Cochlear Specimens Research Articles

A method for accurate and reproducible specimen alignment for insertion tests of cochlear implant electrode arrays

PurposeThe trajectory along which the cochlear implant electrode array is inserted influences the insertion forces and the probability for intracochlear trauma. Controlling the trajectory is especially relevant for reproducible conditions in electrode insertion tests. Using ex vivo cochlear specimens, manual alignment of the invisibly embedded cochlea is imprecise and hardly reproducible. The aim of this study was to develop a method for creating a 3D printable pose setting adapter to align a specimen along a desired trajectory toward an insertion axis.MethodsPlanning points of the desired trajectory into the cochlea were set using CBCT images. A new custom-made algorithm processed these points for automated calculation of a pose setting adapter. Its shape ensures coaxial positioning of the planned trajectory to both the force sensor measuring direction and the insertion axis. The performance of the approach was evaluated by dissecting and aligning 15 porcine cochlear specimens of which four were subsequently used for automated electrode insertions.ResultsThe pose setting adapter could easily be integrated into an insertion force test setup. Its calculation and 3D printing was possible in all 15 cases. Compared to planning data, a mean positioning accuracy of 0.21 ± 0.10 mm at the level of the round window and a mean angular accuracy of 0.43° ± 0.21° were measured. After alignment, four specimens were used for electrode insertions, demonstrating the practical applicability of our method.ConclusionIn this work, we present a new method, which enables automated calculation and creation of a ready-to-print pose setting adapter for alignment of cochlear specimens in insertion test setups. The approach is characterized by a high level of accuracy and reproducibility in controlling the insertion trajectory. Therefore, it enables a higher degree of standardization in force measurement when performing ex vivo insertion tests and thereby improves reliability in electrode testing.

A method for image-guided positioning of cochlear specimens in insertion test benches using 3D printed stands

Abstract Cochlear implants include an electrode array (EA) which needs to be inserted into the cochlea. Insertion tests using artificial cochlear models (ACM) or ex vivo specimens are widely used methods during EA development to characterize EA design properties, including insertion forces. Measured forces are directly linked to the orientation of the cochlear lumen with respect to the insertion axis of the test bench. While desired insertion directions in ACM experiments can be predefined by design, specimens are individually shaped and the cochlear lumen is embedded invisibly. Therefore, a new method for accurate, individual specimen positioning is required. A key element of the proposed method is a customizable pose setting adapter (PSA) used to adjust the specimen’s fine positioning. After rigid fixation of the specimen to a holder featuring spherical registration markers and subsequent cone beam computed tomography the desired insertion direction is planned. The planned data is used to calculate the individual shape of the PSA. Finally, the PSA is 3D printed and mounted between force sensor and specimen holder to correctly align the specimen to the test bench’s insertion axis. All necessary hard- and software have been developed including the specimen holder, a software for registration and trajectory planning, and a custom Matlab script whose output drives a parametric CAD file of the PSA. Positioning accuracy was determined in a first trial using 10 virtual trajectories and was found to be 0.23 ± 0.12 mm and 0.38 ± 0.17°. The presented stereotactic positioning procedure enables high repeatability in future ex vivo insertion experiments due to accurate, image-guided control of the insertion direction.

Anatomical Variations of the Human Cochlea Determined from Micro-CT and High-Resolution CT Imaging and Reconstruction.

Understanding the anatomical variations in the human cochlea is important for cochlear implants. This study examined these variations using a reconstructed fusion model of the skull and temporal bone. The three-dimensional (3D) digital model of the temporal bone was reconstructed from multiple axial micro-computed tomography (CT) scans of temporal bone and high resolution CT of the skull from 15 cadavers. A skull model was reconstructed and merged with the reconstructed temporal bone. The 3D relationship between the cochlea and the skull's mid-sagittal plane was analyzed. The α and β angles of the cochlear autogenous rotation and bottom position, respectively, further subdivided the cochlear spatial orientation. The relationship between the base of the cochlea and the round window was evaluated with the Φ angle. Cochlear size was measured and the relationship was statistically analyzed. Cochlear implant electrode arrays were observed in five cases of right-temporal bone specimens. The α, β, and Φ angles were 46.01 ± 9.65, 56.79 ± 3.58, and 44.41 ± 7.23, respectively. The α angle varied greatly and was negatively correlated to the Φ angle (correlation coefficient = -0.211, P<0.05). Among the five specimens, the α and Φ angle of the 2R and 4R cochlear specimens was lower and higher than the mean value, respectively. These measurements revealed variations in the size and position of the cochlea. Some of these variations may require surgical adjustments for insertion of electrodes with cochlear implants and present a greater challenge for implantation of cochlear electrode implantation. These data also provide a better understanding of variations in human cochlear anatomy. Anat Rec, 301:1086-1095, 2018. © 2017 Wiley Periodicals, Inc.

Morphological Study of Surgically Obtained Human Cochlear Specimens - Technical Aspects

ObjectTo explore the procedures in per-operative harvesting and management of fresh human cochlear specimens for research. MethodsDuring trans-cochlear surgery to remove large petro-clival meningiomas causing life-threatening compression on the brainstem, cochleae are normally destroyed and drilled away in order to reach the apical petrous and clivus region. Instead the cochlea can be dissected out after ethical permission was obtained from the local ethical committee (EPN) and allowance gained from the patients. Surgery is performed by a team consisting of oto- and neurosurgeons as a two-day procedure with total petrosectomy in combination with an inferior re-routing of the facial nerve. Fixation of the cochleae was done in the operating room as soon as the specimens had been separated from the temporal bones. Decalcification began after hours’ to overnight’s fixation for 4 weeks. Sectioning parallel to the modiolus (mid-modiolus) was performed with a cryostat microtome. The sections were subjected to immunofluorescence (IF). ResultsUsing freshly prepared 4% paraformaldehyde (PFD) solution, adequate fixation of fine inner ear structures was achieved with hours’ immersion of the cochlear specimens. Decalcification in 6.2% ethylene diamine-tetracetic acid (EDTA) solution for 4 weeks yielded a thoroughly decalcified cochlea. Experiences in processing 14 human cochleae and analysing main landmarks in five human inner ear plastic/silicone casts showed that the oval window/stapes footplate are backward tilted, at an angle about 15 degrees, from the plane perpendicular to the modiolar axis. The distance from the modiolar apex to the anterior border of the oval window/footplate in these inner ear casts measured between 4 and 5 mm. High quality IF staining was obtained. ConclusionSurgically obtained human cochlear specimen, when properly processed, contains ideally preserved antigenicity for immunohistochemical study. Adequate orientation during sectioning helps obtain optimal mid-modiolar sections showing fine structures of the cochlea.

Ultrastructural pathological changes in the cochlear cells of connexin 26 conditional knockout mice

Mutations in the gene of connexin 26 (Cx26) are the most common cause of human non-syndromic hereditary deafness. The pathogenesis of deafness caused by Cx26 remains uncertain. To explore the basic mechanism underlying Cx26 null mutations, ultrastructural changes and a number of marker proteins in the cochlear sensory epithelium of Cx26 conditional knockout mice were observed in the current study. Cochlear specimens were obtained from Cx26 conditional knockout mice (cCx26ko), while wild-type mice served as controls. Antibodies against the pillar cell marker P75, the supporting cell marker prox1 and hair cell markers myosin 6 and phalloidin were labeled in different cells of the cochlear sensory epithelium of cochlear cryosections. The ultrastructural morphology of cochlear sensory epithelium was observed using transmission electron microscopy. Following the observation of cochlear sensory epithelium cell markers for hair cells and supporting cells, no significant changes were observed at the early stage, while the tunnel of the organ of Corti and Nuel’s space was not developed prior to hearing onset in cCx26 knockout mice. Cell death was observed from postnatal day 10 (P10). The only region of surviving cells observed in the cochlea was the Hensen cell region, where microglia-like cells appeared following P180. Overall, the present study showed an abnormal ultrastructural morphology in the cochlear sensory epithelium in cCx26ko mice. Microglia-like cells may be involved in the process of cell degeneration in cCx26ko mice.

The expression of glutamate aspartate transporter (GLAST) within the human cochlea and its distribution in various patient populations

Glutamate plays an important role in the central nervous system as an excitatory neurotransmitter. However, its abundance can lead to excitotoxicity which necessitates the proper function of active glutamate transporters. The glutamate-aspartate transporter (GLAST) has been shown to exist and function within non-human cochlear specimens regulating the inner ear glutamate concentration. In this study, we examined human cochleas from formalin-fixed celloidin-embedded temporal bone specimens of three different types of patients (Meniere's disease, normal controls, and other otopathologic conditions) and examined the differential expression of GLAST in the spiral ligament of the basal, middle, and apical turns of the cochlea. Immunohistochemical staining was performed with polyclonal antibodies against GLAST and image analysis was carried out with the Image J analysis software. In contrast to other studies with non-human specimens, GLAST was expressed in the spiral ligament fibrocytes but was not detected in the satellite cells of the spiral ganglia or supporting cells of the Organ of Corti in the human cochlea. Our data also showed that GLAST expression significantly differs in the basal and apical turns of the cochlea. Lastly, post-hoc analysis showed a difference in the GLAST immunoreactive area of patients with Meniere's disease when compared to that of patients with other otopathologic conditions—such as presbycusis or ototoxicity. These results may potentially lead to further understanding of different disease states that affect hearing.

Expression of TrkB and BDNF in human cochlea—an immunohistochemical study

Surgical human cochlear specimens were obtained during the removal of large posterior cranial fossa meningioma by a transcochlear approach in which the cochlea was removed for maximal exposure of the tumor and protection of important structures, such as the brainstem, cranial nerves, and pivotal blood vessels. The cochlear tissue was fixed and cryo-sectioned for tyrosine kinase receptor B (TrkB) and brain-derived neurotrophic factor (BDNF) immunohistochemistry. TrkB receptor protein was expressed in both neuronal somata and the processes of human spiral ganglion neurons (SGNs). In the human organ of Corti, TrkB immunoreactivity was mainly present in nerve fibers underneath outer hair cells. BDNF expression was found neither in the organ of Corti nor in the spiral ganglion of human cochlea. For antibody specificity and for control and comparative purposes, TrkB immunocytochemistry was performed in primary cultures of cochlear neuron/glia from adult guinea pig. Confocal laser scanning microscopy showed that TrkB was homogeneously distributed in the cytoplasm of both neuronal somata and axons. Knowledge of the expression of TrkB receptor in human cochlea should help to determine the target structures for neuron preservation in hearing-impaired patients. Our results indicate that the regeneration of SGNs under pathological conditions can be enhanced with BDNF/TrkB-based pharmaceutical or genetic strategies.

Expression of myelin basic protein in the human auditory nerve—An immunohistochemical and comparative study

The aim of this study is to analyse the expression and distribution of myelin basic protein (MBP or Myelin A1 protein) in the human spiral ganglion and auditory nerve. Cryostat sections were made from freshly fixed human cochlear specimens removed at surgery in patients with life-threatening petro-clival meningiomas compressing the brain stem. The sections were subjected to immunohistochemistry using antibodies against MBP, S-100 and Tubulin. The immunoreaction was documented using laser confocal microscopy. Type I spiral ganglion nerve somata (SGN) were surrounded by so-called "satellite glial cells" (SGCs) that lacked expression of MBP consistent with earlier light and electron microscopic findings indicating that these cells are non-myelinating. S-100 labeling showed that the SGCs form a continuous network in the apical region. The pattern of myelination in human spiral ganglion is different from that in other species' spiral ganglion. The striking differences in myelin outline should be investigated further in combination with its influence on signal coding and preservation properties in man.

Inner ear pathology of alpha-galactosidase A deficient mice, a model of Fabry disease

Objective Fabry disease is characterized by genetic alpha-galactosidase A deficiency, resulting in accumulation of glycolipids (GL-3) and tissue damage. Hearing loss is also common and attributed to GL-3 accumulation in the inner ear. The only reported histological studies dealt with murine and human specimens. Accordingly, histopathological studies of the cochlea were performed on an alpha-galactosidase A deficient murine model of Fabry disease, using C57BL6/J mice as the controls. Methods The hearing ability was evaluated using the ABR threshold, while cochlear specimens were observed light microscopically and ultrathin temporal bone sections by TEM. Results HE staining showed no accumulation of GL-3 or abnormal cochlear morphology in the alpha-galactosidase A deficient mice, but toluidine blue staining and TEM revealed GL-3 accumulation in the stria vascularis and kidney. No GL-3 accumulation was detected in the C57BL6/J controls by either HE staining or TEM. The alpha-galactosidase A deficient mice and the controls showed no clear differences in the ABR threshold (hearing acuity), but for older animals the threshold was higher in the C57BL6/J controls. Conclusion In summary, although the alpha-galactosidase A deficient mice showed no clear hearing loss, GL-3 accumulation was demonstrated in the cochlea.

A Mouse Model for Degeneration of the Spiral Ligament

Previous studies have indicated the importance of the spiral ligament (SL) in the pathogenesis of sensorineural hearing loss. The aim of this study was to establish a mouse model for SL degeneration as the basis for the development of new strategies for SL regeneration. We injected 3-nitropropionic acid (3-NP), an inhibitor of succinate dehydrogenase, at various concentrations into the posterior semicircular canal of adult C57BL/6 mice. Saline-injected animals were used as controls. Auditory function was monitored by measurements of auditory brain stem responses (ABRs). On postoperative day 14, cochlear specimens were obtained after the measurement of the endocochlear potential (EP). Animals that were injected with 5 or 10 mM 3-NP showed a massive elevation of ABR thresholds along with extensive degeneration of the cochleae. Cochleae injected with 1 mM 3-NP exhibited selective degeneration of the SL fibrocytes but alterations in EP levels and ABR thresholds were not of sufficient magnitude to allow for testing functional recovery after therapeutic interventions. Animals injected with 3 mM 3-NP showed a reduction of around 50% in the EP along with a significant loss of SL fibrocytes, although degeneration of spiral ganglion neurons and hair cells was still present in certain regions. These findings indicate that cochleae injected with 3 mM 3-NP may be useful in investigations designed to test the feasibility of new therapeutic manipulations for functional SL regeneration.

Effect of a Beta-Stimulant on the Inner Ear Stria Vascularis

In vivo and in vitro experiments were conducted to investigate the effect of alpha-isoproterenol on the inner ear stria vascularis with intracellular cytochrome oxidase activity used as an index. Intraperitoneal injection of alpha-isoproterenol (5 mg/kg) was performed in 10 rats, and that of physiological saline in 4 rats, for 21 consecutive days. After the 3-week treatment, bilateral cochleas were excised for frozen sections and stained for cytochrome oxidase. The staining density of the stria vascularis for the enzyme was analyzed with a computer. Electron microscopic observation was also performed for some specimens. As for the in vitro experiments, bilateral cochleas from 6 normal rats were excised for cell culture. Cochlear cells from the right ear were cultured with medium containing alpha-isoproterenol (10-micromol/L concentration), and those from the left ear with medium alone. After 3-day culture, the enzyme activity of cytochrome oxidase in the stria vascularis was quantified by the same method used for the in vivo experiments. Cytochrome oxidase activity was markedly elevated in the alpha-isoproterenol group. The activity tended to be higher in the lower turns of the cochlea. Electron microscopy revealed that numerous mitochondria were present in marginal cells that protruded into the endolymphatic space. The enzyme activity was also elevated in the stria vascularis from cochlear specimens in the alpha-isoproterenol group of the in vitro experiment. The above results suggest that alpha-isoproterenol accelerated the metabolic activity of the cells that constitute the stria vascularis. The increase in activity was probably attributable to direct pharmaceutical effects of the beta-stimulant, rather than an increase in blood flow. It is possible that the cells that constitute the stria vascularis may have beta-receptors.

Postnatal development of the organ of Corti in cats: a light microscopic morphometric study

This study quantitatively characterizes the development of the major morphological features of the organ of Corti during the first 2 weeks postnatal, the period when the cat auditory system makes the transition from being essentially non-functional to having nearly adult-like responses. Four groups of kittens ( n=3) were studied at one day postnatal (P1), P5, P10, P15, and compared to adults. Measurements were made of the organ of Corti at 3 cochlear locations: 20%, 60% and 85% of basilar membrane length from the base – cochlear locations which in the adult correspond to best frequencies of ≈20 kHz, 2 kHz and 500 Hz, respectively. In addition, measurements of basilar membrane length and opening of the tunnel of Corti were made in 20 cochlear specimens from kittens aged P0–P6. Results indicate that: (i) at P0 the basilar membrane has attained adult length, and the tunnel of Corti is open over approximately the basal one-half of the cochlea; (ii) the initial opening of the tunnel of Corti occurs at a site about 4 mm from the cochlear base (best frequency of ≈25 kHz in the adult cochlea); (iii) the thickness of the tympanic cell layer decreases markedly at the basal 20-kHz location; (iv) the areas of the tunnel of Corti and space of Nuel and the angulation of the inner hair cells (IHC) relative to the basilar membrane all show marked postnatal increases at both the middle and apical locations; (v) IHC are nearly adult-like in length and shape at birth, whereas the OHC (at 2-kHz and 500-Hz locations) undergo marked postnatal changes; (vi) disappearance of the marginal pillars and maturation of the supporting cells are not yet complete by P15.

A technique for protecting delicate specimens during processing

The sensory end organ of the ear, the organ of Corti, rests on a thin basilar membrane which lies between the bone of the central modiolus and the bony wall of the cochlea. In vivo, the organ of Corti is protected by the bony wall which totally surrounds it. In order to examine the sensory epithelium by scanning electron microscopy it is necessary to dissect away the protective bone and expose the region of interest (Fig. 1). This leaves the fragile organ of Corti susceptible to physical damage during subsequent handling. In our laboratory cochlear specimens, after dissection, are routinely prepared by the O-T- O-T-O technique, critical point dried and then lightly sputter coated with gold. This processing involves considerable specimen handling including several hours on a rotator during which the organ of Corti is at risk of being physically damaged. The following procedure uses low cost, readily available materials to hold the specimen during processing ,preventing physical damage while allowing an unhindered exchange of fluids.Following fixation, the cochlea is dehydrated to 70% ethanol then dissected under ethanol to prevent air drying. The holder is prepared by punching a hole in the flexible snap cap of a Wheaton vial with a paper hole punch. A small amount of two component epoxy putty is well mixed then pushed through the hole in the cap. The putty on the inner cap is formed into a “cup” to hold the specimen (Fig. 2), the putty on the outside is smoothed into a “button” to give good attachment even when the cap is flexed during handling (Fig. 3). The cap is submerged in the 70% ethanol, the bone at the base of the cochlea is seated into the cup and the sides of the cup squeezed with forceps to grip it (Fig.4). Several types of epoxy putty have been tried, most are either soluble in ethanol to some degree or do not set in ethanol. The only putty we find successful is “DUROtm MASTERMENDtm Epoxy Extra Strength Ribbon” (Loctite Corp., Cleveland, Ohio), this is a blue and yellow ribbon which is kneaded to form a green putty, it is available at many hardware stores.

Studies of ion distribution in the inner ear: scanning electron microscopy and x-ray microanalysis of freeze dried cochlear specimens

Secondary and backscattered scanning electron imaging were combined with energy dispersive X-ray analysis (EDXA) to investigate ion distribution in freeze-dried chinchilla inner ears. Fluid residues obtained from the perilymphatic and endolymphatic spaces consist of characteristically shaped crystals associated with an amorphous matrix. EDXA spectra obtained from crystals are consistent with the expected ion composition of the fluid of origin. In particular, potassium levels in perilymph and sodium levels in endolymph are very low. This indicates that the integrity of the major fluid compartments of the inner ear is maintained during freeze-drying, and that uncontaminated samples can routinely be obtained by microdissection. Spectra obtained from crystals on the underside of the tectorial membrane are typical of endolymph, strong evidence that this fluid occupies the subtectorial spaces. The spectra of vestibular endolymph crystals are essentially identical to those obtained from cochlear endolymph, indicating no difference in ionic composition of endolymph in the two divisions of the labyrinth. Fluid in the spaces of the organ of Corti does not form crystals, which suggests that the ‘cortilymph’ may contain more protein than perilymph or endolymph. Secondary scanning electron imaging of cochlear tissues revealed little or no disruption of cell membranes of most cochlear tissues, including outer hair cells and stria vascularis. The gross interior features of tissue, such as interior cell borders and cell nuclei, appeared well preserved when backscattered scanning electron imaging was employed. Backscattered imaging was found to be necessary for the identification of crystals of fluid origin upon the surfaces of tissue specimens.

Horseradish peroxidase as a label of injured cells

The present study is concerned with artifacts likely to occur in a horseradish peroxidase exclusion test. Incubation of murine peritoneal macrophages and lymphocytes with the peroxidase showed a close relationship between the number of living cells and the percentage of cells excluding the tracer. The penetration of the cytoplasm by horseradish peroxidase is attributed to an increase in the permeability of the cell membrane during the incubation (ranging from 10 to 120 min). It was not increased by the presence of tracer throughout the incubation period. However, concomitant fixation of the cell in the presence of horseradish peroxidase caused an increase in the influx of the tracer. The horseradish peroxidase exclusing test applied to the guinea-pig organ of Corti has proved to be valid provided that: (a) mechanical lesions prior to the tracer incubation are avoided; (b) incubation is terminated by removal of the extracellular tracer; (c) fixation is carried out as soon as possible; (d) a low concentration of horseradish peroxidase is used; and (e) specimens are incubated in diaminobenzidine-H2O2 medium for the shortest possible period. Although fixation-induced cytoplasmic infiltration by horseradish peroxidase was not detected in cochlear specimens, the findings call attention to possible sources of error and define the level of significance of the test. Horseradish peroxidase does not appear to be a cytotoxic agent under the conditions used.

A Technique for Preparation of Cochlear Specimens for Assessment with the Scanning Electron Microscope

(1978). A Technique for Preparation of Cochlear Specimens for Assessment with the Scanning Electron Microscope. Acta Oto-Laryngologica: Vol. 85, No. sup351, pp. 3-23.

A Technique for Preparation of Large Cochlear Specimens for Assessment with the Transmission Electron Microscope

A Technique for Preparation of Large Cochlear Specimens for Assessment with the Transmission Electron Microscope

Phosphotungstic acid staining of the organ of Corti for electron microscopy.

AbstractPhosphotungstic acid (PTA) 1% in absolute alcohol was used, according to Bloom and Aghajanian's ('66) method, during the final dehydration stage of glutaraldehyde‐fixed cochlear specimens which were processed for electron microscopic study without osmium treatment. Familiar structures emerged with additional characteristics. PTA stained supporting elements. Synaptic areas were emphasized and the synaptic bar of hair cells opposite afferent endings was densely stained. Dark clumps were found near the synaptic surface of efferent nerve endings that seemed to correspond to the material sometimes seen between vesicles near the synaptic cleft. A dense layer was present in hair cells opposite efferent terminals between the outer layer of the double membrane and the synaptic membrane. Formations resembling synapses between the nerve endings of outer hair cells were emphasized by PTA staining, but were difficult to find because of their rarity. It seems unlikely that synaptic interaction between cochlear nerve endings is vital in the basic physiology of hearing.PTA staining permits recognition of cochlear nerve ending type independent of hair cell features which will make it a useful aid for the study of the development of the inner ear and of pathological ear conditions.

DISTRIBUTION OF SOME OXIDIZING ENZYMES IN THE COCHLEA.

The cochlear specimens prepared by the freezing-method may be subjected successfully to the precise histochemical examination on the distribution of succinic dehydrogenase and/or DPN-diaphorase. The following results were obtained by this method.1. Concentration of both enzymes of both the stria vascularis and lower portions of the spiral ligament is larger in the lower turns than in the higher turns.2. There are no differences in the enzymic activities of the organ of Corti between each turn.