Histological Examination of Mitochondrial Morphology in a Parkinson's Disease Model
June 23rd, 2023
This study presents a method to analyze the morphology of mitochondria based on immunostaining and image analysis in mouse brain tissue in situ. It also describes how this allows one to detect changes in mitochondrial morphology induced by protein aggregation in Parkinson's disease models.
Our laboratory investigates the cellular mechanism involving protein aggregation in the context of Parkinson's disease. In addition, we use cellular and animal models for testing potential treatments against Parkinson's disease. In this article, we propose a protocol for the study of mitochondrial morphology in situ as an indicator of their health based on immunostaining and image analysis in paraffin-embedded tissue sections.
Mitochondrial morphology has been extensively studied in Parkinson's disease and other diseases in vitro. Nevertheless, protocols for studying mitochondrial morphology in vivo are still lacking. This protocol provides a robust method for staining mitochondria in brain tissue, and it explains in details how to perform the morphological analysis.
This study shows that immunostaining combined with image analysis is a reliable method for understanding mitochondrial morphology. In fact, it allows to quantify the number of mitochondria as well as other morphological parameters, such as the aspect ratio in both cell culture and tissue. Importantly, as this method is immunofluorescence based, it is possible to relate the morphologies back to specific cellular populations.
Here we present a protocol for studying mitochondrial morphology in Parkinson's disease, but this protocol can be applied to any in vivo disease model. In addition, it may aid screening lead compounds for the treatment of not only Parkinson disease, but also any other disease characterized by mitochondrial dysregulation. To begin, take the paraffin brain sections of PFF-injected C57 black six mice.
Dewax the slides by dipping them in xylene substitute and incubating them for two minutes. For rehydration, dip the slides in 100%95%and 70%ethanol, and then two times in deionized water. Transfer the samples into a microwaveable container along with double distilled water.
Warm the 100 times citrate buffer with pH six at four degrees Celsius. Then prepare 350 milliliters of fresh citrate buffer. Pour the prepared citrate buffer into a plastic container with a lid.
Place the slides into the citrate buffer container and secure the lid. Microwave the samples at 700 watts for four minutes, and then allow time for a five minute rest. Microwave for another 1.5 minutes, followed by a five minute rest period, and replenish the citrate buffer.
Microwave for 1.5 minutes, and let it rest for five minutes. Then cool the samples and citrate buffer on ice for 20 minutes and wash them with double distilled water. Dry the sample slides using a paper towel without touching the tissue.
Using a pap pen, draw rectangles around the pieces of tissue. Now transfer all slides to a slide immune-staining box. Gently wash them multiple times with TBS-T ensuring TBS-T drops stay within the pap pen rectangles.
Tap the slides on a paper towel to remove TBS-T. Add 50 microliters of blocking to each rectangle and incubate for one hour at room temperature. At the end of the incubation, using a paper towel, remove the blocking solution from the samples.
Gently pipette 50 microliters of the primary antibody mixture in TBS-T to each rectangle and incubate the samples overnight at four degrees Celsius. Then wash the slides with TBS-T. Remove the TBS-T by tapping on a paper towel.
Repeat this process three times. Next, add 50 microliters of secondary antibody mixture at one to 1000 dilution in TBS-T to each rectangle and incubate at 37 degrees Celsius for one hour in the dark. Afterward, wash the sample three times with TBS-T.
Then incubate the sample with DAPI at a concentration of two micrograms per milliliter and TBS-T for one minute. Remove the DAPI solution by tapping on a paper towel and wash three times with TBS-T. To mount the glass cover slip, add two drops per slide of mounting reagent to the samples.
Press the cover slip gently to eliminate bubbles and let the samples dry for one hour at room temperature. Image at least 50 cells in various fields using a structured illumination fluorescence imaging system equipped with a 60 times oil objective or a confocal technology. To analyze the cells, isolate the region of interest by drawing a contour around the positive or negative cell and using the crop function.
Next, activate shape descriptors and limit to threshold options in the set measurement function. Adjust the threshold level by selecting the threshold function in the adjust menu. Finally, use the analyze particles tool and set an appropriate size to capture the mitochondria.
For example, set the size to 25 infinity, then activate pixel units and select show masks. Substantia nigra pars compacta of PFF injected mice co-immunostained for tyrosine Hydroxylase, VDAC1, PS129 alpha-synuclein and DAPI are shown. Anti PS129 alpha-synuclein staining allowed to discriminate cells that harbored PS129 lesions from healthy cells.
Image analysis of VDAC1 staining of tyrosine Hydroxylase positive neurons revealed a reduction of both mitochondrial number counts and aspect ratio between neurons bearing PS129 lesions and neurons lacking them.
Overview
This study presents a protocol for analyzing mitochondrial morphology in situ using immunostaining and image analysis on mouse brain tissue. It focuses on detecting morphological changes induced by protein aggregation in Parkinson's disease models.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Parkinson's Disease Research
Background
- Mitochondrial morphology is critical for understanding cellular health.
- Protein aggregation is a key feature in Parkinson's disease pathology.
- Existing protocols for in vivo mitochondrial studies are lacking.
- Immunostaining can relate mitochondrial health to specific cell populations.
Purpose of Study
- To provide a reliable method for assessing mitochondrial morphology.
- To enable the quantification of mitochondrial parameters in brain tissue.
- To study the impacts of protein aggregation on mitochondria in a disease model.
Methods Used
- Utilized immunofluorescence staining on paraffin-embedded tissue sections.
- Focused on paraffin brain sections from PFF-injected C57 black six mice.
- The study includes detailed steps for preparing, staining, and imaging samples.
- Image analysis was performed to quantify mitochondrial morphology.
Main Results
- Reduction in mitochondrial counts and altered morphology were observed in neurons with PS129 lesions compared to healthy neurons.
- The method successfully discriminated between healthy and affected neuronal populations.
- Quantification of morphological parameters highlighted significant differences related to disease state.
Conclusions
- This study establishes a robust protocol for mitochondrial analysis in disease models, which can facilitate screening of treatment candidates.
- The method provides insights into mitochondrial health related to Parkinson's disease.
- Outcomes may enhance understanding of cellular mechanisms involved in neurodegeneration.
Frequently Asked Questions
Executive Industry Relevance
Quantitative assessment of mitochondrial morphology in situ enables early detection of neurodegenerative pathology and supports mechanistic de-risking in CNS drug discovery. This protocol provides a robust workflow for linking mitochondrial health to disease-relevant neuronal populations, informing target validation and lead prioritization in Parkinson's disease models. Integrating high-content morphological analysis strengthens predictive confidence at the discovery and preclinical interface.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of mitochondrial dysfunction as a mechanistic driver in neurodegeneration.
- Supports functional target validation by quantifying morphological changes in disease-relevant neurons.
- Facilitates biological de-risking by distinguishing affected from healthy cell populations.
- Provides quantitative endpoints for hypothesis-driven portfolio triage.
Screening & Assay Development
- Prepares validated tissue-based systems for downstream compound screening.
- Delivers reproducible, quantitative morphological outputs for assay standardization.
- Enables high-content screening readiness for evaluating lead compounds affecting mitochondrial health.
- Supports platform reuse across neurodegenerative disease models with mitochondrial involvement.
Translational & Preclinical Research
- Aligns morphological readouts with disease-relevant biomarkers in preclinical models.
- Ensures continuity from discovery-stage findings to in vivo validation of therapeutic hypotheses.
- Informs risk-adjusted advancement decisions by linking cellular pathology to functional outcomes.
- Provides predictive de-risking for translational biomarker strategies in CNS portfolios.
Pipeline & Workflow Integration
This protocol integrates into the discovery-to-preclinical continuum, bridging early mechanistic studies with in vivo validation in Parkinson's disease models.
- Discovery Biology: Supports hypothesis testing on mitochondrial dysfunction and its role in neurodegeneration.
- Screening: Provides quantitative, reproducible morphological metrics for compound evaluation.
- Analytics: Enables statistical comparison of mitochondrial parameters across experimental conditions.
- Translational Research: Connects cellular pathology to disease-relevant endpoints in animal models.
- Enterprise Reuse: Offers a standardized, scalable workflow adaptable to other models of mitochondrial dysregulation.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in neurodegenerative target validation.
- Operational Value: Delivers standardized, reproducible, and scalable tissue-based morphological analysis.
- Strategic Value: Improves go/no-go decisions and capital efficiency by providing robust quantitative endpoints.
- Portfolio Impact: Enables risk-adjusted prioritization and advancement of CNS therapeutic candidates.
Implementation Considerations
- Requires expertise in immunostaining, fluorescence imaging, and image analysis.
- Needs access to structured illumination or confocal microscopy platforms.
- Demands cross-team standardization of antibody selection and image quantification parameters.
- Adaptable to various in vivo disease models with mitochondrial involvement.
- Potential limitations include antibody specificity and tissue heterogeneity affecting measurement accuracy.
