Source Paper
V. Singh, S. Roth, G. Llovera, R. Sadler, D. Garzetti et al.
Journal of Neuroscience • 2016
We have identified a bidirectional communication along the brain-gut microbiota-immune axis and show that the gut microbiota is a central regulator of immune homeostasis. Acute brain lesions induced dysbiosis of the microbiome and, in turn, changes in the gut microbiota affected neuroinflammatory and functional outcome after brain injury. The microbiota impact on immunity and stroke outcome was transmissible by microbiota transplantation. Our findings support an emerging concept in which the gut microbiota is a key regulator in priming the neuroinflammatory response to brain injury. These findings highlight the key role of microbiota as a potential therapeutic target to protect brain function after injury.
Objective: To assess the causal impact of microbiota composition on stroke outcome by recolonizing germ-free mice with dysbiotic or normal microbiota and measuring lesion volume and functional deficits
This is a Germ-Free Mouse Recolonization protocol using mouse as the model organism. The procedure involves 10 procedural steps, 3 equipment items, 2 materials. Extracted from a 2016 paper published in Journal of Neuroscience.
Model and subjects
mouse • Not specified in provided text • unknown • Not specified in provided text • Not specified in provided text
Study window
Estimated timing pending
Core workflow
Establish germ-free mouse colony • Collect microbiota samples • Characterize microbiota composition
Primary readouts
Key equipment and reagents
Verified items
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Obtain or maintain germ-free mice for the recolonization experiment
Note: Germ-free mice serve as the baseline population to be recolonized
“Recolonizing germ-free mice with dysbiotic poststroke microbiota exacerbates lesion volume and functional deficits”
Collect dysbiotic microbiota from stroke-affected mice and normal microbiota from control mice
Note: Dysbiotic microbiota characterized by reduced species diversity and bacterial overgrowth of bacteroidetes
“Reduced species diversity and bacterial overgrowth of bacteroidetes were identified as hallmarks of poststroke dysbiosis”
Perform next-generation sequencing to characterize the composition of dysbiotic and normal microbiota
Note: Identifies species diversity and bacterial composition differences between dysbiotic and normal microbiota
“Using two distinct models of acute middle cerebral artery occlusion, we show by next-generation sequencing that large stroke lesions cause gut microbiota dysbiosis”
Administer dysbiotic poststroke microbiota to germ-free mice
Note: Dysbiotic microbiota group for comparison with normal microbiota recolonization
“Recolonizing germ-free mice with dysbiotic poststroke microbiota exacerbates lesion volume and functional deficits after experimental stroke”
Administer normal control microbiota to germ-free mice as control group
Note: Control group for comparison with dysbiotic microbiota recolonization
“Recolonizing germ-free mice with dysbiotic poststroke microbiota exacerbates lesion volume and functional deficits after experimental stroke compared with the recolonization with a normal control microbiota”
Perform stroke induction using two distinct models of acute middle cerebral artery occlusion
Note: Conducted on recolonized mice to assess stroke outcome differences between dysbiotic and normal microbiota groups
“Using two distinct models of acute middle cerebral artery occlusion, we show by next-generation sequencing that large stroke lesions cause gut microbiota dysbiosis”
Perform in vivo intestinal bolus tracking to assess intestinal motility in recolonized mice
Note: Determines if dysbiotic microbiota affects intestinal barrier function and motility
“intestinal barrier dysfunction and reduced intestinal motility as determined by in vivo intestinal bolus tracking”
Analyze proinflammatory T-cell polarization in the intestinal immune compartment and ischemic brain
Note: Dysbiotic microbiota induces proinflammatory T-cell polarization compared to normal microbiota
“recolonization of mice with a dysbiotic microbiome induces a proinflammatory T-cell polarization in the intestinal immune compartment and in the ischemic brain”
Use in vivo cell-tracking studies to demonstrate migration of intestinal lymphocytes to the ischemic brain
Note: Demonstrates the mechanistic link between microbiota composition and neuroinflammatory response
“Using in vivo cell-tracking studies, we demonstrate the migration of intestinal lymphocytes to the ischemic brain”
Quantify brain lesion volume and assess functional deficits in recolonized mice after stroke
Note: Primary outcome measures comparing dysbiotic versus normal microbiota recolonization groups
“Recolonizing germ-free mice with dysbiotic poststroke microbiota exacerbates lesion volume and functional deficits after experimental stroke compared with the recolonization with a normal control microbiota”
This section explains what the experiment is doing, which readouts matter, what the data artifacts usually look like, and how the analysis should flow from raw capture to reported result.
To assess the causal impact of microbiota composition on stroke outcome by recolonizing germ-free mice with dysbiotic or normal microbiota and measuring lesion volume and functional deficits
Objective
To assess the causal impact of microbiota composition on stroke outcome by recolonizing germ-free mice with dysbiotic or normal microbiota and measuring lesion volume and functional deficits
Subjects
From papermouse • Not specified in provided text • unknown • Not specified in provided text • Not specified in provided text
Cohort notes
From paperGerm-free mice were recolonized with either dysbiotic poststroke microbiota or normal control microbiota
Establish germ-free mouse colony (Not specified in provided text)
Collect microbiota samples (Not specified in provided text)
Characterize microbiota composition (Not specified in provided text)
Recolonize germ-free mice with dysbiotic microbiota (Not specified in provided text)
Brain lesion volume after stroke
From paperNext-generation sequencing used to characterize microbiota composition; in vivo cell-tracking and intestinal bolus tracking used to assess mechanistic pathways
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Functional deficits after stroke
From paperNext-generation sequencing used to characterize microbiota composition; in vivo cell-tracking and intestinal bolus tracking used to assess mechanistic pathways
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Gut microbiota composition (species diversity, bacterial overgrowth)
From paperNext-generation sequencing used to characterize microbiota composition; in vivo cell-tracking and intestinal bolus tracking used to assess mechanistic pathways
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Intestinal barrier function
From paperNext-generation sequencing used to characterize microbiota composition; in vivo cell-tracking and intestinal bolus tracking used to assess mechanistic pathways
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Brain lesion volume after stroke
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Functional deficits after stroke
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Gut microbiota composition (species diversity, bacterial overgrowth)
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Intestinal barrier function
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
Preprocessing / cleaning
Next-generation sequencing used to characterize microbiota composition; in vivo cell-tracking and intestinal bolus tracking used to assess mechanistic pathways
Scoring or quantification
Quantify the primary readouts for this experiment: Brain lesion volume after stroke; Functional deficits after stroke; Gut microbiota composition (species diversity, bacterial overgrowth); Intestinal barrier function.
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Brain lesion volume after stroke, Functional deficits after stroke, Gut microbiota composition (species diversity, bacterial overgrowth), Intestinal barrier function.
Source links and direct wording from the methods section for validation and deeper review.
Citation
V. Singh et al. (2016). Microbiota Dysbiosis Controls the Neuroinflammatory Response after Stroke. Journal of Neuroscience
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