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
Materials & Equipment Checklist
6 items
Gather these items before starting the experiment. Check off items as you prepare.
Equipment3
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Protocol Steps
View Abstract
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.
1
Establish germ-free mouse colony
Obtain or maintain germ-free mice for the recolonization experiment
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Note: Germ-free mice serve as the baseline population to be recolonized
View evidence from paper
“Recolonizing germ-free mice with dysbiotic poststroke microbiota exacerbates lesion volume and functional deficits”
2
Collect microbiota samples
Collect dysbiotic microbiota from stroke-affected mice and normal microbiota from control mice
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Note: Dysbiotic microbiota characterized by reduced species diversity and bacterial overgrowth of bacteroidetes
View evidence from paper
“Reduced species diversity and bacterial overgrowth of bacteroidetes were identified as hallmarks of poststroke dysbiosis”
3
Characterize microbiota composition
Perform next-generation sequencing to characterize the composition of dysbiotic and normal microbiota
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Note: Identifies species diversity and bacterial composition differences between dysbiotic and normal microbiota
View evidence from paper
“Using two distinct models of acute middle cerebral artery occlusion, we show by next-generation sequencing that large stroke lesions cause gut microbiota dysbiosis”
4
Recolonize germ-free mice with dysbiotic microbiota
Administer dysbiotic poststroke microbiota to germ-free mice
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Note: Dysbiotic microbiota group for comparison with normal microbiota recolonization
View evidence from paper
“Recolonizing germ-free mice with dysbiotic poststroke microbiota exacerbates lesion volume and functional deficits after experimental stroke”
5
Recolonize germ-free mice with normal microbiota
Administer normal control microbiota to germ-free mice as control group
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Note: Control group for comparison with dysbiotic microbiota recolonization
View evidence from paper
“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”
6
Induce acute middle cerebral artery occlusion
Perform stroke induction using two distinct models of acute middle cerebral artery occlusion
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Note: Conducted on recolonized mice to assess stroke outcome differences between dysbiotic and normal microbiota groups
View evidence from paper
“Using two distinct models of acute middle cerebral artery occlusion, we show by next-generation sequencing that large stroke lesions cause gut microbiota dysbiosis”
7
Measure intestinal motility
Perform in vivo intestinal bolus tracking to assess intestinal motility in recolonized mice
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Note: Determines if dysbiotic microbiota affects intestinal barrier function and motility
View evidence from paper
“intestinal barrier dysfunction and reduced intestinal motility as determined by in vivo intestinal bolus tracking”
8
Assess T-cell polarization
Analyze proinflammatory T-cell polarization in the intestinal immune compartment and ischemic brain
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Note: Dysbiotic microbiota induces proinflammatory T-cell polarization compared to normal microbiota
View evidence from paper
“recolonization of mice with a dysbiotic microbiome induces a proinflammatory T-cell polarization in the intestinal immune compartment and in the ischemic brain”
9
Track intestinal lymphocyte migration
Use in vivo cell-tracking studies to demonstrate migration of intestinal lymphocytes to the ischemic brain
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Note: Demonstrates the mechanistic link between microbiota composition and neuroinflammatory response
View evidence from paper
“Using in vivo cell-tracking studies, we demonstrate the migration of intestinal lymphocytes to the ischemic brain”
10
Measure lesion volume and functional deficits
Quantify brain lesion volume and assess functional deficits in recolonized mice after stroke
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Note: Primary outcome measures comparing dysbiotic versus normal microbiota recolonization groups
View evidence from paper
“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”
Subjects / Specimens
Species
mouse
Strain
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Age
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Sex
unknown
Weight
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Germ-free mice were recolonized with either dysbiotic poststroke microbiota or normal control microbiota