Semi-automated quantification of hair cells in the mature mouse utricle

Cathy Yea Won Sung; Melanie Barzik; Tucker Costain; Lizhen Wang; Lisa L Cunningham

doi:10.1016/j.heares.2021.108429

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Semi-automated quantification of hair cells in the mature mouse utricle

Journal article

Open access

Peer reviewed

Semi-automated quantification of hair cells in the mature mouse utricle

Cathy Yea Won Sung, Melanie Barzik, Tucker Costain, Lizhen Wang and Lisa L Cunningham

Hearing research, Vol.416, pp.108429-108429

03/15/2022

DOI: 10.1016/j.heares.2021.108429

PMCID: PMC9034969

PMID: 35081508

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https://doi.org/10.1016/j.heares.2021.108429View

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Abstract

The mouse utricle model system is the best-characterized ex vivo preparation for studies of mature mammalian hair cells (HCs). Despite the many advantages of this model system, efficient and reliable quantification of HCs from cultured utricles has been a persistent challenge with this model system. Utricular HCs are commonly quantified by counting immunolabeled HCs in regions of interest (ROIs) placed over an image of the utricle. Our data indicate that the accuracy of HC counts obtained using this method can be impacted by variability in HC density across different regions of the utricle. In addition, the commonly used HC marker myosin 7a results in a diffuse cytoplasmic stain that is not conducive to automated quantification and must be quantified manually, a labor-intensive task. Furthermore, myosin 7a immunoreactivity is retained in dead HCs, resulting in inaccurate quantification of live HCs using this marker. Here we have developed a method for semi-automated quantification of surviving HCs that combines immunoreactivity for the HC-specific transcription factor Pou4f3 with labeling of activated caspase 3/7 (AC3/7) to detect apoptotic HCs. The discrete nuclear Pou4f3 signal allowed us to utilize the binary or threshold function within ImageJ to automate HC quantification. To further streamline this process, we created an ImageJ macro that automates the process from raw image loading to a final quantified image that can be immediately evaluated for accuracy. Within this quantified image, the user can manually correct the quantification via an image overlay indicating the counted HC nuclei. Pou4f3-positive HCs that also express AC3/7 are subtracted to yield accurate counts of surviving HCs. Overall, we present a semi-automated method that is faster than manual HC quantification and identifies surviving HCs with high accuracy.

Animals

Gene Expression Regulation

Hair

Hair Cells, Auditory

Mice

Saccule and Utricle

Transcription Factors

Details

Title: Subtitle: Semi-automated quantification of hair cells in the mature mouse utricle
Creators: Cathy Yea Won Sung - National Institute on Deafness and Other Communication Disorders
Melanie Barzik - National Institute on Deafness and Other Communication Disorders
Tucker Costain - National Institute on Deafness and Other Communication Disorders
Lizhen Wang - National Institute on Deafness and Other Communication Disorders
Lisa L Cunningham - National Institute on Deafness and Other Communication Disorders
Resource Type: Journal article
Publication Details: Hearing research, Vol.416, pp.108429-108429
DOI: 10.1016/j.heares.2021.108429
PMID: 35081508
PMCID: PMC9034969
NLM abbreviation: Hear Res
ISSN: 0378-5955
eISSN: 1878-5891
Grant note: ZIA DC000079 / Intramural NIH HHS
Language: English
Date published: 03/15/2022
Academic Unit: Biology
Record Identifier: 9984961047502771

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