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.
Highlights
In the United States, nearly 800,000 people have a stroke annually, making it the 5th leading cause of death and the leading cause of adult disability (Benjamin et al, 2018)
While much research has been performed on post-stroke neuroplasticity and neuroinflammation (Gelderblom et al, 2009; Bachmann et al, 2014), these studies have been limited by conventional imaging methods
STPT, a modern microscopy technique that produces large-scale, 3-D images of intact, uncleared brain tissue, is expanding the capacity to investigate brain-wide circuit mechanisms associated with brain injury, as well as models of neurological and psychiatric disorders
Summary
In the United States, nearly 800,000 people have a stroke annually, making it the 5th leading cause of death and the leading cause of adult disability (Benjamin et al, 2018). Given that leukocytes modulate axonal growth after injury through the secretion of cytokines and growth factors (Wang et al, 2018), it is possible that migration of immune cells, such as CD8+ T cells, to remote brain regions could affect post-stroke plasticity far from the stroke lesion. This highlights the need to understand where neuroplasticity and neuroinflammation occur in the brain after stroke, and if the two phenomena are interconnected. Our results indicate that mesoscale whole-brain imaging reveals important information about region-specific changes in both neural connectivity and neuroinflammation that may be key to developing long-term therapies to promote functional recovery after stroke
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