@inproceedings {Schulze1833_2016,
	year = {2016},
	author = {Schulze, Jennifer and Nolte, Lena and Lyutenski, Stefan and Tinne, Nadine and Ripken, Tammo and Willaredt, Marc and Nothwang, Hans Gerd and Lenarz, Thomas and Warnecke, Athanasia},
	title = {Combination of SLOT and Immunohistochemistry to Define Complex Morphological Phenotype Changes in Cochlear Structures, Exemplary Shown on Cav1.3 Knock-Out Mice},
	booktitle = {Assoc. Res. Otolaryng. MidWinter Meeting (ARO)},
	URL = {http://c.ymcdn.com/sites/www.aro.org/resource/resmgr/Abstract_Archives/UPDATED_2016_ARO_Abstract_Bo.pdf},
	abstract = {Overview
The present study focuses on the identification of
morphological differences of Cav1.3 knock-out mice in
comparison to wild type mice by application of different
methods. Cav1.3 knock-out mice lack the voltage-gated L-type
Ca2+ channels Cav1.3. This channel controls vesicle fusion
and subsequent neurotransmitter release from cochlear inner
hair cells to afferent auditory nerve fibers. Cav1.3 knock-out
mice display a developmental failure of cochlear structures.
This study aimed at the characterization of early and late
stage of the degenerative process in the inner ear in order
to identify suitable time-point for therapeutic interventions
concerning preservation of hair cell function and regeneration
of the auditory nerve.
Methods
Wild type mice and knock-out mice were used for the
isolation of the cochleae and fixed by immersion of PFA (4%).
For immunocytochemistry, the cochleae were dissected into
basal, medial and apical sections and these were stained
with CtBP2, Otoferlin and DAPI for counting the number of
synaptic ribbons per inner hair cell. For scanning laser optical
tomography (SLOT), decalcification, dehydration, optical
clearing and antibody staining of the whole cochlea were
performed. For visualization of myelinated nerve fibers, the
cochleae were stained with osmium.
Results
Coupling of SLOT and immunocytochemistry allowed the
overview of whole cochleae to show specific anatomical
structures and selective mapping of cellular structures (hair
cells and afferent nerve fibers respectively). Accordingly, the
number of synaptic ribbons in the Cav1.3 knock-out mice is
decreased along the different cochlear turns compared to
wild type mice as shown previously. The decrease starts
around postnatal day (P) 9 in the basal and medial turn of the
cochlea and around P20 in the apical turn. A slightly reduced
myelination and density of afferent auditory fibers was shown
by osmium staining of the cochlea at P18
Conclusions
This study reveals that SLOT is a suitable tool in the field of
otology for in toto visualization of the internal structure of the
cochlea. By means of the different methods, we showed that
the degenerative processes start as early as P9 in the basal
and medial turn of the cochleae of Cav1.3 knock-out mice and
proceed to the apical turn at about P20.
Funding
Cluster of Excellence “Hearing4all”}
}