The authors have nothing to disclose.

All procedures performed were approved by the Experimental Animal Ethics Committee of Zhejiang University. All instruments and materials used in surgery are sterile.&#160;The TBI proceudre takes about 20 minutes.
1. Animal care
<ol>
	<li>Use 5- to 6-week-old male C57BL/6J mice (20-25 g of weight) in this experiment.</li>
	<li>Maintain mice on a 12 h/12 h light/dark cycle, and make sure they receive food and water ad libitum. Provide the same amounts of food and water to both the sham and TBI...

<ol>
	<li>Cheng, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Trends in traumatic brain injury mortality in China, 2006-2013: A population-based longitudinal study. 14, e1002332 (2017).</li><li>Maas, A. I. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+brain+injury:+integrated+approaches+to+improve+prevention,+clinical+care,+and+research.">Traumatic brain injury: integrated approaches to improve prevention, clinical care, and research.</a> The Lancet Neurology. 16, 987-1048 (2017).</li><li>Gaddam, S. S., Buell, T., Robertson, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+manifestations+of+traumatic+brain+injury.">Systemic manifestations of traumatic brain injury.</a> Handbook of Clinical Neurology. 127, 205-218 (2015).</li><li>Kabadi, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid-percussion-induced+traumatic+brain+injury+model+in+rats.">Fluid-percussion-induced traumatic brain injury model in rats.</a> Nature Protocols. 5, 1552-1563 (2010).</li><li>Fung, T. C., Olson, C. A., Hsiao, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+the+microbiota,+immune+and+nervous+systems+in+health+and+disease.">Interactions between the microbiota, immune and nervous systems in health and disease.</a> Nature Neuroscience. 20, 145-155 (2017).</li><li>Collins, S. M., Surette, M., Bercik, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interplay+between+the+intestinal+microbiota+and+the+brain.">The interplay between the intestinal microbiota and the brain.</a> Nature Reviews Microbiology. 10, 735-742 (2012).</li><li>Cryan, J. F., Dinan, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mind-altering+microorganisms:+the+impact+of+the+gut+microbiota+on+brain+and+behaviour.">Mind-altering microorganisms: the impact of the gut microbiota on brain and behaviour.</a> Nature Reviews Neuroscience. 13, 701-712 (2012).</li><li>Nicholson, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moderate+Traumatic+Brain+Injury+Alters+the+Gastrointestinal+Microbiome+in+a+Time-Dependent.">Moderate Traumatic Brain Injury Alters the Gastrointestinal Microbiome in a Time-Dependent.</a> Shock. , (2018).</li><li>Houlden, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+injury+induces+specific+changes+in+the+caecal+microbiota+of+mice+via+altered+autonomic+activity+and+mucoprotein+production.">Brain injury induces specific changes in the caecal microbiota of mice via altered autonomic activity and mucoprotein production.</a> Brain, Behavior, and Immunity. 57, 10-20 (2016).</li><li>Treangen, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+Brain+Injury+in+Mice+Induces+Acute+Bacterial+Dysbiosis+Within+the+Fecal+Microbiome.">Traumatic Brain Injury in Mice Induces Acute Bacterial Dysbiosis Within the Fecal Microbiome.</a> Frontiers in Immunology. 9, 2757 (2018).</li><li>Alder, J., Fujioka, W., Lifshitz, J., Crockett, D. P., Thakker-Varia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+fluid+percussion:+model+of+traumatic+brain+injury+in+mice.">Lateral fluid percussion: model of traumatic brain injury in mice.</a> Journal of Visualized Experiments. , (2011).</li><li>Thompson, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+fluid+percussion+brain+injury:+a+15-year+review+and+evaluation.">Lateral fluid percussion brain injury: a 15-year review and evaluation.</a> Journal of Neurotrauma. 22, 42-75 (2005).</li><li>Pang, W., Vogensen, F. K., Nielsen, D. S., Hansen, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Faecal+and+caecal+microbiota+profiles+of+mice+do+not+cluster+in+the+same+way.">Faecal and caecal microbiota profiles of mice do not cluster in the same way.</a> Laboratory Animals. 46, 231-236 (2012).</li></ol>

Presented here is a simple and efficient protocol to determine changes in cecal microbiota after TBI in mice. Induction of brain injury and collection of caecum content samples are critical parts of the protocol.
Despite researchers having studied the changes of gut microbiota following TBI, the brain injury used in these studies were CCI-8 and weight drop/impact-induced models9. However, the CCI model mostly replicates brain contusion, and the w...

Traumatic brain injury (TBI) is a global public health problem and the leading cause of death and disability in young adults1,2. TBI causes many deaths every year, and survivors experience a variety of physical, psychiatric, emotional, and cognitive disabilities. Therefore, TBI is a heavy burden to a patient's family and societal resources. TBI involves both the primary brain injury that occurs at the time of trauma and any secondary brain injuries that develop hours to months following initial injury. Secondary brain injury is mediated by several biochemical cascades, which are not only detrimental to the brain but also have significant negative effects on various organ systems, including the gastrointestinal system3.
Currently, there are three models to induce TBI in animal experiments: fluid percussion injury, control cortical impact (CCI), and weight drop acceleration. Lateral fluid percussion injury (LFPI) is the most commonly used model to establish diffuse brain injury (DAI)4. The device produces brain injury through a craniectomy by applying a brief fluid pressure pulse to the intact dura. This pulse is created by the strike of the pendulum. LFPI is a reproducible and controllable modeling method for TBI research. 
The microbiome is defined as the collective genomes of all microorganisms that reside in the human body. Intestinal microbes in particular not only play an important role in intestinal homeostasis and function but also regulate many aspects of host physiology and the functioning of other organs5. In recent years, there is increasing evidence that indicates that gut microbiota regulate brain development and function via brain-gut axes6. Disruption of the gut microbiota has been linked to several brain function disorders including Parkinson's disease, mood disorders, and autism7. Recently, preclinical studies have also reported that acute brain injury can induce changes in gut microbiota8,9. 
A study by Treangen et al.10 found significant decreases in three microbial species and increases in two microbial species after CCI-induced TBI. This evidence indicates that modulation of gut microbiota may be a therapeutic method in TBI management. However, the mechanisms underlying brain injury-induced gut microbiota changes remain unknown. For this reason, a relatively simple and efficient model of studying the changes in gut microbiota after TBI is required. Therefore, the present study presents a protocol to examine alterations in gut microbiota after TBI in mice.

<table border="0" cellpadding="0" cellspacing="0" style="width:958px;" width="959">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:222px;">DNA isolation kit&#160;</td>
			<td style="width:212px;">QIAGEN</td>
			<td style="width:354px;">51604</td>
			<td style="width:171px;">For fast purification of genomic DNA from stool samples</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gene analysis service</td>
			<td>GENEWIZ</td>
			<td></td>
			<td>Gene analyse service</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heating pad</td>
			<td>Shanghai SAFE Biotech Co.</td>
			<td>TR-200</td>
			<td>heating pad</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Injector</td>
			<td style="width:212px;">The First Affiliated Hospital, School of Medicine, Zhejiang University</td>
			<td></td>
			<td>injector</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">LFPI device</td>
			<td style="width:212px;">Virginia 
			Commonwealth University</td>
			<td>FP302</td>
			<td>LFPI device</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Micro cranial drill</td>
			<td>RWD Life Science</td>
			<td>78061</td>
			<td>Micro cranial drill</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Povidone Iodine</td>
			<td style="width:212px;">The First Affiliated Hospital, School of Medicine, Zhejiang University</td>
			<td></td>
			<td>Povidone Iodine</td>
		</tr>
	</tbody>
</table>

investigating alterations in caecum microbiota after traumatic brain injury in mice

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also demonstrate that traumatic brain injuries cause changes to the gut microbiota. However, mechanisms underlying the bidirectional regulation of the brain-gut axis remain unknown. Currently, few models exist for studying the changes in gut microbiota after traumatic brain injury. Therefore, the presented study combines protocols for inducing traumatic brain injury using a lateral fluid percussion device and analysis of caecum samples following injury for investigating alterations in the gut microbiome. Alterations of the gut microbiota composition after traumatic brain injury are determined using 16S-rDNA sequencing. This protocol provides an effective method for studying the relationships between enteric microorganisms and traumatic brain injury.

Establishment of TBI is shown in Figure 1. After anesthesia and disinfection, the scalp was incised sagittally (Figure 1A). A craniotomy (3 mm in diameter) was trephined into the skull over the right parietal cortex with an electric drill, the dura was kept intact (Figure 1B,C). A plastic injury cannula was placed over the bone window and cemented to the skull using dental acrylic (<...

Watch this Scientific Journal Video about Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice at JoVE.com

Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice

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.

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also ...

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.

Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice

Abstract