Short-Chain Fatty Acids Improve Poststroke Recovery via Immunological Mechanisms.

Recovery after stroke is a multicellular process encompassing neurons, resident immune cells, and brain-invading cells. Stroke alters the gut microbiome, which in turn has considerable impact on stroke outcome. However, the mechanisms underlying gut-brain interaction and implications for long-term recovery are largely elusive. Here, we tested the hypothesis that short-chain fatty acids (SCFAs), key bioactive microbial metabolites, are the missing link along the gut-brain axis and might be able to modulate recovery after experimental stroke. SCFA supplementation in the drinking water of male mice significantly improved recovery of affected limb motor function. Using in vivo wide-field calcium imaging, we observed that SCFAs induced altered contralesional cortex connectivity. This was associated with SCFA-dependent changes in spine and synapse densities. RNA sequencing of the forebrain cortex indicated a potential involvement of microglial cells in contributing to the structural and functional remodeling. Further analyses confirmed a substantial impact of SCFAs on microglial activation, which depended on the recruitment of T cells to the infarcted brain. Our findings identified that microbiota-derived SCFAs modulate poststroke recovery via effects on systemic and brain resident immune cells.SIGNIFICANCE STATEMENT Previous studies have shown a bidirectional communication along the gut-brain axis after stroke. Stroke alters the gut microbiota composition, and in turn, microbiota dysbiosis has a substantial impact on stroke outcome by modulating the immune response. However, until now, the mediators derived from the gut microbiome affecting the gut-immune-brain axis and the molecular mechanisms involved in this process were unknown. Here, we demonstrate that short-chain fatty acids, fermentation products of the gut microbiome, are potent and proregenerative modulators of poststroke neuronal plasticity at various structural levels. We identified that this effect was mediated via circulating lymphocytes on microglial activation. These results identify short-chain fatty acids as a missing link along the gut-brain axis and as a potential therapeutic to improve recovery after stroke.

SpinalTRAQ: A novel volumetric cervical spinal cord atlas identifies the corticospinal tract synaptic projectome in healthy and post-stroke mice.

Descending corticospinal tract (CST) connections to the neurons of the cervical spinal cord are vital for performance of forelimb-specific fine motor skills. In rodents, CST axons are almost entirely crossed at the level of the medullary decussation. While specific contralateral axon projections have been well-characterized using anatomic and molecular approaches, the field currently lacks a cohesive imaging modality allowing rapid quantitative assessment of the entire, bilateral cervical cord projectome at the level of individual laminae and cervical levels. This is potentially important as the CST is known to undergo marked structural remodeling in development, injury, and disease. We developed SpinalTRAQ (Spinal cord Tomographic Registration and Automated Quantification), a novel volumetric cervical spinal cord atlas and machine learning-driven microscopy acquisition and analysis pipeline that uses serial two-photon tomography- images to generate unbiased, region-specific quantification of the fluorescent pixels of anterograde AAV-labeled CST pre-synaptic terminals. In adult mice, the CST synaptic projectome densely innervates the contralateral hemicord, particularly in laminae 5 and 7, with sparse, monosynaptic input to motoneurons in lamina 9. Motor pools supplying axial musculature in the upper cervical cord are bilaterally innervated. The remainder of the ipsilateral cord has sparse labeling in a distinct distribution compared to the contralateral side. Following a focal stroke of the motor cortex, there is a complete loss of descending corticospinal axons from the injured side. Consistent with prior reports of axon collateralization, the CST spinal projectome increases at four weeks post-stroke and continues to elevate by six weeks post stroke. At six weeks post-stroke, we observed striking synapse formation in the denervated hemicord from the uninjured CST in a homotopic distribution. Additionally, CST synaptic reinnervation increases in the denervated lamina 9 in nearly all motoneuron pools, exhibiting novel patterns of connectivity. Detailed level- and lamina-specific quantification of the bilateral cervical spinal cord synaptic projectome reveals previously undescribed patterns of CST connectivity in health and injury-related plasticity.

Using Operant Reach Chambers to Assess Mouse Skilled Forelimb Use After Stroke.

Skilled forelimb reaching and grasping are important components of rodent motor performance. The isometric pull task can serve as a tool for quantifying forelimb function following stroke or other CNS injury as well as in forelimb rehabilitation. This task has been extensively developed for use in rats. Here, we describe methods of setup and training of an operant reach chamber for mice. Using a reward of peanut oil, mice are adaptively trained to pull a handle positioned slightly outside of an operant chamber, with automated recording of the number of attempts, force generated, success rate, and latency to maximal force.

Forelimb Cortical Stroke Reduces Precision of Motor Control in Mice.

Background and Objective. Rodent models of stroke impairment should capture translatable features of behavioral injury. This study characterized poststroke impairment of motor precision separately from strength in an automated behavioral assay. Methods. We measured skilled distal forelimb reach-and-grasp motions within a target force range requiring moderate-strength. We assessed whether deficits reflected an increase in errors on only one or both sides of the target force range after photothrombotic cortical stroke. Results. Pull accuracy was impaired for 6 weeks after stroke, with errors redistributing to both sides of the target range. No decrease in maximum force was measured. Conclusions. This automated reach task measures sustained loss of motor precision following cortical stroke in mice.

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

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.