections from auditory ganglia can be highly altered. This results in a clear reduction of auditory input and an alteration of connectivity of terminals on cochlear nuclei neurons. Such a process could stimulate the reorganization of the neural circuits and neuroplasticity. Cochlea removal has been demonstrated to be a good model in which to analyse brainstem neuroplasticity, particularly with regard to the cochlear nuclei. After cochlea removal three main periods of degeneration and regeneration were observed. Early effects, during the first week post lesion, involved acute degeneration with nerve ending oedema and degeneration. During the second and, probably, the third post lesion weeks, degeneration was still present, even though a limited and diffuse expression of GAP-43 started. Around 1 month post lesion, degeneration at the cochlear nuclei progressively disappeared and a relevant GAP-43 expression was found. We conclude that neuroplasticity leads neurons to modify their activity and/or their synaptic tree as a consequence of animal adaptation to learning and memory. For the human being neuroplasticity is involved in language learning and comprehension, particularly the acquisition of a second language. Neuroplasticity is important for therapeutic strategies, such as hearing aids and cochlear implants.

Cochlear microphonic audiometry: a new hearing test for objective diagnosis of deafness.

CONCLUSIONS: Objective audiometric tests could constitute a valuable tool for detection of deafness. This could be especially useful in children (universal newborn hearing screening) and non-collaborative patients, who are especially difficult candidates for classic audiometry. The cochlear microphonic audiometry (CMA) technique offers the possibility of obtaining objective audiometric profiles, highly correlated with those obtained by pure tone audiometry (PTA). Therefore, CMA could be used as an alternative test to obtain the audiometric profile of these patients. OBJECTIVE: The main purpose of the present study was to demonstrate that CMA provides objective audiometric profiles by avoiding active participation by the patient. Subjects and methods. CMA specific equipment, improved for non-invasive recording of cochlear microphonic potentials, was used. This tool plots the recordings obtained as the classic audiogram. Verification of the method was carried out in adult patients by comparing the PTA with the CMA audiometric profiles obtained for each patient. RESULTS: Our findings showed that audiometric profiles obtained from CMA are highly correlated, without statistical differences, to those obtained with PTA. More than 81% of patients explored (91.67% at 250 Hz) exhibited differences below 10 dB(HL) between tests at all exploration frequencies, while a low number of cases showed differences over 20 dB(HL).

Effect of recombinant human growth hormone administration on body composition and vascular function and structure in old male Wistar rats.

The process of ageing affects negatively both cardiovascular system and body composition. On the other hand, the hormones of the somatotrophic axis, growth hormone (GH) and insulin-like growth factor-I (IGF-I), whose production is reduced by age, are involved in the regulation of the cardiovascular system. The aim of this study was to investigate the effect of GH on body composition, vascular function and structure in old male rats. Old (20 months) and adult (4 months) male Wistar rats were used. One group of old animals was treated with GH for 4 weeks. Periepididimary fat weight, Specific Gravity Index (SGI), dose responses to Acetylcholine (ACh), Isoproterenol (Iso), Phenylephrine (Phe) and ACh in the presence of NG-nitro-L: -arginine metylester (L-NAME; ACh + L-NAME), as well as vascular morphology in aortic rings, were studied. Old rats showed increased fat weight and decreased SGI as compared to adult animals. GH increased SGI and tended to reduce fat weight. Old rats showed an impairment in the vasodilator response to ACh and Iso; GH significantly improved the vasodilatation induced by Iso, whereas the response to ACh was not significantly enhanced by GH treatment. There were no significant differences between adult and old rats in the contractile response to Phe, and GH did not show any effect. Contraction induced by ACh + L-NAME was higher in old rats as compared to adults, and treatment with GH significantly reduced this response. Aortic media area was increased in old rats, and GH administration reduced this parameter. In conclusion, GH shows beneficial effects on body composition, as well as on vascular function and morphology in old male rats.

Effects of aging on cochlear monoamine turnover.

The aging of the cochlear dopaminergic system has been analyzed by quantifying the levels of dopamine (DA) and its metabolites (3,4-dihydroxyphenylacetic acid, DOPAC, and homovanillic acid, HVA) in adult rats aged 3, 12 or 24 months. The main results were an increase in DA, DOPAC and HVA basal concentrations in aged females with respect to the adults (3 or 12 months old), while just DA and DOPAC increased in aged males. A higher synthesis of DA in aged animals could support these findings, which could indicate some kind of compensatory mechanism related to presbycusis.

Effects of aging on C57BL/6J mice: an electrophysiological and morphological study.

Presbycusis is a progressive hearing loss associated with aging that manifests as deafness linked to cochlear morphological degeneration. The effects of aging on the auditory system were studied in C57BL/6J mice using electrophysiological (brainstem auditory evoked potentials; BAEP) and morphological techniques. Cochleae of animals aged 1, 6, 9, 12, 15, 18, 21, or 24 months old were used for that purpose. The BAEP showed a progressive increase in latency and a reduction in amplitude. Morphological studies demonstrated total degeneration of the organ of Corti, which was replaced by a single epithelial layer. An affinity histochemistry study demonstrated minor modifications of glycoconjugates in the organ of Corti during the aging process.