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Automated quantification of dopaminergic immunostained neurons in substantia nigra using freely available software

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Abstract

Computerized techniques for image analysis are critical for progress in cell biology. The complexity of the data in current methods eliminates the need for manual image analysis and usually requires the application of multiple algorithms sequentially to the images. Our aim was to develop a software for immunohistochemical analysis of brain dopaminergic neurons combining several computational approaches to automatically analyze and quantify their number in the substantia nigra after a neurotoxic injury. For this purpose, we used a Parkinson’s disease animal model to test our application. The dopaminergic neurotoxin, 6-hydroxydopamine, was administered in adult male rats to damage dopaminergic neurons in substantia nigra and to induce hemiparkinsonism. The lesion was corroborated by behavioral evaluation in response to apomorphine and amphetamine. The animals were euthanized and their brains processed for tyrosine hydroxylase immunohistochemistry for dopamine neuron identification. Neurons positive for tyrosine hydroxylase were evaluated in substantia nigra by light microscopy. The images were used to show quantification applicability. To test our software counting accuracy and validity, automatic dopamine neuron number was correlated with the data obtained by three independent observers. Several parameters were used to depict neuronal function in dataset images from control and lesioned brains. In conclusion, we could perform an automated quantification of dopaminergic neurons and corroborate the validity and accuracy of a freely available software.

Highlights

Automated technique developed using free software

Application to enhance efficiency and scope of neuroscience studies based on immunostaining

Automated computational method for cellular and molecular brain research

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Acknowledgements

We thank Franco Emiliano Nieto Grimalt for his support in the data curation and visualization; JMJ for animal care; and CM, SEG, and Instituto de Bioingeniería (IBio) for their expert technical support.

Funding

Dirección de Investigaciones, Universidad de Mendoza, Mendoza, Argentina. Resolutions 70/2016, 81/2016 and 159/2017, grants N° 0038 and 0013; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) PIP 11220100100126/11, and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) PICT 2015–1052.

Author information

Author notes

  1. Susana Ruth Valdez and Ricardo Jorge Cabrera have made equal contribution.

Authors and Affiliations

  1. Instituto de Investigaciones Biomédicas (INBIOMED), Universidad de Mendoza, Huarpes 630, 5500, Mendoza, Argentina

    María Paula Bonaccorso Marinelli & Ricardo Jorge Cabrera

  2. Instituto de Medicina y Biología Experimental de Cuyo (IMBECU)-CONICET, Universidad Nacional de Cuyo, at Av. Dr. Adrián Ruiz Leal, 5500, Mendoza, Argentina

    María Paula Bonaccorso Marinelli & Ricardo Jorge Cabrera

  3. Instituto de Bioingeniería-Facultad de Ingeniería-Universidad de Mendoza, Av. Boulogne Sur Mer 683, 5500, Mendoza, Argentina

    María Paula Bonaccorso Marinelli

  4. Laboratorio de Neurofarmacología, (IIBYT-CONICET), Universidad Nacional de Córdoba, Facultad de Ciencias Químicas, Universidad Católica de Córdoba, Córdoba, Argentina

    Gustavo Baiardi

  5. Laboratorio de Reproducción y Lactancia, Instituto de Medicina y Biología Experimental de Cuyo (IMBECU)-CONICET, Universidad Nacional de Cuyo, Av. Dr. Adrián Ruiz Leal, 5500, Mendoza, Argentina

    Susana Ruth Valdez

  6. Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Cuyo, at Padre J. Contreras 1300, 5500, Mendoza, Argentina

    Susana Ruth Valdez

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  1. María Paula Bonaccorso Marinelli
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  2. Gustavo Baiardi
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  3. Susana Ruth Valdez
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  4. Ricardo Jorge Cabrera
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Contributions

María Paula Bonaccorso Marinelli: conceptualization, software, formal analysis, investigation, writing original draft and editing, visualization.

Gustavo Baiardi: writing review, supervision, final approval.

Susana Ruth Valdez: methodology, investigation, resources, project administration, funding acquisition.

Ricardo Jorge Cabrera: conceptualization, methodology, investigation, resources, writing review and editing, supervision, funding acquisition.

Corresponding author

Correspondence to Ricardo Jorge Cabrera.

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Bonaccorso Marinelli, M.P., Baiardi, G., Valdez, S.R. et al. Automated quantification of dopaminergic immunostained neurons in substantia nigra using freely available software. Med Biol Eng Comput 60, 2995–3007 (2022). https://doi.org/10.1007/s11517-022-02643-8

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  • DOI: https://doi.org/10.1007/s11517-022-02643-8

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