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

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

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article number1055
JournalFrontiers in Neuroscience
Volume13
DOIs
StatePublished - Oct 4 2019

Fingerprint

Photons
Stroke
Tomography
Brain
Forelimb
Middle Cerebral Artery Infarction
Suid Herpesvirus 1
Neuronal Plasticity
Workflow
Atlases
Intramuscular Injections
Motor Cortex
Brain Diseases
Green Fluorescent Proteins
Brain Injuries
Microscopy
Software
Lymphocytes
Pathology
Viruses

Keywords

  • CD8 T cells
  • corticospinal tract
  • neural connectivity
  • neuroinflammation
  • pseudorabies virus
  • serial two-photon tomography
  • stroke
  • whole brain imaging

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

Visualization and Quantification of Post-stroke Neural Connectivity and Neuroinflammation Using Serial Two-Photon Tomography in the Whole Mouse Brain. / 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.; Stowe, Ann M.

In: Frontiers in Neuroscience, Vol. 13, 1055, 04.10.2019.

Research output: Contribution to journalArticle

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. ; Stowe, Ann M. / Visualization and Quantification of Post-stroke Neural Connectivity and Neuroinflammation Using Serial Two-Photon Tomography in the Whole Mouse Brain. In: Frontiers in Neuroscience. 2019 ; Vol. 13.
@article{e1fd4997f23d4755ae3fa25f3b86d631,
title = "Visualization and Quantification of Post-stroke Neural Connectivity and Neuroinflammation Using Serial Two-Photon Tomography in the Whole Mouse Brain",
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.",
keywords = "CD8 T cells, corticospinal tract, neural connectivity, neuroinflammation, pseudorabies virus, serial two-photon tomography, stroke, whole brain imaging",
author = "Katherine Poinsatte and Dene Betz and Torres, {Vanessa O.} and Ajay, {Apoorva D.} and Shazia Mirza and Selvaraj, {Uma M.} and Plautz, {Erik J.} and Xiangmei Kong and Sankalp Gokhale and Meeks, {Julian P.} and Ramirez, {Denise M.O.} and Goldberg, {Mark P.} and Stowe, {Ann M.}",
year = "2019",
month = "10",
day = "4",
doi = "10.3389/fnins.2019.01055",
language = "English (US)",
volume = "13",
journal = "Frontiers in Neuroscience",
issn = "1662-4548",
publisher = "Frontiers Research Foundation",

}

TY - JOUR

T1 - Visualization and Quantification of Post-stroke Neural Connectivity and Neuroinflammation Using Serial Two-Photon Tomography in the Whole Mouse Brain

AU - Poinsatte, Katherine

AU - Betz, Dene

AU - Torres, Vanessa O.

AU - Ajay, Apoorva D.

AU - Mirza, Shazia

AU - Selvaraj, Uma M.

AU - Plautz, Erik J.

AU - Kong, Xiangmei

AU - Gokhale, Sankalp

AU - Meeks, Julian P.

AU - Ramirez, Denise M.O.

AU - Goldberg, Mark P.

AU - Stowe, Ann M.

PY - 2019/10/4

Y1 - 2019/10/4

N2 - 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.

AB - 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.

KW - CD8 T cells

KW - corticospinal tract

KW - neural connectivity

KW - neuroinflammation

KW - pseudorabies virus

KW - serial two-photon tomography

KW - stroke

KW - whole brain imaging

UR - http://www.scopus.com/inward/record.url?scp=85073698328&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85073698328&partnerID=8YFLogxK

U2 - 10.3389/fnins.2019.01055

DO - 10.3389/fnins.2019.01055

M3 - Article

C2 - 31636534

AN - SCOPUS:85073698328

VL - 13

JO - Frontiers in Neuroscience

JF - Frontiers in Neuroscience

SN - 1662-4548

M1 - 1055

ER -