Visualization of the Membranous Labyrinth and Nerve Fiber Pathways in Human and Animal Inner Ears Using MicroCT Imaging

Design and implantation of bionic implants for restoring impaired hair cell function relies on accurate knowledge about the microanatomy and nerve fiber pathways of the human inner ear and its variation. Non-destructive isotropic imaging of soft tissues of the inner ear with lab-based microscopic X-ray computed tomography (microCT) offers high resolution but requires contrast enhancement using compounds with high X-ray attenuation. We evaluated different contrast enhancement techniques in mice, cat, and human temporal bones to differentially visualize the membranous labyrinth, sensory epithelia, and their innervating nerves together with the facial nerve and middle ear. Lugol’s iodine potassium iodine (I2KI) gave high soft tissue contrast in ossified specimens but failed to provide unambiguous identification of smaller nerve fiber bundles inside small bony canals. Fixation or post-fixation with osmium tetroxide followed by decalcification in EDTA provided superior contrast for nerve fibers and membranous structures. We processed 50 human temporal bones and acquired microCT scans with 15 μm voxel size. Subsequently we segmented sensorineural structures and the endolymphatic compartment for 3D representations to serve for morphometric variation analysis. We tested higher resolution image acquisition down to 3.0 μm voxel size in human and 0.5 μm in mice, which provided a unique level of detail and enabled us to visualize single neurons and hair cells in the mouse inner ear, which could offer an alternative quantitative analysis of cell numbers in smaller animals. Bigger ossified human temporal bones comprising the middle ear and mastoid bone can be contrasted with I2KI and imaged in toto at 25 μm voxel size. These data are suitable for surgical planning for electrode prototype placements. A preliminary assessment of geometric changes through tissue processing resulted in 1.6% volume increase caused during decalcification by EDTA and 0.5% volume increase caused by partial dehydration to 70% ethanol, which proved to be the best mounting medium for microCT image acquisition

Human Cochlear Nerve Model: Data Collection and Simulation

Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheMotivation. Neurone des menschlichen Hörsystems weisen mikroanatomische Besonderheiten auf, welche sie von denen anderer Säugetierarten signifikant unterscheiden. Ihre unmyelinisierten Zellsomata gemeinsam mit ihrem gelegentlichen Auftreten als Cluster mit direktem Zellmembrankontakt führen zu Vermutungen, dass diese die neuronalen Muster bei der Signalweiterleitung erheblich beeinflussen. Des Weiteren wird die Anregbarkeit der Neurone bei extrazellulärer Stimulation mittels Cochlea-Implantate durch den inhomogenen anatomischen Aufbau der Cochlea als auch durch den 3D Verlauf der Neurone selbst, stark beeinflusst. Histologische Methoden, kombiniert mit bildgebenden Verfahren und Visualisierungstechniken wurden in der vorliegenden Arbeit genutzt, um morphometrische Eigenschaften des menschlichen Hörnervs zu eruieren. Diese geometrischen Parameter wurden anschließend in ein Computermodell eines auditiven Neurons implementiert um seine Anregbarkeit sowie sein Feuerungsverhalten bei intra- als auch extrazellulärer Stimulation zu analysieren. Methodik. Die präsentierten anatomischen Ergebnisse basieren auf mehreren menschlichen Hörschnecken und bilden das Fundament für die anschließend durchgeführten Simulationen. Da gesunde und lebende menschliche Hörneurone experimentell nicht zugänglich sind, bilden Computersimulationen eine adäquate Methode um mehr über ihr Verhalten zu erfahren. Immunhistochemische Färbungen in Kombination mit Konfokalmikroskopie ermöglichen die Akquirierung von 3D-Daten von Hörneuronen, um verschiedenste geometrische Parameter systematisch entlang der Cochlea zu quantifizieren. Diese Methode hat jedoch den großen Nachteil, dass die Probe in dünne Schnitte aufgeteilt werden muss, was einerseits zu Informationsverlust, andererseits eine computergestützte Rekonstruktion nahezu unmöglich gestaltet. Deshalb wurde eine menschliche Cochlea in einem hochauflösendem mikroCT gescannt. Datensegmentierung und Visualisierung ermöglichten die 3D-Rekonstruktion von Nervenverläufen. Der CT Datensatz wurde weiters verwendet, um ein anatomisch präzises Finite-Elemente-Modell der menschlichen Cochlea zu entwickeln. Dieses wurde genutzt um die von aktiven Elektroden induzierten elektrischen Potentiale entlang der rekonstruierten Faserverläufe zu bestimmen. Die resultierenden Daten wurden anschließend in das biophysikalische Modell implementiert, um die Anregbarkeit und das zeitliche Verhalten eines Neurons bei verschiedenen Stimulationsstrategien zu testen. Resultate Hierarchische Clusteranalyse an volumetrischen Daten von unmyelinisierten Hörneuronen lieferte Hinweise auf die Existenz von vier verschiedenen Populationen beim Menschen. Die rekonstruierten Volumina variieren erheblich was zu unterschiedlichen Laufzeiten von Aktionspotentialen über das Zellsoma führt. Dies impliziert wiederum unterschiedliche zeitliche Parameter bei der Signalweiterleitung ins zentrale Nervensystem. Die zeitlichen Parameter als auch die Geschwindigkeit der generierten elektrischen Signale sind vor allem vom Durchmesser des peripheren- und zentralen Axons und von der Neuronlänge abhängig. Aufgrund der sich nach oben hin verengenden Anatomie der Hörschnecke, zeigen apikale Neurone einen sehr spiraligen Verlauf und haben signifikant längere periphere Axone verglichen mit Neuronen aus dem mittleren bzw. basalen Bereich. Aus diesem Grund weisen die durchgeführten Finite-Elemente- und Kompartmentmodell Analysen auf unterschiedliche Anregungsprofile bei Neuronen aus verschiedenen Frequenzbereichen hin. Außerdem wurde die Verletzung des tonotopischen Prinzips von aktiven Elektroden, welche sich im mittleren Bereich der Hörschnecke befinden, festgestellt.Objectives. Human auditory neurons show micro-anatomical peculiarities which differ considerably from other mammalian species. Their unmyelinated cell bodies as well as their appearance in clusters featuring direct cell-to-cell contact, compose unique morphological characteristics rising questions concerning the spiking pattern of these human sensory neurons. Besides, the anatomical composition of the cochlea representing an inhomogeneous structure as well as the three dimensional (3D) pathways of the action potential (AP) transmitting neural parts mainly affect the excitability of neurons to micro-stimulation induced by electrodes of cochlear implants (CI). Histological methods combined with imaging and visualization techniques were used to gain morphometrical features of human cochlear neurons which were subsequently implemented to a computational model of an auditory neuron to test its excitability and spiking pattern to intra- as well as extracellular stimulation. Methods. The anatomical findings presented in this thesis are based on several human cochleae and compose the fundament of the subsequent computer simulations which provide an appropriate method for single cell analysis since vital human auditory neurons are not experimentally accessible. Immunohistochemical staining and confocal laser scanning microscopy enabled to acquire 3D image stacks of cochlear neurons where geometrical parameters were quantified systematically along the cochlear spiral. However, this method requires sectioning of the specimen which causes loss of information of the whole analyzed structure making accurate computational reconstruction virtually impossible. To overcome this limitation, one human inner ear was digitalized using ultra-high resolution micro-CT imaging. Visualization and segmentation of data enabled to reconstruct the 3D pathways of tonotopical aligned cochlear neurons. CT data was subsequently used to develop an anatomical exact finite element model (FEM) of the human cochlea which was deployed to calculate the resulting distribution of electrical potentials induced by electrodes along the reconstructed auditory nerve fibers. Implementation of finite element data to a compartment model of a human cochlear neuron enabled to analyze its excitability and temporal behavior to different stimulation strategies. Results. Hierarchical cluster analysis of acquired volumetric data of unmyelinated human auditory cell somata indicates the existence of four distinct populations of auditory neurons within the human hearing organ. The volume varies enormously resulting in delay differences of the generated AP while passing the somatic region which consequently influences the temporal parameters of signal transduction to the central nervous system. Moreover, these temporal parameters as well as the conduction velocity of APs are additionally depending on peripheral- and central process diameters and process length. Due to the narrowing anatomy of this coiled organ, apical neurons show highly spiral 3D pathways and feature significantly longer peripheral processes compared to middle- or basal turn neurons. As a consequence, finite element- and compartment model analysis suggests varying excitation profiles for nerve fibers originating from different frequency regions and the breach of the tonotopical principle by active electrodes of CIs located in the middle turn region. Conclusion. The combination of biological data collection methods, visualization of their results, mathematical modeling and computer simulation becomes increasingly important in the field of neuroscience or when refining bionic devices. Taking the human anatomical occurrences for CIs into account will tremendously improve the development of new electrode array configurations and stimulation strategies to restore the hearing sense more accurate. However, the results presented in this thesis provide new insights into the morphometrical and anatomical structure of the human cochlea and demonstrate the behavior of cochlear neurons in an anatomically precise computational environment.17

Compartment models for SGNs.

<p>(A) Type I cells, rectified: Myelinated segments are shown in gray. Excitable (active) membranes with high ion channel densities (red segments) in the peripheral terminal and in the nodes of Ranvier are needed for spike amplification. In contrast to feline cells, in man the pre- and postsomatic compartments are longer, the soma is larger and not myelinated and the peripheral as well as the central axons are longer. (B) According to Ohm's law the sum of all currents to the center of a compartment is zero. The currents are defined by extracellular potential V_e, intracellular potential V_i, membrane capacitance C_m, membrane conductance G_m and intracellular resistance R. Natural excitation by synaptic current from a hair cell ribbon synapse is simulated as current injection into the first compartment (peripheral terminal). In this case extracellular potentials V_e are assumed to be zero. For nonmyelinated type II cells the same modeling approach was used with uniform ion channel densities as in the original Hodgkin-Huxley model and with constant compartment lengths in the axons. (C) The same neural pathway of a human type I cell model as used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079256#pone.0079256-Rattay3" target="_blank">[7]</a> is placed over a cross section of a feline cochlea demonstrating a possible position of a scala tympani electrode relative to a target cell. The length relations are the same as in the rectified versions in A. Extracellular potentials are calculated for a homogeneous infinite medium which causes spherical isopotentials, indicated by dashed lines. Note that the cat soma is much closer to the electrode than the human one.</p

Immunofluorescence of MBP, TuJ1 and peripherin in cat (A, B) and human (C, D) spiral ganglion.

<p>(A, middle turn region) shows a myelinated type II neuron in a cat inner ear. The arrow highlights the peripherin positive cell body of a type II cochlear neuron; the arrow head depicts its isolating myelin. (B, basal region) illustrates two unmyelinated type II neurons (white arrows) which were (partly) surrounded by myelin of neighboring type I neurons. Additionally, the arrow head points to a type I SGN which was fully surrounded by myelin. Note the absence of these surrounding myelin layers at the depicted type II neurons. A myelinated type I neuron (white arrow head) and an unmyelinated type II neuron (white arrow) of the human spiral ganglion are shown in (C, basal turn). D (middle turn) presents a human type II cell body surrounded by myelin (white arrow) and type I neurons surrounded by myelin (white arrow heads). Scale bars 30 µm.</p

Computed SGN spike conduction times with additional delay Δt per 1 µm soma diameter increase.

<p>d1 and d2 represent peripheral and central axon diameters; nmsoma denotes the number of surrounding single membrane layers in the soma region including the pre- and postsomatic segments. t1, t2, t3 and t4 denote postsynaptic delay, spike conduction time in the peripheral axon, presomatic delay and spike conduction time in the central axon, respectively. t_total  = t1+t2+t3+t4, Δt|dsoma+1 µm denotes the enlargement step of the presomatic delay when dsoma is 1 µm increased.</p

Temporal profiles of transmembrane voltages and extracellular potentials of an extracellularly stimulated feline type I cell.

<p>A small ball electrode simulates the situation of monopolar cathodic stimulation with a cochlear implant for a situation shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079256#pone-0079256-g001" target="_blank">Figure 1C</a>. (A) During application of the 100 µs stimulus pulse the voltage across the membrane is influenced in each compartment. For this electrode placement the threshold is reached in the peripheral terminal and therefore the SGN excitation is similar to natural signaling. The transmembrane voltage lines, shifted vertically according to their distance along the neural path, show AP conductance; myelinated compartment responses in dark gray, compartments with voltage sensitive ion channels in red. (B) The short spike duration is demonstrated with the redrawn transmembrane voltage of the presomatic compartment. (C) Simulated extracellular potential for the position of the center of the stimulating electrode. (D) Simulated recorded signal for natural synaptic excitation, modeled as current injection into the first compartment (E) Simulated (blue, copy of C) and experimentally recorded (black) intracochlear voltage profiles generated with a cochlear implant show similar temporal characteristics although the simulated single cell activity is compared with a compound action potential recording. The black curve is redrawn from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079256#pone.0079256-Miller1" target="_blank">[51]</a> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079256#pone-0079256-g001" target="_blank">Figure 1</a>, intracochlear recording, cathodic pulse −11.1 dB rel. 1 mA). Simulated situations correspond to scala tympani stimulation in the basal turn. Electrode position and neural path as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079256#pone-0079256-g001" target="_blank">Figure 1C</a>; homogeneous extracellular medium with extracellular resistivity of 300 Ohm.cm and other data as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079256#pone.0079256-Rattay3" target="_blank">[7]</a>.</p

Peripheral and central process diameters of type I neurons based on 3 human specimens and 3 cat specimens.

<p>Data according to their location along the cochlea spiral.</p

SGN response to strong and weak synaptic stimuli.

<p>(A) Postsynaptic currents from rat experiments are characterized by amplitude, time to peak and time constant for decay. (B–E) responses of a type I SGN with parameters from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079256#pone-0079256-g008" target="_blank">Figure 8A</a> are shown for compartments # 1, 3, 5 and 25. Reduction from a typical synaptic current amplitude (B) to threshold (C) caused an essentially longer delay. Including ion current fluctuations (noisy membrane current model, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079256#pone.0079256-Rattay3" target="_blank">[7]</a>) in all compartments with active channels resulted in sharply synchronized responses for strong stimulation (D) and in late responses with large jitter (E). Compartment 25 is the fifth postsomatic node of Ranvier in the central process and represents the main part of the expected jitter at the proximal axon ending.</p

Transmission electron microscopy images of human SGNs.

<p>(A) Cell body of a putative type I SGN completely enwrapped with myelin. Additionally, the process of the SGN shows continuous myelination (white arrow heads). The standard human SGN is shown in B. White arrows highlight an unmyelinated cell body encircled by a satellite glial cell and the myelin lacking process of a SGN. The myelination of the central process starts after about 7 µm pointed by the white arrow head. Scale bar 10 µm.</p