Optimised immunofluorescence method on cleared intact Mongolian gerbil cochlea.

Immunofluorescence on cleared intact cochlea allows detailed analysis of the cochlear ultrastructure, while avoiding the problems of dissection and serial sections. Protocols have been developed for mice and Mongolian gerbils. This technical note proposes a detailed and optimised immunofluorescence protocol in the Mongolian gerbil comprising significant quantitative and qualitative improvements. This protocol sequentially comprises: fixation (1 day), decalcification (6 days), pre-treatment (7.5hours), immunolabelling (42hours), dehydration and clearing (23hours), followed by mounting and laser scanning confocal microscopy acquisition. This protocol has been optimised in terms of duration (10 days versus 13 days) with a reduction of the number of steps, improvement of the specificity of immunolabelling and optimisation of the quality of the results obtained. This technical note provides a detailed description of this protocol.

Azimuthal sound source localization of various sound stimuli under different conditions.

AIM: To evaluate azimuthal sound-source localization performance under different conditions, with a view to optimizing a routine sound localization protocol. MATERIAL AND METHOD: Two groups of healthy, normal-hearing subjects were tested identically, except that one had to keep their head still while the other was allowed to turn it. Sound localization was tested without and then with a right ear plug (acute auditory asymmetry) for each of the following sound stimuli: pulsed narrow-band centered on 250Hz, continuous narrowband centered on 2000Hz, 4000Hz and 8000Hz, continuous 4000Hz warble, pulsed white noise, and word ("lac" (lake)). Root mean square error was used to calculate sound-source localization accuracy. RESULTS: With fixed head, localization was significantly disturbed by the earplug for all stimuli (P<0.05). The most discriminating stimulus was continuous 4000Hz narrow-band: area under the ROC curve (AUC), 0.99 [95% CI, 0.95-1.01] for screening and 0.85 [0.82-0.89] for diagnosis. With mobile head, localization was significantly better than with fixed head for 4000 and 8000Hz stimuli (P<0.05). The most discriminating stimulus was continuous 2000Hz narrow-band: AUC, 0.90 [0.83-0.97] for screening and 0.75 [0.71-0.79] for diagnosis. In both conditions, pulsed noise (250Hz narrow-band, white noise or word) was less difficult to localize than continuous noise. CONCLUSION: The test was more sensitive with the head immobile. Continuous narrow-band stimulation centered on 4000Hz most effectively explored interaural level difference. Pulsed narrow-band stimulation centered on 250Hz most effectively explored interaural time difference. Testing with mobile head, closer to real-life conditions, was most effective with continuous narrow-band stimulation centered on 2000Hz.

Sound source localization.

Sound source localization is paramount for comfort of life, determining the position of a sound source in 3 dimensions: azimuth, height and distance. It is based on 3 types of cue: 2 binaural (interaural time difference and interaural level difference) and 1 monaural spectral cue (head-related transfer function). These are complementary and vary according to the acoustic characteristics of the incident sound. The objective of this report is to update the current state of knowledge on the physical basis of spatial sound localization.

Management of multiple tumors in neurofibromatosis type 2 patients.

INTRODUCTION: Neurofibromatosis type 2 is characterized by the presence of bilateral vestibular schwannomas. However, other nervous system tumors may also occur. Therefore, the management of NF2 patients is complex and requires a multidisciplinary discussion in a specialized center. MATERIALS AND METHODS: All recent articles concerning tumors other than vestibular schwannoma in NF2 disease were reviewed, using PubMed databases. RESULTS: Intracranial meningiomas occur in 50% of NF2 patients, and are often multiple. Surgery remains the main treatment and should be performed in cases of growing tumors. The role of antiangiogenic therapy is currently under evaluation and the role of radiosurgery still remains to be defined in NF2 disease. Spinal tumors occur in about half of NF2 patients. Surgery should be discussed when radiological tumor progression is demonstrated, even if spinal tumors are asymptomatic, in order to preserve neurological function and good quality of life. As regards lower cranial nerve schwannomas, radiosurgery appears to be a more appropriate treatment for growing tumor with a small volume in order to avoid post-operative complications, especially swallowing disorders. Facial nerve schwannomas may appear, on MRI, like vestibular schwannomas. The diagnosis should be suspected when the facial palsy is an early symptom during cerebello-pontine tumor progression. Trigeminal schwannomas are frequent in NF2 disease and fortunately they are often asymptomatic. Among major neurofibromatosis types, peripheral nerve sheath schwannomas are only present in patients with NF2 disease and schwannomatosis. Surgical resection is required when the cutaneous schwannomas is painful or when tumor progression is observed and causes symptoms. CONCLUSION: Tumors other than vestibular schwannoma are also associated with a poor prognosis in NF2 patients. Surgery remains the main treatment in most cases. Each treatment decision in NF2 disease requires a complete evaluation of all cranial and spinal locations of the disease in order to establish surgical priorities and strategies.

Imaging and cell count in cleared intact cochlea in the Mongolian gerbil using laser scanning confocal microscopy.

OBJECTIVES: To draw up a clearing protocol for Mongolian gerbil cochlea, and to assess the feasibility of quantifying and analyzing 3D cell architecture in the transparent cochleae. MATERIALS AND METHODS: Freshly dissected inner ears were prepared on a 13-day protocol: fixation, microdissection, post-fixation, decalcification, pretreatment (signal enhancement, permeabilization and blocking), fluorescent labeling (indirect immunolabeling and direct labeling), dehydration, clearing in Spalteholz solution (MSBB: methyl salicylate and benzyl benzoate) and mounting. Image acquisition used laser scanning confocal microscopy. ImageJ software was used to measure the length of the organ of Corti thus available for analysis and to count inner and outer hair cells. RESULTS: Four cochleas underwent imaging. 3D reconstruction enabled organ of Corti length to be measured, at a mean 1269±346µm. Mean inner and outer hair-cell count per organ of Corti length was 142±44 and 400±122, respectively. CONCLUSION: Cochlear clearing by MSBB was feasible in Mongolian gerbils and provided high-resolution immunofluorescence-labeled inner-ear images. To our knowledge, this was the first application of the technique in this species. Cell count could thus be performed along the organ of Corti length without traumatic dissection.

How to perform 3D reconstruction of skull base tumours.

The surgical management of skull base lesions is difficult due to the complex anatomy of the region and the intimate relations between the lesion and adjacent nerves and vessels. Minimally invasive approaches are increasingly used in skull base surgery to ensure an optimal functional prognosis. Three-dimensional (3D) computed tomography (CT) reconstruction facilitates surgical planning by visualizing the anatomical relations of the lesions in all planes (arteries, veins, nerves, inner ear) and simulation of the surgical approach in the operating position. Helical CT angiography is performed with optimal timing of the injection in terms of tumour and vessel contrast enhancement. 3D definition of each structure is based on colour coding by automatic thresholding (bone, vessels) or manual segmentation on each slice (tumour, nerves, inner ear). Imaging is generally presented in 3 dimensions (superior, coronal, sagittal) with simulation of the surgical procedure (5 to 6 reconstructions in the operating position at different depths).

Impact of modulating phase duration on electrically evoked auditory brainstem responses obtained during cochlear implantation.

Objective To investigate the effect of increasing phase duration (pulse width, T-pulse) using a biphasic pulse composed of an initial anodic active phase followed by a balancing cathodic phase on the electrically evoked auditory brainstem responses (eABRs) recorded at the time of cochlear implantation. Design eABRs recorded during 188 surgeries for cochlear implantation from 1999 to 2006 in a single center were retrospectively reviewed by two independent observers. All patients were fitted with a NEURELEC cochlear implant (CI) device, initially DIGISONIC(®) then DIGISONIC SP(®) (2004-2006). Result Immediately following cochlear implantation, stimulation by the CI resulted in reliable wave III and V eABR waveforms (mean wave III latency 2.23 ± 0.38 ms SD and wave V latency 4.28 ± 0.42 ms SD). Latencies followed an apical to basal gradient (0.32 ms increase in mean eV latency and 0.12 ms for eIII latency). With increasing phase duration, wave III and wave V latencies significantly decreased in association with a shortening of the eIII-eV interwave gap, while amplitudes of both waves increased. Conclusion The impact of increasing phase duration on latency and amplitude of brainstem responses in a large set of patients implanted with NEURELEC CIs was reported.

Endonasal endoscopic approach for removal of intranasal nasal glial heterotopias.

BACKGROUND: Nasal Glial Heterotopias also called Nasal Gliomas (NG) are rare congenital tumours of the midline frontonasal space arising from a normal neurectodermal tissue entrapped during the closure of the anterior neuropore. Historically, such tumours were approached using a frontal craniotomy. The study aims to evaluate a fully endonasal endoscopic approach for intranasal NG removal. METHODS: We report a retrospective study of intranasal and mixed NG treated using endonasal endoscopic techniques and computer assisted navigation system from 1997 to 2010 in two tertiary referral centres of Paediatric Otolaryngology. All tumours were investigated using two imaging modalities: craniofacial MRI and CT-scan. RESULTS: Fifteen patients were included (0 to 14 years of age). All tumours were totally removed and no recurrence was observed after a mean follow-up of 32 months. A skull base plasty was done in 13 cases to cover a bony defect or to treat a cerebrospinal leak. Nasal packing was usually removed 24 hours after surgery and all children were discharged home after 2 to 4 days. CONCLUSION: Removal of intranasal NGs using an endonasal endoscopic approach and a dedicated computer assisted navigation system is a safe and efficient procedure. Early management is recommended to treat neonatal airway obstruction.