Visualization and Quantification of Post-Stroke Neural Connectivity and Neuroinflammation Using Serial Two-Photon Tomography in the Whole Mouse Brain

Authors

Katherine Poinsatte, University of Texas Southwestern Medical Center
Dene Betz, University of Texas Southwestern Medical Center
Vanessa O. Torres, University of Texas Southwestern Medical Center
Apoorva D. Ajay, University of Texas Southwestern Medical Center
Shazia Mirza, University of Texas Southwestern Medical Center
Uma M. Selvaraj, University of Texas Southwestern Medical Center
Erik J. Plautz, University of Texas Southwestern Medical Center
Xiangmei Kong, University of Texas Southwestern Medical Center
Sankalp Gokhale, University of Texas Southwestern Medical Center
Julian P. Meeks, University of Texas Southwestern Medical Center
Denise M. O. Ramirez, University of Texas Southwestern Medical Center
Mark P. Goldberg, University of Texas Southwestern Medical Center
Ann M. Stowe, University of KentuckyFollow

Abstract

Whole-brain volumetric microscopy techniques such as serial two-photon tomography (STPT) can provide detailed information on the roles of neuroinflammation and neuroplasticity throughout the whole brain post-stroke. STPT automatically generates high-resolution images of coronal sections of the entire mouse brain that can be readily visualized in three dimensions. We developed a pipeline for whole brain image analysis that includes supervised machine learning (pixel-wise random forest models via the “ilastik” software package) followed by registration to a standardized 3-D atlas of the adult mouse brain (Common Coordinate Framework v3.0; Allen Institute for Brain Science). These procedures allow the detection of cellular fluorescent signals throughout the brain in an unbiased manner. To illustrate our imaging techniques and automated image quantification, we examined long-term post-stroke motor circuit connectivity in mice that received a motor cortex photothrombotic stroke. Two weeks post-stroke, mice received intramuscular injections of pseudorabies virus (PRV-152), a trans-synaptic retrograde herpes virus driving expression of green fluorescent protein (GFP), into the affected contralesional forelimb to label neurons in descending tracts to the forelimb musculature. Mice were sacrificed 3 weeks post-stroke. We also quantified sub-acute neuroinflammation in the post-stroke brain in a separate cohort of mice following a 60 min transient middle cerebral artery occlusion (tMCAo). Naive e450⁺-labeled splenic CD8⁺ cytotoxic T cells were intravenously injected at 7, 24, 48, and 72 h post-tMCAo. Mice were sacrificed 4 days after stroke. Detailed quantification of post-stroke neural connectivity and neuroinflammation indicates a role for remote brain regions in stroke pathology and recovery. The workflow described herein, incorporating STPT and automated quantification of fluorescently labeled features of interest, provides a framework by which one can objectively evaluate labeled neuronal or lymphocyte populations in healthy and injured brains. The results provide region-specific quantification of neural connectivity and neuroinflammation, which could be a critical tool for investigating mechanisms of not only stroke recovery, but also a wide variety of brain injuries or diseases.

Document Type

Article

Publication Date

10-4-2019

Notes/Citation Information

Published in Frontiers in Neuroscience, v. 13, article 1055.

© 2019 Poinsatte, Betz, Torres, Ajay, Mirza, Selvaraj, Plautz, Kong, Gokhale, Meeks, Ramirez, Goldberg and Stowe.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Digital Object Identifier (DOI)

https://doi.org/10.3389/fnins.2019.01055

Funding Information

This study was funded by grants to AS from NIH/NINDS (NS088555), to AS from the Dana Foundation David Mahoney Neuroimaging Program, to VT from the NIH Intergrative Immunology Training Program (5T32AI005284-40) the Haggerty Center for Brain Injury and Repair, and the Texas Institute for Brain Injury and Repair (TIBIR) (UTSW). SG was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under award Number KL2TR001103. PRV-152 was generously provided by Dr. Lynn Enquist (Princeton University, Princeton, NJ) through the Center for Neuroanatomy with Neurotropic Viruses (NIH P40 OD010996).

Related Content

All raw data is immediately available upon request.

Repository Citation

Poinsatte, Katherine; Betz, Dene; Torres, Vanessa O.; Ajay, Apoorva D.; Mirza, Shazia; Selvaraj, Uma M.; Plautz, Erik J.; Kong, Xiangmei; Gokhale, Sankalp; Meeks, Julian P.; Ramirez, Denise M. O.; Goldberg, Mark P.; and Stowe, Ann M., "Visualization and Quantification of Post-Stroke Neural Connectivity and Neuroinflammation Using Serial Two-Photon Tomography in the Whole Mouse Brain" (2019). Neurology Faculty Publications. 54.
https://uknowledge.uky.edu/neurology_facpub/54