Data_Sheet_1_Visualization of the Membranous Labyrinth and Nerve Fiber Pathways in Human and Animal Inner Ears Using MicroCT Imaging.pdf

<p>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.</p

Data_Sheet_2_Visualization of the Membranous Labyrinth and Nerve Fiber Pathways in Human and Animal Inner Ears Using MicroCT Imaging.pdf

<p>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.</p

Primary antibodies used for immunohistochemistry.

<p>Primary antibodies used for immunohistochemistry.</p

Tumour microtissue formation.

<p>SEM pictures were taken after ten days: Monocultures of A549 and SV80 were seeded in a ratio of 2500 cells/40 μl, whereas Colo699 monocultures were cultured in form of 1250 cells/40 μl. All co-cultures were seeded in a carcinoma cell: fibroblast ratio of 1∶2/40 μl (A549 co-cultures: 2500 cancer cells +5000 fibroblasts / Colo699 co-cultures: 1250 cancer cells +2500 fibroblasts). (A/B) A549 monocultures, (C/D) A549 co-cultures; (E/F) Colo699 monocultures, (G/H) Colo699 co-cultures; (I/J) SV80 monocultures. All co-cultures displayed a more homogenous and rounder microtissue surface compared to monocultures.</p

Cytokeratin 7 expression pattern.

<p>IHC staining of A549, SV80 monocultures and A549 co-cultures after ten days (Bar in 1–3: 100 μm). Cytokeratin 7 staining could only be observed in cancer cells but not in SV80 cells.</p

SV80 microtissue protein expression pattern.

<p>IHC slices of SV80 in monocultures after ten days (Bar in A: 100 μm); SV80 microtissue staining is only shown representatively after ten days because no difference in the staining pattern between five and ten days was detected. Microtissues were completely positive for vimentin and Ki67, whereas no E-Cadherin and α-SMA expression was observed.</p

A549 microtissue protein expression pattern.

<p>IHC slices of A549 in mono- and co-cultures after five and ten days (Bar in A-D: 100 μm, bar in Insert: 25 μm); an increase in vimentin and a simultaneous decrease in E-Cadherin is displayed in co-cultures. Also an upregulation of Ki67 expression in co-cultures was detected.</p

Growth curve and Cell proliferation/activation pattern.

<p><b>A</b>: Microtissue volumes were calculated by the formula V = 4/3*Π*r3, where r = ½*√ d1*d2. Mean volumes are displayed in μm3 with their corresponding standard deviations. <b>B</b>: Mean and standard deviation of Ki67 positivity was calculated by counting Ki67 positive and negative cell nuclei on slices of three different spheroids representatively. Thereafter, the percentage of positive cell nuclei to whole cell cumber was calculated. Co-cultivation of both tumour cell lines with a fibroblast cell line led to a significant upregulation (p<0,05) of Ki67 expression in all microtissues.</p

Extracellular matrix expression pattern.

<p>Fibronectin was displayed in microtissues after ten days. (Bar in A549/SV80 monocultures: 50 μm, bar in all other microtissues: 100 μm); Secretion of fibronectin could be observed in microtissues containing fibroblasts, whereas in all tumour cell monocultures no fibronectin secretion could be detected.</p

Summary of IHC protein expression pattern. +/- displays the presence/absence of the indicated protein.

<p>Summary of IHC protein expression pattern. +/- displays the presence/absence of the indicated protein.</p