3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Carla M. Cabral; Anita A. Koshy

doi:10.3791/52237

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Method Article

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

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DOI:

10.3791/52237

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December 9th, 2014

Carla M. Cabral¹ , Anita A. Koshy¹^,²^,³

¹Bio5 Institute, University of Arizona, ²Department of Neurology, University of Arizona, ³Department of Immunobiology, University of Arizona

Erratum Notice

Important: There has been an erratum issued for this article. View Erratum Notice

Summary

Using this protocol, we were able to image 160 µm thick brain sections from mice infected with the parasite Toxoplasma gondii, which enables visualization and analysis of the spatial relationship between the encysting parasite and the infected neuron.

Abstract

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 µm) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.

Introduction

The overall goal of this method is to obtain high resolution, three-dimensional images of individual neurons that are infected by the obligate intracellular parasite Toxoplasma gondii.

Toxoplasma is often considered one of the most successful parasites because of its large intermediate host range, which includes humans and rodents. In both humans and rodents, after acute infection through ingestion of contaminated food or water, Toxoplasma is able to cause a persistent infection of the CNS by converting from its fast replicating form (the tachyzoite) to its slow-replicating and encysting form (the bradyzoite). In ....