Voelcker Academy

Research Symposium 2011


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Pavan Patamalla

Design-based Stereological Analysis of Degeneration of Dopaminergic Neurons in the MitoPark Mouse Model of Parkinson’s Disease

Pavan Patamalla

Mentor(s): Dr. Senlin Li MD

Parkinson’s disease (PD) affects over 1 million Americans and is a neurodegenerative disorder that is characterized by progressive degeneration of the dopaminergic neurons located in a region of the brain known as the substantia nigra. Most animal models of PD used in academic research involve the use of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP is a neurotoxin and kills dopaminergic neurons quickly. Because of the rapidity with which the neurotoxin takes hold, tracking the cell loss in conjunction with the symptoms and the progression of the disease is quite difficult. The MitoPark mouse model provides the advantage of modeling a prolonged course of neurodegeneration that shows the typical phenotype of the progression of the disease. By inactivation of mitochondrial transcription factor A, a critical protein for mitochondrial DNA expression, dopamine neurons are selectively rendered respiratory chain deficient.

To further characterize this model, design based stereology was developed with The Zeiss AxioImager.M2 microscope and MicroBrightField’s Stereo Investigator software. Results obtained with design-based stereology are independent of the shape, size, spatial orientation, and spatial distribution of the cells, representing a methodology that can limit bias to a minimum in quantitative neuromorphology. The optical fractionator is a probe specifically designed for unbiased counting of cells through the NvVref method, which doesn’t require a reference volume, making it the method of choice. The method uses mean cell density within the unbiased virtual counting spaces by using random sampling for the investigated counting spaces, thereby minimizing the coefficient of error.

Two groups of 3 mice were used to examine the progressive degeneration of dopamine neurons in the substantia nigra. The mice were sacrificed at 28 weeks of age, brain sectioned and immunostained for dopamine neuron markers. Designed based stereological analysis was performed on both the control group and the MitoPark group of mice. The data revealed neuronal loss of 51.04% in the substantia nigra of MitoPark mice, compared with that of control group (averaging 4,660 versus 9,518.69 neurons neurons). The results are consistent with other estimates in the literature. Therefore, this unbiased method for neuron counting will certainly strengthen our on-going research in treatment of the progression of Parkinson’s disease.

Acknowledgements: Special thanks for Wenrui for stereological training, Dr. Chen for help with serial sectioning and immunostaining, and Senlin Li for overall support by being an amazing mentor and providing the calibration slide for the Zeiss AxioImager.M2 microscope.