Name: systems analysis of the neuroinflammatory and hemodynamic response to traumatic brain injury
Uploaded: 2022-05-27T13:15:00
Description: Watch this Scientific Journal Video about Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury at JoVE.com

This project was supported by the National Institutes of Health R21 NS104801 (EMB)&#160;&#160;and R01 NS115994 (LBW/EB) and Children&#8217;s Healthcare of Atlanta Junior Faculty Focused Award (EMB). This work was also supported by the U.S. Department of Defense through the Congressionally Directed Medical Research Programs under Award No. W81XWH-18-1-0669 (LBW/EMB). Opinions, interpretations, conclusions and recommendations are those of the author and are not necessarily endorsed by the Department of Defense.&#160;This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. 1937971. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.

All animal procedures are approved by Emory University Institutional Animal Care and Use Committee (IACUC) and followed the NIH Guidelines for the Care and Use of Laboratory Animals.
1. Weight-drop model of mild traumatic brain injury
<ol>
	<li>Prepare the weight-drop setup. Mount a vise on a flat surface with a 1 m guide tube (2.54 cm inner diameter) aligned vertically (check using a level). Use a 54 g bolt (0.95 cm basic body diameter, 2 cm head diameter, 10.2 cm length) for the impact.</l...

<ol>
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Herein we detail methods for assessment of the hemodynamic and neuroinflammatory response to repetitive mild traumatic brain injury. Further, we have shown how to integrate these data as part of a multivariate systems analysis using partial least squares regression. In the text below we will discuss some of the key steps and limitations associated with the protocol as well as the advantages/disadvantages of the methods over existing methods.
Weight-drop model of mild traumatic brain in...

Overview 
Mild traumatic brain injuries (mTBIs) impact ~1.6-3.8 million athletes annually1. These injuries, including sub-concussive and concussive injuries, can leave patients with transient physical, emotional, psychological and cognitive symptoms2. Moreover, repetitive mTBI (rmTBI) sustained within a &#8220;window of vulnerability&#8221; can lead to cumulative severity and duration of cognitive consequences that last longer than the effects of a single mTBI alone3, and ultimately even to permanent loss of function4,5,6. Although many patients recover within a relatively short time frame (&#60;1 week), 10-40% of patients suffer longer lasting effects of mTBI for &#62; 1 month, with some lasting up to 1 year3,7,8,9. Despite the high prevalence and lasting consequences of these injuries, injury mechanisms are poorly understood and no effective treatment strategies exist.
Given the high variability in outcomes after mTBI/rmTBI, one challenge in identifying early-stage molecular triggers from tissue obtained in terminal mTBI/rmTBI studies is the lack of longitudinal data demonstrating definitive &#34;acute molecular links&#34; of these molecular triggers to longer-term outcomes. To overcome this challenge, our group has discovered that acutely reduced cerebral blood flow&#160;measured acutely using&#160;an optical tool called diffuse correlation spectroscopy (DCS), strongly correlates with longer-term cognitive outcome in a mouse model of rmTBI10. Using this hemodynamic biomarker, we showed that mice with acutely low cerebral blood flow (and, by extension, worse predicted long-term outcome) have concomitant acute increases in neuronal phospho-signaling within both MAPK and NF&#954;B pathways, increases in neuronal expression of pro-inflammatory cytokines, and increases in expression of the phagocyte/microglial marker Iba111. These data suggest a possible role for neuronal phospho-signaling, cytokine expression, and microglial activation in both the acute regulation of cerebral blood flow post injury as well as in triggering a signaling cascade that leads to neuronal dysfunction and worse cognitive outcome. Herein, we detail our approach to simultaneously probe both the hemodynamic and neuroinflammatory environment after rmTBI and how to integrate these complex datasets. Specifically, we outline procedures for four key steps to this comprehensive approach: (1) a weight-drop model of mild traumatic brain injury, (2) assessment of cerebral blood flow with diffuse correlation spectroscopy, (3) quantification of the neuroinflammatory environment, and (4) data integration (Figure 1). Below, we provide a brief introduction to each of these key steps to help guide readers through the rationale behind our methods. The remainder of the manuscript provides a detailed protocol for each of these key steps.
Weight-drop model of mild traumatic brain injury 
Although many excellent preclinical models of repetitive mild TBI exist12,13,14,15,16,17,18, we employ a well-established and clinically relevant weight-drop closed head injury model. Key features of this model include (1) blunt impact of the intact skull/scalp followed by unrestricted rotation of the head about the neck, (2) no overt structural brain injury, edema, blood&#8211;brain barrier damage, acute cell death, or chronic brain tissue loss, and (3) persistent (up to 1 year) cognitive deficits that emerge only after multiple hits19 (Figure 2).
Assessment of cerebral blood flow with diffuse correlation spectroscopy 
Diffuse correlation spectroscopy (DCS) is a non-invasive optical technique that measures blood flow5,20,21. In DCS, a near-infrared light source is placed on the tissue surface. A detector is placed at a fixed distance from the source on the tissue surface to detect light that has multiply scattered through the tissue (Figure 3). Scattering off moving red blood cells causes the detected light intensity to fluctuate with time. A simple analytical model known as correlation diffusion theory is used to relate these intensity fluctuations to an index of blood flow (CBFi, Figure 4). Although the units of CBFi (cm2/s) are not the traditional units of flow (mL/min/100 g), a previous study in mice has shown that CBFi strongly correlates with cerebral blood flow measured by arterial spin labeled MRI21.
For reference, the DCS instrument used here was built in-house and is comprised of an 852 nm long coherence-length laser, an array of 4 photon counting avalanche photodiodes, and a hardware autocorrelator board (single tau, 8 channel, 100 ns minimum sample time)21,22. Data is acquired with homemade software written in LabView. The animal interface for the device consists of a 400 &#956;m multimode source fiber (400-2200 nm wavelength range, pure silica core, TECS Hard Cladding) and a&#160;780 nm single mode detector fiber (780-970 nm wavelength range, pure silica core, TECS Hard Cladding, 730 &#177; 30 nm second mode cut-off) spaced 6 mm apart and embedded in a black 3D-printed sensor (4 mm x 8 mm, Figure 3).
Quantification of the neuroinflammatory environment 
Although neuroinflammation is regulated by diverse cellular processes, two key relevant mechanisms are extracellular signaling by cytokines/chemokines and intracellular signaling by phospho-proteins. To investigate the neuroinflammatory environment of the brain post-injury, brains are extracted from mice, microdissected, and cytokines/chemokines and phospho-proteins are quantified using Luminex (Figure 5, Figure 6, Figure 7). Luminex multiplexed immunoassays enable simultaneous quantification of a diverse collection of these proteins by coupling enzyme-linked immunosorbent assays (ELISAs) to fluorescently tagged magnetic beads. Distinct fluorescent tags are used for each protein of interest, and beads of each tag are functionalized with a capture antibody against that particular protein. Hundreds of beads for capturing each protein are mixed together, placed in a 96 well plate, and incubated with sample. After sample incubation, a magnet is used to trap the beads in the well while the sample is washed out. Next, biotinylated detection antibody binds to the analyte of interest to form an antibody-antigen sandwich similar to a traditional ELISA, but with the ELISA for each protein occurring on a different fluorescently tagged bead. Adding phycoerythrin-conjugated streptavidin (SAPE) completes each reaction. The Luminex instrument then reads the beads and separates the signal according to each fluorescent tag/protein.
Data integration 
Because of the large number of analytes (e.g., cytokines) measured in the Luminex assay, data analysis can be difficult to interpret if each quantified protein is analyzed individually. To simplify analysis and to capture trends observed among analytes, we use a multivariate analysis method called partial least squares regression (PLSR, Figure 8)23. PLSR works by identifying an axis of weights corresponding to each measured protein (i.e., cytokines or&#160;phospho-proteins, referred to as &#8220;predictor variables&#8221;) that together optimally explain co-variance of the measured proteins with a response variable (e.g., cerebral blood flow). The weights are referred to as &#8220;loadings&#8221; and are assembled into a vector known as a latent variable (LV). By projecting (referred to as &#8220;scoring&#8221;) the measured protein data on each of two LVs, the data can be re-plotted in terms of these LVs. After computing the PLSR, we use a varimax rotation to identify a new LV that maximizes the covariance between the sample projections onto the LV and the predictor variable24. This approach allows us to define LV1 as the axis for which the variance of the response variable is best explained. LV2 maximizes co-variance between the response variable and LV1 residual data, which may be associated with biological or technical variability between samples. Lastly, we conduct a Leave One Out Cross Validation (LOOCV) to ensure that the PLSR model is not heavily dependent upon any one sample23.
In this protocol, we detail methods to characterize the neuroinflammatory and hemodynamic tissue response to mTBI. The general workflow is outlined in Figure 1. In this protocol, mice are subject to one or more mTBIs using a weight-drop closed head injury model. Cerebral blood flow is measured longitudinally before and at multiple time points after injury. At the time point of interest for interrogation of neuroinflammatory changes, the animal is euthanized, and the brain is extracted. Brain regions of interest are isolated via microdissection and then lysed to extract protein. Lysates are then used for both Luminex multiplexed immunoassays of cytokine and phospho-protein expression as well as Western blot. Finally, this holistic dataset is integrated using a partial least squares regression analysis.

<table>
	<tbody>
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			<td>Adjustable pipettes</td>
			<td></td>
			<td></td>
			<td>any adjustable pipette</td>
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			<td>Aluminum foil</td>
			<td>VWR</td>
			<td>89107-726</td>
			<td></td>
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		<tr>
			<td>Bio-Plex cell lysis kit</td>
			<td>C Bio-Rad</td>
			<td>171304012</td>
			<td></td>
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		<tr>
			<td>BRAND BRANDplates pureGrade Microplates, Nonsterile</td>
			<td>BrandTech</td>
			<td>781602 96</td>
			<td></td>
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		<tr>
			<td>Complete mini protease inhibitor tablet</td>
			<td>Sigma-Aldrich</td>
			<td>11836153001</td>
			<td></td>
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			<td>Depilatory cream</td>
			<td>Amazon</td>
			<td>Nair</td>
			<td></td>
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		<tr>
			<td>DiH2O</td>
			<td>VWR</td>
			<td>VWRL0200-1000</td>
			<td></td>
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		<tr>
			<td>Handheld magnetic separator block for 96 well flat bottom plates</td>
			<td>Millipore Sigma Catalogue</td>
			<td>40-285</td>
			<td></td>
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		<tr>
			<td>Hardware Autocorrelator Board</td>
			<td>www.correlator.com</td>
			<td>Flex05-8ch</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane 250 mL</td>
			<td>MED-VET INTERNATIONAL</td>
			<td>RXISO-250</td>
			<td></td>
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		<tr>
			<td>Kimwipe (11.2 x 21.3 cm)</td>
			<td>VWR</td>
			<td>21905-026</td>
			<td></td>
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		<tr>
			<td>Laboratory vortex mixer</td>
			<td>VWR</td>
			<td>10153-838</td>
			<td></td>
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		<tr>
			<td>LabView</td>
			<td>National Instruments</td>
			<td>LabVIEW</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminex 200, HTS, FLEXMAP 3D, or MAGPIX with xPONENT software</td>
			<td>Luminex Corporation</td>
			<td></td>
			<td></td>
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			<td>Luminex Drive Fluid</td>
			<td>Luminex</td>
			<td>MPXDF-4PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminex sheath fluid</td>
			<td>EMD Millipore</td>
			<td>SHEATHFLUID</td>
			<td></td>
		</tr>
		<tr>
			<td>MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel - Premixed 32 Plex - Immunology Multiplex Assay</td>
			<td>Millipore Sigma</td>
			<td>MCYTMAG-70K-PX32</td>
			<td></td>
		</tr>
		<tr>
			<td>MILLIPLEX MAPK/SAPK Signaling 10-Plex Kit-Cell Signaling Multiplex Assay</td>
			<td>Millipore Sigma</td>
			<td>48-660MAG</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini LabRoller rotator</td>
			<td>VWR</td>
			<td>10136-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride</td>
			<td>Sigma-Aldrich</td>
			<td>P7626-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered Saline (PBS)</td>
			<td>VWR</td>
			<td>97064-158</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate Sealer</td>
			<td>VWR</td>
			<td>82050-992</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene microfuge tubes</td>
			<td>VWR</td>
			<td>20901-547</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini LabRoller</td>
			<td>Millipore Sigma</td>
			<td>Z674591</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent Reservoirs</td>
			<td>VWR</td>
			<td>89094-668</td>
			<td></td>
		</tr>
		<tr>
			<td>R Programming Language</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RStudio</td>
			<td>www.rstudio.com</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Titer plate shaker</td>
			<td>VWR</td>
			<td>12620-926</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>1 m acrylic guide tube</td>
			<td>McMaster-Carr</td>
			<td>49035K85</td>
			<td></td>
		</tr>
		<tr>
			<td>4 photon counting avalanche photodiode</td>
			<td>Perkin-Elmer</td>
			<td>SPCM-AQ4C-IO</td>
			<td></td>
		</tr>
		<tr>
			<td>400 um multimode source fiber</td>
			<td>Thorlabs Inc.</td>
			<td>FT-400-EMT</td>
			<td></td>
		</tr>
		<tr>
			<td>54 g bolt</td>
			<td>Ace Hardware</td>
			<td></td>
			<td>0.95 cm basic body diameter, 2 cm head diameter, 10.2 cm length</td>
		</tr>
		<tr>
			<td>780 nm single mode detector fiber</td>
			<td>Thorlabs Inc.</td>
			<td>780HP</td>
			<td></td>
		</tr>
		<tr>
			<td>852 nm long-coherence length laser</td>
			<td>TOPTICA Photonics</td>
			<td>iBeam smart</td>
			<td></td>
		</tr>
	</tbody>
</table>

systems analysis of the neuroinflammatory and hemodynamic response to traumatic brain injury

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional deficits. Numerous studies by our group and others have shown that mTBI stimulates cytokine expression and activates microglia, decreases cerebral blood flow and metabolism, and impairs cerebrovascular reactivity. Moreover, several works have reported an association between derangements in these neuroinflammatory and hemodynamic markers and cognitive impairments. Herein we detail methods to characterize the neuroinflammatory and hemodynamic tissue response to mTBI in mice. Specifically, we describe how to perform a weight-drop model of mTBI, how to longitudinally measure cerebral blood flow using a non-invasive optical technique called diffuse correlation spectroscopy, and how to perform a Luminex multiplexed immunoassay on brain tissue samples to quantify cytokines and immunomodulatory phospho-proteins (e.g., within the MAPK and NF&#954;B pathways) that respond to and regulate activity of microglia and other neural immune cells. Finally, we detail how to integrate these data using a multivariate systems analysis approach to understand the relationships between all of these variables. Understanding the relationships between these physiologic and molecular variables will ultimately enable us to identify mechanisms responsible for mTBI.

Previously collected data were taken from prior work in which a group of eight C57BL/6 mice were subjected to three closed-head injuries (Figure 2) spaced once daily11. In this work, cerebral blood flow was measured with diffuse correlation spectroscopy 4 h after the last injury (Figure 3, Figure 4). After post-injury CBF assessment, the animals were euthanized, and brain tissue was extracted for quantificati...

Watch this Scientific Journal Video about Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury at JoVE.com

Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury

This protocol presents methods to characterize the neuroinflammatory and hemodynamic response to mild traumatic brain injury and to integrate these data as part of a multivariate systems analysis using partial least squares regression.

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional ...

These methods can be used to characterize neuroinflammatory and hemodynamic responses to brain injury as part of a multivariate system analysis using partial least squares regression. These techniques are used to give more of a holistic view of the brain's response to injury. To induce an injury, confirm a lack of response to toe pinch in an anesthetized mouse and place mouse in the prone position on the center of a thin membrane.Using both hands to hold the tissue taught, secure the mouse's tail under a thumb and position to mouse head under the guide tube. When the mouse is in position, aiming for the impact between the back of the eyes and the front of the ears, drop the bolt from the top of the guide tube onto the dorsal aspect of the mouse's head. Upon impact, the mouse will break through the tissue, allowing rapid acceleration of the head about the neck.After the injury, allow the mouse to recover in the supine position on a 37-degree Celsius warming pad. After a two-minute period of stabilization, gently rest the DCS sensor over the right hemisphere of the mouse's skull, such that the top edge of the optical sensor lines up with the back of the eye and the side of the sensor lines up along the midline. Cup a hand over the sensor to shield it from room light and acquire five seconds of data.Then, reposition the sensor over the left hemisphere and acquire five seconds of left hemisphere data. To assess multiplexed cytokine and phospho-protein production, add 150 microliters of mixed lysis buffer per approximately three milligrams of harvested animal brain tissue. And use a 1, 000-microliter pipette tip to mechanically triturate the tissue 15 to 20 times.Place the homogenized samples on a rotator for 30 minutes at four degrees Celsius before collecting the tissue debris by centrifugation. Transfer the supernatants into new tubes and centrifuge the samples to remove any remaining precipitate. Transfer the supernatants to new tubes and determine the protein concentration by BCA assay according to standard protocols.Prepare 25 microliters of each sample at the optimal protein concentration as determined by the linear range analysis in new tubes. Using assay buffer to normalize the total volume of each sample to 25 microliters. To prepare an assay plate, add 200 microliters of wash buffer to each well of a 96-well plate and shake the plate on a shaker for 10 minutes at 750 revolutions per minute.At the end of the incubation, decant the wash buffer and tap the plate onto a paper towel to remove any residue. Next, add 25 microliters of assay buffer to each well, followed by 25 additional microliters of buffer to the background wells. Add 25 microliters of the diluted samples to the appropriate sample wells and vortex the via of multiplexed magnetic beads for one minute, before adding 25 microliters of the thoroughly re-suspended beads to each well.When all of the beads have been added, seal the plate with a plate sealer and cover the plate with foil for an overnight incubation with shaking at two to eight degrees Celsius. The next morning, place the plate on a magnetic separator, making sure that the wells are aligned with the magnets. After two minutes, decant the well contents with the plate still attached to the magnetic separator and add 200 microliters of wash buffer to each well.After keeping it shaking for two minutes, place the plate onto the magnetic separator for two minutes. Then, decant the well contents with the plate still attached to the magnetic separator and wash the plate with a fresh 200 microliters of wash buffer per well, as just demonstrated. After the second wash, add 25 microliters of detection antibody per well and re-cover with foil for a one-hour incubation at 750 revolutions per minute at room temperature.At the end of the incubation, add 25 microliters streptavidin phycoerythrin to each well and return the plate to the shaker for an additional 30 minutes at room temperature. At the end of the incubation, place the plate onto the magnetic separator for two minutes before decanting the well contents. Wash the plate two times with 200 microliters of fresh washing buffer per wash and add 75 microliters of the appropriate drive fluid to each well.Then, re-suspend the beads on the plate shaker for five minutes at room temperature and read the plates on the analyzer according to the manufacturer's instructions. In this analysis, the cerebral blood flow was measured with diffuse correlation spectroscopy four hours after the last injury. The cerebral blood flow index and mean cerebral flow index for each hemisphere can then be determined.A linear range analysis can be conducted to determine an appropriate protein loading mass prior to collecting data from all samples. Cytokine data can be prepared by subtracting the background measurements from the sample data, then computing Z-score data for each anolyte. Partial least squares regression can be conducted by using a phagocyte microglial activation marker or other variable of interest as the response variable, and the cytokine measurements as the predictor variables.Varimax rotation can be performed to maximize the covariance of the data on latent variable one, with the activation marker measurements. High loading weights in latent variable one correspond with the cytokine expressions most associated with high expression of the activation marker. Linear regressions between the activation marker and cytokines illustrate that those cytokines with the greatest loading weights in latent variable one were also statistically significant for this analysis.Although we focused this protocol on traumatic brain injury, these methods are widely generalizable to the study of a plethora of pathological conditions that affect the brain. We use these techniques to help us identify tractable targets to modulate in future experiments to establish causal mechanistic relationships and ultimately with the goal of developing novel therapeutic strategies for mild traumatic brain injury.

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Neuroscience

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 ...

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 ...

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 ...

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.

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 ...

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.

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

Murine Model of Controlled Cortical Impact for the Induction of Traumatic Brain Injury

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the United States. In fact, TBI is a contributing factor to over a third of all injury-related mortality. Nonetheless, the cellular and molecular mechanisms underlying the pathophysiology of TBI are poorly understood. Thus, preclinical models of TBI capable of replicating the injury mechanisms pertinent to TBI in human patients are a critical research need. The controlled cortical impact (CCI) model of TBI utilizes a mechanical device to directly impact the exposed cortex. While no model can full recapitulate the disparate injury patterns and heterogeneous nature of TBI in human patients, CCI is capable of inducing a wide range of clinically applicable TBI. Furthermore, CCI is easily standardized allowing investigators to compare results across experiments as well as across investigative groups. The following protocol is a detailed description of applying a severe CCI with a commercially available impacting device in a murine model of TBI.

Here we describe a protocol for the induction of murine traumatic brain injury via an open-head controlled cortical impact.

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

A Metric Test for Assessing Spatial Working Memory in Adult Rats Following Traumatic Brain Injury

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of human studies, animal models provide suitable alternatives to test treatment methods, and to study the mechanisms and related complications of the condition. Experimental rodent models have historically been the most widely used due to their accessibility, low cost, reproducibility, and validated approaches. A metric test, which tests the ability to recall the placement of two objects at various distances and angles from one another, is a technique to study impairment in spatial working memory (SWM) after TBI. The significant advantages of metric tasks include the possibility of dynamic observation, low cost, reproducibility, relative ease of implementation, and low stress environment. Here, we present a metric test protocol to measure impairment of SWM in adult rats after TBI. This test provides a feasible way to evaluate physiology and pathophysiology of brain function more effectively.

Traumatic brain injury (TBI) is commonly associated with memory impairment. Here, we present a protocol to assess spatial working memory after TBI via a metric task. A metric test is a useful tool to study spatial working memory impairment after TBI.

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of ...