title: Spiral ganglion neuron quantification in the guinea pig cochlea using Confocal Laser Scanning Microscopy compared to embedding methods.
abstract: Neuron counting in the cochlea is a crucial but time-consuming operation for which various methods have been developed. To improve simplicity and efficiency, we tested an imaging method of the cochlea, and based on Confocal Laser Scanning Microscopy (CLSM), we visualised Rosenthal's Canal and quantified the spiral ganglion neurons (SGN) within. Cochleae of 8 normal hearing guinea pigs and one implanted with a silicone filament were fixed in paraformaldehyde (PFA), decalcified, dehydrated and cleared in Spalteholz solution. Using the tissue's autofluorescence, CLSM was performed at 100 fold magnification generating z-series stacks of about 20 slices of the modiolus. In 5 midmodiolar slices per cochlea the perimeters of the Rosenthal's Canal were surveyed, representative neuron diameters were measured and the neurons first counted manually and then software-assisted. For comparison, 8 normal hearing guinea pig cochleae were embedded in paraffin and examined similarly. The CLSM method has the advantage that the cochleae remain intact as an organ and keep their geometrical structure. Z-stack creation is nearly fully-automatic and frequently repeatable with various objectives and step sizes and without visible bleaching. The tissue shows minimal or no shrinking artefacts and damage typical of embedding and sectioning. As a result, the cells in the cleared cochleae reach an average diameter of 21 μm and a density of about 18 cells/10,000 μm(2) with no significant difference between the manual and the automatical counts. Subsequently we compared the CLSM data with those generated using the established method of paraffin slides, where the SGN reached a mean density of 9.5 cells/10,000 μm(2) and a mean soma diameter of 13.6 μm. We were able to prove that the semi-automatic CLSM method is a simple and effective technique for auditory neuron count. It provides a high grade of tissue preservation and the automatic stack-generation as well as the counter software reduces the effort considerably. In addition this visualisation technique offers the potential to detect the position and orientation of cochlear implants (CI) within the cochlea and tissue growing in the scala tympani around the CI and at the position of the cochleostomy due to the fact that the implant does not have to be removed to perform histology as in case of the paraffin method.
authors: Department of Otolaryngology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Electronic address: wrzeszcz.antonina@mh-hannover.de.
authors: Antonina
authors: A
authors: Wrzeszcz
authors: Günter
authors: G
authors: Reuter
authors: Ingo
authors: I
authors: Nolte
authors: Thomas
authors: T
authors: Lenarz
authors: Verena
authors: V
authors: Scheper
mesh_terms: D000818:Animals; D001331:Automation; D003051:Cochlea / Q000473:pathology*; D003054:Cochlear Implants; D003638:Deafness / Q000473:pathology; D004492:Edetic Acid / Q000737:chemistry; D016057:Evoked Potentials, Auditory, Brain Stem / Q000502:physiology; D005557:Formaldehyde / Q000737:chemistry; D006168:Guinea Pigs; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal; D009474:Neurons / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D012533:Scala Tympani / Q000473:pathology; D012984:Software; D013136:Spiral Ganglion / Q000378:metabolism / Q000473:pathology*
chemical_list: D011108:Polymers; D005557:Formaldehyde; D004492:Edetic Acid; C003043:paraform
keywords: CI; CLSM; Confocal Laser Scanning Microscopy; EDTA; HE; MSBB; PFA; SGN; aABR; acoustically evoked auditory brainstem response; cochlear implant; ethylenediamine tetraacetic acid-disodium salt; hematoxylin and eosin; methyl salicylate benzyl benzoate; paraformaldehyde; spiral ganglion neurons