Neuroscience
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Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice
Chapters
Summary 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.
Transcript
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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