Spiral ganglion neuron quantification in the guinea pig cochlea using Confocal Laser Scanning Microscopy compared to embedding methods

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• 作者：Antonina Wrzeszcz ; G眉nter Reuter ; Ingo Nolte ; Thomas Lenarz ; Verena Scheper
• 关键词：CLSM ; Confocal Laser Scanning Microscopy ; SGN ; spiral ganglion neurons ; CI ; cochlear implant ; PFA ; paraformaldehyde ; aABR ; acoustically evoked auditory brainstem response ; EDTA ; ethylenediamine tetraacetic acid-disodium salt ; MSBB ; methyl salicylate benzyl benzoate ; HE ; hematoxylin and eosin
• 刊名：Hearing Research
• 出版年：December, 2013
• 年：2013
• 卷：306
• 期：Complete
• 页码：145-155
• 全文大小：3152 K

文摘

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 100fold 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聽渭m and a density of about 18聽cells/10,000聽渭m² 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聽cells/10,000聽渭m² and a mean soma diameter of 13.6聽渭m.

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.