Name: investigating alterations in caecum microbiota after traumatic brain injury in mice
Uploaded: 2019-09-19T13:00:00.000+00:00
Duration: 4 min 29 s
Description: Watch this Scientific Journal Video about Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice at JoVE.com

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 groups throughout the study.</li>
	<li>Make all efforts to minimize the animal pain and discomfort.</li>
</ol>
2. Induction of traumatic brain injury
<ol>
	<li>Inject&#160;Ketamine (80-100 mg/kg)/Xylazine (10 mg/kg) IP&#160;for anesthesia. Test the depth of anesthesia using an eye reflex or pain reflex. Use artificial tears or lubricant eye ointment to keep the eyes from drying.</li>
	<li>After anesthesia, put the mouse in prone position. Use a temperature-controlled heating pad to maintain the temperature at 37 &#176;C during the surgery and for 30 min after TBI.</li>
	<li>Shave the hair of the incision area.</li>
	<li>Disinfect the scalp with 70% ethanol using three alternating scrubs, then incise the scalp in a sagittal plane.</li>
	<li>Use forceps to retract the incision on both sides and separate the periosteum slightly.</li>
	<li>Use a marker to draw a circle (3 mm diameter) on the right parietal area of the skull, 2 mm away from the midline.</li>
	<li>Drill the skull with an electric drill. Ensure that this step is operated carefully to protect the dura from being damaged.</li>
	<li>Remove the bone flap and expose a small bone window (3 mm in diameter).</li>
	<li>Place a plastic injury cannula (internal diameter = 2.5 mm, length = 8 mm) over the craniotomy and cement the cannula to the skull using a dental acrylic.</li>
	<li>Fill the cannula with sterile 0.9% NaCl (normal saline) using a syringe (5 mL) to ensure that there are no bubbles in the cannula.</li>
	<li>Turn on the oscilloscope and amplifier and ensure that the high-pressure tube of the lateral fluid percussion injury (LFPI) device is free of air bubbles. Test the device by delivering about 10 pulses until it gives a steady signal. Adjust the angle of the pendulum starting position to reach a pulse intensity of about 2.0 atm.</li>
	<li>Connect the injury cannula to the LFPI device. Induce brain injury by pulling the trigger and releasing the pendulum. Then, obtain a pulse and transmit it to the dura through the entire closed fluid-filled tubing system.</li>
	<li>Operate the mice in the sham group with the same surgical procedure. Do not perform the LFPI.</li>
</ol>
3. Post-surgery treatment
<ol>
	<li>After inducing the brain injury, remove the plastic cannula and suture the incision.&#160;During surgery administer buprenorphine (2mg/kg) SQ or IP and thereafter every 6-12 h for three days.</li>
	<li>Lay the mouse on a heating pad until it is ambulatory. Set the temperature of the heating pad to 37 &#176;C to accelerate anesthetic resuscitation.</li>
	<li>Put the mouse back in the cage and administer food and water ad libitum.</li>
</ol>
4. Laparotomy and sample collection from the caecum
<ol>
	<li>Euthanize the mice by CO2 followed by cervical dislocation at the corresponding time points. 
	NOTE: In this experiment, the chosen time points were 1 h, 6 h, 1 d, 3 d, and 7 d post-traumatic brain injury to analyze the dynamic evolution of gut microbiota.</li>
	<li>Remove the hair from the surface of the abdomen. Disinfect the abdomen with 70% ethanol.</li>
	<li>Place a sterile drape over the mouse. Make an incision from the lower abdomen midline, just above the prepuce in the male mice.</li>
	<li>After the intestines are exposed, locate the cecum and gently separated it from other intestinal tracts. Avoid grasping the cecum with toothed or sharp forceps. Use atraumatic forceps, such as Adson forceps with serrations.</li>
	<li>Cut the caecum with sharp scissors.</li>
	<li>Extract the caecum contents manually onto sterile dressing and store the contents in 1.5 mL microcentrifuge tubes.</li>
	<li>Store the caecum contents at -80 &#176;C until microbiome analysis.</li>
</ol>
5. DNA extraction and 16S-rDNA sequencing and data analysis
<ol>
	<li>Isolation of DNA from feces 
	NOTE: A commercially available DNA isolation kit (Table of Materials) was used for this experiment.
	<ol>
		<li>Use a scalpel to scrape 300 mg of feces in a 2 mL microcentrifuge tube and place the tube on ice.</li>
		<li>Add 1 mL of inhibit buffer to each sample. Vortex continuously for 1 min or until the feces sample is thoroughly homogenized.</li>
		<li>Centrifuge the sample at the maximum speed for 1 min to pellet the feces particles.</li>
		<li>Pipette 2 &#181;L of proteinase K into a new 2 mL microcentrifuge tube. Pipet 600 &#181;L of the supernatant from step 5.1.3 into the 2 mL microcentrifuge tube containing proteinase K. Then, add 600 &#181;L of Buffer 1 and vortex for 15 s.</li>
		<li>Incubate the sample at 70 &#176;C for 10 min.</li>
		<li>Add 600 &#181;L of 100% ethanol to the lysate (1:1 ratio) and mix by vortexing. Centrifuge at the maximum speed briefly to remove drops from the inside of the tube lid.</li>
		<li>Apply 600 &#181;L of the lysate to the spin column. Centrifuge at the maximum speed for 1 min. Discard the flow-through. Repeat this step one more time. Then, transfer the column into a new 2 mL collection tube.</li>
		<li>Open the spin column and add 500 &#181;L of Buffer 2. Centrifuge at the maximum speed for 1 min. Remove the column and place it in a new 2 mL collection tube.</li>
		<li>Add 500 &#181;L of Buffer 3 into the column. Centrifuge at the maximum speed for 3 min. Discard the flow-through. Repeat the centrifugation process once to ensure the Buffer 3 is completely eluted.</li>
		<li>Place the spin column into a new tube 2 mL collection tube and pipette 200 &#181;L of Buffer 4 directly onto the membrane. Incubate for 1 min at room temperature (RT), then centrifuge at the maximum speed for 1 min to elute DNA.</li>
	</ol>
	</li>
	<li>16S-rDNA sequencing and data analysis
	<ol>
		<li>Use 20-30 ng of DNA to generate amplicons.</li>
		<li>Use commercially available primers designed for the relatively conserved regions bordering the V3 and V4 hypervariable regions of bacteria 16S rDNA. The forward primers containing the sequence &#34;CCTACGGRRBGCASCAGKVRVGAAT&#34; and reverse primers containing the sequence &#34;GGACTACNVGGGTWTCTAATCC&#34; were used in the present study.</li>
		<li>Make the PCR reactions mixture by adding 2.5 &#181;L of Buffer 1, 2 &#181;L of dNTPs, 1 &#181;L of each primer, 0.5 &#181;L of DNA polymerase, and 20 ng of template DNA in a tube. Use ddH2O to adjust the reaction system to 25 &#181;L.</li>
		<li>Set the PCR reaction parameters as follows: perform the pre-denaturation at 94 &#176;C for 3 min once. Perform denaturation at 94 &#176;C for 5 s, anneal at 57 &#176;C for 90 s, extend at 72&#176;C for 10 s, and repeat this 24x.</li>
		<li>Perform PE250/300 paired-end sequencing according to the manufacturer&#39;s instruction and use QIIME data analysis package for 16S rRNA data analysis. 
		NOTE: In this experiment, the DNA sequencing and data analysis were primarily done by a professional sequencing company.</li>
	</ol>
	</li>
</ol>

<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>

The authors sincerely thank Baohong Wang for her technical guidance.

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 weight drop model may suffer from some inaccuracies. Among all existing brain injury models, LFPI is the most commonly used model, which includes focal and diffuse brain injury11,12 and replicates many important features observed in clinical TBI patients. Key steps throughout the process include protection of the dura and keeping the cannula airtight. However, both craniotomy and conglutination require the operation of a skilled performer, which may be a limitation of this protocol. However, LFPI is still a simple, stable, and accurate method. Therefore, TBI in mice was induced using LFPI in this study, which makes the protocol more representative and valuable in brain injury research.
For gut microbiota analysis, fecal samples were used for its non-invasiveness and convenience. Nevertheless, there are some limitations of fecal sampling. First, the amount of feces collected in each mouse at a certain time point is small, and the amount of feces may be insufficient to perform 16S-rDNA analysis. Second, due to post-traumatic gastrointestinal dysfunction, feces samples are difficult to collect during the acute phase after brain injury (1 h post-injury in this study). In addition, evidence has shown that fecal microbiota composition is different from microbiota in other intestinal segments13. Therefore, caecum contents were collected for gut microbiota analysis in this study. Caecum content sampling requires that mice are sacrificed, and this method allows for the sampling of intestinal tract for histological and immunological examinations, which are important research aspects in studies of brain injury-induced gut dysfunction. Additionally, this method can be used to investigate microbiota changes in other gastrointestinal tracts including the jejunum, ileum, and colon. In conclusion, this protocol is an ideal method for studying TBI-induced changes in gut microbiota.

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><tbody><tr><td>DNA isolation kit&amp;nbsp;</td><td>QIAGEN</td><td>51604</td><td>For fast purification of genomic DNA from stool samples</td></tr><tr><td>Gene analysis service</td><td>GENEWIZ</td><td></td><td>Gene analyse service</td></tr><tr><td>Heating pad</td><td>Shanghai SAFE Biotech Co.</td><td>TR-200</td><td>heating pad</td></tr><tr><td>Injector</td><td>The First Affiliated Hospital, School of Medicine, Zhejiang University</td><td></td><td>injector</td></tr><tr><td>LFPI device</td><td>Virginia&lt;br/&gt; Commonwealth University</td><td>FP302</td><td>LFPI device</td></tr><tr><td>Micro cranial drill</td><td>RWD Life Science</td><td>78061</td><td>Micro cranial drill</td></tr><tr><td>Povidone Iodine</td><td>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 (Figure 1D).
The procedure of lateral fluid percussion is shown in Figure 2. Before starting the device was tested by delivering about 10 pulses until it gives a steady signal. The angle of the pendulum was adjusted to the starting position to reach a pulse intensity of about 2.0 atm (Figure 2A). The cannula was filled with the sterile normal saline. Then the cannula was connected to the LFPI device (Figure 2B). Brain injury was induced by creating an impulse into the closed cranial cavity (Figure 2C).
Laparotomy and caecum fecal sample collection are shown in Figure 3. The laparotomy was performed on the lower abdomen and along the midline (Figure 3A). The caecum was identified and gently separated (Figure 3B,C). The caecum is usually located in the lower right part of the abdomen. It was then incised with sharp scissors (Figure 3D). The contents of caecum (Figure 3E) were extracted and stored in 1.5 mL tubes (Figure 3F). Fecal samples were immediately stored at -80 &#176;C before further use.
16S-rDNA sequencing demonstrated reduced diversity of caecum microbiota in mice 3 days after TBI, the most abundant taxa in caecum contents of sham and TBI groups were showed in Figure 4. The Wilcoxon rank sum test was performed to evaluate microbiota differences between TBI and sham groups in the 16S sequencing analysis, and the p value was less than 0.05. The non-metric multi-dimensional scaling (NMDS) also showed changed composition of caecum microbiota after TBI (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59410/59410fig1.jpg" /> 
Figure 1:&#160;Establishment of TBI. (A) After anesthesia and disinfection, the scalp was incised. (B) Operate a circinate craniotomy on the skull over the right parietal cortex. (C) Keep the dura intact. (D) Cement the plastic injury cannula to the skull using dental acrylic. <a href="https://www.jove.com/files/ftp_upload/59410/59410fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59410/59410fig2.jpg" /> 
Figure 2: The procedure of lateral fluid percussion. (A) Adjustment of the pendulum starting position angle. (B) Filling of the cannula with sterile normal saline, then connection of the cannula to the LFPI device. (C) Brain injury induction by release of the pendulum and creation of an impulse into the closed cranial cavity. <a href="https://www.jove.com/files/ftp_upload/59410/59410fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59410/59410fig3.jpg" /> 
Figure 3: Laparotomy and caecum fecal sample collection. (A) Beginning of the laparotomy from lower abdomen and along the midline. (B,C) Identification of the caecum and subsequent (gentle) removal. (D) Cutting of the caecum with sharp scissors. (E) Extraction of the contents of caecum. (F) Storing of the caecum content samples in 1.5 mL Eppendorf tubes. <a href="https://www.jove.com/files/ftp_upload/59410/59410fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59410/59410fig4.jpg" /> 
Figure 4: The comparison of caecum microbiota diversity. Most abundant taxa in caecum content of sham and TBI groups demonstrated reduced diversity of caecum microbiota in mice 3 days after TBI. <a href="https://www.jove.com/files/ftp_upload/59410/59410fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59410/59410fig5.jpg" /> 
Figure 5: The NMDS analysis. Non-metric multi-dimensional scaling (NMDS) showed changed composition of caecum microbiota after TBI. <a href="https://www.jove.com/files/ftp_upload/59410/59410fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

investigating-alterations-caecum-microbiota-after-traumatic-brain

Nanyang Technological University

Research

JoVE Journal

Neuroscience

6.2K Views.  Zhejiang University. 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.

Video: Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Medicine

A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal neurological deficits and even amnesia. Athletes participating in contact sports are at high risk of exposure to large number of mTBIs. In terms of the level of injury in a sporting athlete, a mTBI is defined as a mild injury that does not cause gross pathological changes, but does cause short-term neurological deficits that are spontaneously resolved. Despite previous attempts to model mTBI in mice and rats, many have reported gross adverse effects including skull fractures, intracerebral bleeding, axonal injury and neuronal cell death. Herein, we describe our highly reproducible animal model of mTBI that reproduces clinically relevant symptoms. This model uses a custom made pneumatic impactor device to deliver a closed-head trauma. This impact is made under precise velocity and deformation parameters, creating a reliable and reproducible model to examine the mechanisms that contribute to effects of single or repetitive concussive mTBI.

Athletes absorb several hundred mild traumatic brain injuries (mTBI)/concussions every year; however, the consequence of these on the brain is poorly understood. Therefore, an animal model of single and repetitive mTBI that consistently replicates clinically relevant symptoms provides the means to advance the study of mTBI and concussion.

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal ...

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging.

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Murine Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal cancer, inflammatory bowel disease, and Parkinson&#8217;s disease. To study the function of the appendix, a gut disease-associated murine appendectomy model has been established and its step-by-step protocol is described here. This report introduces a facile protocol for caecal patch removal in mice followed by the chemical induction of chronic colitis-associated colorectal cancer using a combination of dextran sulfate sodium (DSS) and azoxymethane (AOM). IgA specific cells and IgA concentration were significantly reduced upon removal of the caecal patch in male C57BL/6 mice&#160;compared to those in the sham group. Simultaneously administering 2% DSS and AOM resulted in nearly 80% mice survival in both sham and appendectomy groups without significant body weight loss. Histological results confirmed colonic inflammation and different degrees of adenocarcinoma. This model can be used for the study of the functional role of the appendix in maintaining gut microbiota homeostasis and pathogenesis of gut colitis and malignancies, as well as for the potential development of drug targeting therapies.

The presented protocol describes a facile surgical removal of the appendix (caecal patch) in a mouse followed by the induction of inflammatory bowel disease-associated colorectal cancer. This murine appendectomy model enables investigation of the biological role of the appendix in the pathogenesis of human gastrointestinal disease.

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal ...

Intracerebroventricular Delivery of Gut-Derived Microbial Metabolites in Freely Moving Mice

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have revealed that gut microbiota-derived metabolites modulate brain-mediated physiological functions through intricate gut-brain pathways in the host. Short-chain fatty acids (SCFAs) are the major bacteria-derived metabolites produced during dietary fiber fermentation by the gut microbiome. Secreted SCFAs from the gut can act at multiple sites in the periphery, affecting the immune, endocrine, and neural responses due to the vast distribution of SCFAs receptors. Therefore, it is challenging to differentiate the central and peripheral effects of SCFAs through oral and intraperitoneal administration of SCFAs. This paper presents a video-based method to interrogate the functional role of SCFAs in the brain via a guide cannula in freely moving mice. The amount and type of SCFAs in the brain can be adjusted by controlling the infusion volume and rate. This method can provide scientists with a way to appreciate the role of gut-derived metabolites in the brain.

Gut-derived microbial metabolites have multifaceted effects leading to complex behavior in animals. We aim to provide a step-by-step method to delineate the effects of gut-derived microbial metabolites in the brain via intracerebroventricular delivery via a guide cannula.

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

rmTBI Induction in Mice via a Closed-Head Injury Method

Modified Mouse Model of Repetitive Mild Traumatic Brain Injury Incorporating Thinned-Skull Window and Fluid Percussion

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models with well-defined pathologies are urgently needed for studying the mechanisms of neuropathology after mild TBI and testing therapeutics. Replicating the entire sequelae of TBI in animal models has proven to be a challenge. Therefore, the availability of multiple animal models of TBI is necessary to account for the diverse aspects and severities seen in TBI patients. CHI is one of the most common methods for fabricating rodent models of rmTBI. However, this method is susceptible to many factors, including the impact method used, the thickness and shape of the skull bone, animal apnea, and the type of head support and immobilization utilized. The aim of this protocol is to demonstrate a combination of the thinned-skull window and fluid percussion injury (FPI) methods to produce a precise mouse model of CHI-associated rmTBI. The primary objective of this protocol is to minimize factors that could impact the accuracy and consistency of CHI and FPI modeling, including skull bone thickness, shape, and head support. By utilizing a thinned-skull window method, potential inflammation due to craniotomy and FPI is minimized, resulting in an improved mouse model that replicates the clinical features observed in patients with mild TBI. Results from behavior and histological analysis using hematoxylin and eosin (HE) staining suggest that rmTBI can lead to a cumulative injury that produces changes in both behavior and gross morphology of the brain. Overall, the modified CHI-associated rmTBI presents a useful tool for researchers to explore the underlying mechanisms that contribute to focal and diffuse pathophysiological changes in rmTBI.

Author Spotlight: Optimizing Rodent Models for Investigating the Mechanisms and Rehabilitation in Repeated Mild Traumatic Brain Injury

This protocol presents a modified mouse model of repetitive mild traumatic brain injury (rmTBI) induced via a closed-head injury (CHI) method. The approach features a thinned-skull window and fluid percussion to reduce the inflammation commonly caused by meninges exposure, along with improved reproducibility and accuracy in modeling rmTBI in rodents.

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models ...