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 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 ...

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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

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

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 ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous variability and heterogeneity (age, gender, type of trauma, other possible pathologies, etc.), animal models play a key role in unraveling factors that are limitations in clinical research. They provide a standardized and controlled setting to investigate the biological mechanisms of injury and repair following TBI. However, not all animal models mimic the diffuse and subtle nature of mTBI effectively. For example, the commonly used controlled cortical impact (CCI) and lateral fluid percussion injury (LFPI) models make use of a craniotomy to expose the brain and induce widespread focal trauma, which are not commonly seen in mTBI. Therefore, these experimental models are not valid to mimic mTBI. Thus, an appropriate model should be used to investigate mTBI. The Marmarou weight drop model for rats induces similar microstructural alterations and cognitive impairments as seen in patients who sustain mild trauma; therefore, this model was selected for this protocol. Conventional computed tomography and magnetic resonance imaging (MRI) scans commonly show no damage following a mild injury, because mTBI induces often only subtle and diffuse injuries. With diffusion weighted MRI, it is possible to investigate microstructural properties of brain tissue, which can provide more insight into the microscopic alterations following mild trauma. Therefore, the goal of this study is to obtain quantitative information of a selected region-of-interest (i.e., hippocampus) to follow up disease progression after obtaining a mild and diffuse brain injury.

The overall goal of this procedure is to obtain quantitative microstructural information of the hippocampus in a rat with mild traumatic brain injury. This is done using an advanced diffusion-weighted magnetic resonance imaging protocol and region-of-interest based analysis of parametric diffusion maps.

Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice

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Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

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

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Automated Gait Analysis in Mice with Chronic Constriction Injury

A Novel In Vitro Model of Blast Traumatic Brain Injury

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury