Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice
September 19th, 2019
Presented here is a protocol to induce diffuse traumatic brain injury using a lateral fluid percussion device followed by the collection of the caecum content for gut microbiome analysis.
Simultaneousness and the lines interactions between the brain and the gut microbiota remains unknown. Use this technique, alterations in the gut microbiota after traumatic brain injury in mice can be examine. The main advantage of this technique is that it is relatively simple and efficient.
Demonstrating the procedure will be Wendong You, a graduate student from my laboratory. For traumatic brain injury induction, confirm a lack of response to toe pinch in an anesthetized mouse, and apply ointment to the animal's eyes. After shaving, disinfect the exposed scalp skin with 70%ethanol before incising the scalp in a sagittal plane.
Using forceps, retract the incision on both sides, and separate the periosteum slightly. Use a marker to draw a three-millimeter-diameter circle on the right parietal area of the skull two millimeters away from the midline. Use an electric drill to carefully penetrate the skull without damaging the dura.
Remove the bone flap to expose a three-millimeter bone window and place a plastic injury cannula over the craniotomy. After cementing the cannula to the skull with dental acrylic, use a five-milliliter syringe to fill the cannula with sterile 0.9%normal saline, taking care to avoid bubbles. Turn on the oscilloscope and amplifier.
Confirm that the high-pressure tube of the lateral fluid percussion injury device is free of air bubbles, and deliver about 10 test pulses, until the device gives a steady signal. Adjust the angle of the pendulum starting position to reach a pulse intensity of about two standard atmosphere and connect the injury cannula to the lateral fluid percussion injury device. Pull the trigger to release the pendulum, inducing the brain injury.
Then obtain and transmit a pulse to the dura through the entire closed, fluid-filled tubing system. After brain injury induction, remove the cannula and suture the incision. Then place the mouse on a heating pad with monitoring until it is ambulatory, before returning the animal to its home cage with ad libitum access to food and water.
At the indicated experimental time points, expose the intestines according to standard protocols and use atraumatic forceps to gently separate the caecum from the other intestinal tracts. When the tissue has been located, cut the caecum with sharp scissors and manually extract the caecum contents onto a sterile dressing. Then transfer the contents to a 1.5-milliliter microcentrifuge tube, for minus 80 degree Celsius storage until microbiome analysis.
16S recombinant DNA sequencing demonstrates a reduced diversity in caecum microbiota in mice three days after traumatic brain injury, with the most abundant taxa within the caecum contents of the sham and traumatic brain injury groups shown in the figure. Further, non-metric, multidimensional scaling reveals changes in the composition of the caecum microbiota after traumatic brain injury. Remember to keep dura intact when drilling the skull, and to be gentle when locating and separating the caecum from other intestinal tracts.
Following this procedure, hematoxylin and eosin staining and analyze can be perform to examine the histological change and inflammatory response within intestinal tracts after traumatic brain injury.
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Overview
This study presents a protocol for inducing diffuse traumatic brain injury (TBI) using a lateral fluid percussion device in mice. Following injury, the study investigates changes in gut microbiota by collecting caecum contents for analysis. The approach aims to elucidate the interactions between brain injury and gut microbiota composition.
Key Study Components
Area of Science
- Neuroscience
- Microbiome analysis
- Traumatic brain injury research
Background
- Understanding the relationships between the brain and gut microbiota is critical in TBI research.
- Current knowledge of how TBI affects gut microbiota is limited.
- The use of mice as a model organism allows for controlled experimental conditions.
Purpose of Study
- To develop a reliable protocol for inducing TBI and analyzing subsequent changes in the gut microbiome.
- To investigate potential correlations between neurological injury and gut microbiota diversity.
- To provide insights into the impacts of TBI on overall health and microbiome interactions.
Methods Used
- A lateral fluid percussion device was utilized to induce TBI in anesthetized mice.
- Careful surgical procedures were undertaken, including skull penetration and cannula implantation.
- Post-injury, caecum contents were collected for microbiome analysis and stored appropriately.
- 16S rDNA sequencing was employed to determine changes in microbiota diversity and composition.
Main Results
- Analysis indicated reduced diversity in caecum microbiota within three days post-TBI.
- Non-metric multidimensional scaling demonstrated significant shifts in microbiota composition following injury.
- The study highlights the need for caution when handling tissues and maintaining sterile conditions.
Conclusions
- This study establishes a methodology for exploring the gut-brain axis in the context of TBI.
- Findings suggest that TBI may disrupt gut microbiota, which could have implications for recovery.
- Enhancement of knowledge regarding gut-brain interactions could inform future therapeutic strategies for TBI.
Frequently Asked Questions
Executive Industry Relevance
This protocol enables mechanistic de-risking of the microbiota-gut-brain axis in traumatic brain injury research by providing a reproducible model to assess microbiome alterations post-injury. It supports target validation and biomarker discovery by linking neurological trauma to quantifiable shifts in caecum microbiota composition. The approach enhances predictive confidence in preclinical studies by standardizing TBI induction and microbiome sampling workflows.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of gut-brain axis hypotheses by measuring microbiota diversity changes after controlled TBI induction.
- Operational Value: Provides a standardized surgical and sampling procedure for consistent caecum microbiota collection across experimental groups.
Screening & Assay Development
- Scientific Value: Generates quantitative 16S rDNA sequencing outputs to profile microbial compositional shifts in caecum samples.
- Operational Value: Establishes a reproducible workflow for microbiome analysis that can be integrated into screening pipelines for gut-targeted therapeutics.
Translational & Preclinical Research
- Scientific Value: Supports disease-relevant system modeling by correlating TBI severity with microbiome alterations in a murine model.
- Operational Value: Enables longitudinal tracking of microbiota changes to inform risk-adjusted advancement decisions in preclinical programs.
Pipeline & Workflow Integration
The method bridges discovery biology and preclinical validation by standardizing TBI induction and gut microbiome analysis for target de-risking.
- Discovery Biology: Facilitates hypothesis testing on microbiota-gut-brain interactions through controlled injury and microbiome profiling.
- Screening: Delivers standardized caecum microbiota samples suitable for downstream 16S sequencing and diversity analysis.
- Analytics: Enables quantitative comparison of microbial composition via non-metric multidimensional scaling and diversity metrics.
- Translational Research: Connects neurological injury models to microbiome biomarkers for preclinical continuity.
- Enterprise Reuse: Establishes a reusable platform for studying gut-brain axis modulation in neurological disease models.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity in gut-brain axis research by providing quantifiable microbiota readouts post-TBI.
- Operational Value: Ensures reproducibility through standardized surgical, sampling, and sequencing protocols.
- Strategic Value: Improves go/no-go decisions by linking TBI models to microbiome-based biomarker signatures.
- Portfolio Impact: Supports risk-adjusted prioritization of compounds targeting gut-brain interactions in TBI recovery.
Implementation Considerations
- Requires expertise in murine surgical procedures and sterile tissue handling.
- Depends on access to lateral fluid percussion injury devices and 16S rDNA sequencing infrastructure.
- Necessitates cross-team standardization between neuroscience and microbiome analysis groups.
- Involves adaptation considerations for caecum sampling across different mouse strains or injury models.
- Limited by the need for aseptic technique during intestinal tissue dissection to prevent contamination.
