The authors would like to thank Elliot Bourgeois for his technical assistance. This work was funded by the National Institutes of Health grants R01HD059288 and R01NS069629.

1. Lateral Fluid Percussion Injury
<ol>
 <li>Anaesthetize the mouse using a mixture of ketamine and xylazine given intraperitoneally. Then prepare the mouse's head for incision using an iodine scrub.</li>
 <li>Perform a craniectomy over the right parietal area using 3 mm (outside diameter) trephine.</li>
 <li>Secure Luer-loc needle hub (inside diameter 3 mm) over the craniectomy using cyanoarylate and dental acrylic.</li>
 <li>24 hr later, anaesthetize the mouse using isoflurane via inhalation.</li>
 <li>Once normal breathing resumes, but before the mouse becomes sensitive to stimulation, deliver 20 msec pulse of saline into the skull via the fluid percussion injury device.</li>
 <li>Immediately after injury remove the hub, reanaesthetize the mouse using isoflurane, and suture the scalp closed. 
 Sham-operated controls will receive an identical procedure with step 1.5 omitted.</li>
</ol>
2. Behavioral Analysis - Conditioned Fear Response
<ol>
 <li>Handle mice on two consecutive days prior to conditioned fear response (CFR) training.</li>
 <li>Place mouse in conditioning chamber for 3 min before administering 1.5 mA floor shock for 2 sec. Leave mouse in chamber for an additional 30 sec.</li>
 <li>After delay period (usually 24 hr), return mouse to conditioning chamber for 5 min. Assess freezing at 5 sec intervals. 
 Analysis consists of comparing the relative amount of time freezing in two populations, in our case brain-injured mice and sham-operated controls. Lower freezing rates (compared to control) indicate inability to retain the association between the context and the floor shock, an indication of memory impairment and cognitive dysfunction.</li>
</ol>
3. Preparing Acute Hippocampal Slices
*Note: Anesthetization using a Bell Jar can only be implemented for terminal procedures (such as the brain dissection described herein).
<ol>
 <li>7 days after injury, make 1 L of artificial cerebral spinal fluid (aCSF) and 250 ml sucrose cutting solution.</li>
 <li>Use ice liberally to ensure that all instruments and solutions used during brain slice preparation are ice cold.</li>
 <li>Anaesthetize mouse using isoflurane. Quickly and gently remove brain from mouse and place in sucrose.</li>
 <li>Trim brain and place on cutting surface on a drop of superglue in front of an agar block.</li>
 <li>Cut 350 &mu;m thick coronal slices, you should be able to get 4 or 5 slices with the intact hippocampal circuit.</li>
 <li>Incubate slices for at least 1 hr at 37 &deg;C.</li>
</ol>
4. Extracellular Field Potential Recording
<ol>
 <li>Pull borosilicate glass electrodes to 2-5 M&Omega;s using vertical puller.</li>
 <li>Place slice in chamber and insert electrode onto electrode holder.</li>
 <li>Lower stimulating electrode into slice in an axonal tract such as the perforant path or Schaffer collaterals. Lower recording electrode into position while monitoring output traces to ensure that electrodes are at the same z-level (depth). When a maximal response is achieved, holding stimulation current level constant, electrodes are at the same z-level.</li>
 <li>The resultant trace consists of three components: the presynaptic fiber volley, the field extracellular postsynaptic potential (fEPSP), and the postsynaptic population spike. The components may overlap temporally in some preparations, making analysis more difficult. If there is ambiguity between whether responses are presynaptic or postsynaptic, APV and CNQX can be added to the bath to block excitatory transmission, and all remaining signals will be of presynaptic origin.</li>
 <li>Stimulation protocols vary to produce three distinct experiment types: input/output curves, paired pulse recordings, and long-term plasticity experiments. 
 Analysis typically consists of measurements comparing the slope of the fEPSP across in both brain-injured mice and sham-operated controls, controlling for the input strength by measuring the fiber volley.</li>
</ol>
5. Visualized Patch-clamp Recording
<ol>
 <li>Prepare slices, electrodes, and rig to record as previously described above up to step 4.2.</li>
 <li>Only consider cells deeper than 80 &mu;m into the tissue as cells nearer the surface will be dead and/or have reduced connectivity. Approach a cell with positive pressure on the electrode to ensure that it does not get clogged as it moves down through the tissue. When the electrode gently touches the cell apply negative pressure in order to create a 'gigaseal' between the electrode and the plasma membrane.</li>
 <li>Apply short bursts of negative pressure in order to rupture the plama membrane under the electrode and achieve whole-cell configuration.</li>
 <li>Eliminate capacitance transients using amplifier and compensate for series resistance. Compensating for the series resistance of the cell is essential to ensure accurate measurements.</li>
 <li>Two modes of single cell recordings can be performed: current clamp (measuring membrane voltage) and voltage clamp (measuring current passing through ion channels in the membrane). 
 Possible types of analysis are numerous but in our case mostly consist of biophysical analysis quantifying the rate and size of spontaneous synaptic currents in both brain-injured mice and sham-operated controls.</li>
</ol>
6. Voltage Sensitive Dye Imaging (VSD)
<ol>
 <li>Prepare slices and rig to record as previously described above up to step 4.2.</li>
 <li>Prepare dye stock by mixing 1 mg di-3-ANEPPDHQ into 50 &mu;l ethanol; dispense 2 &mu;l aliquots into foil wrapped tubes, store at -20 &deg;C. Make working dye solution daily, by diluting 1:200 in aCSF.</li>
 <li>Incubate slice on aCSF moistened filter paper and stain with 90 &mu;l of dye for 16 min; rinse with aCSF and place in interface recording chamber.</li>
 <li>Adjust light stimulation intensity until response is centered in middle of camera range. Images are typically acquired at a rate of 500-1,000 frames per second.</li>
 <li>Trigger shutter for light stimulation 200 msec before electrical stimulation to allow initial rapid photobleaching to stabilize. Alternate acquisition trials between electrically stimulated, and non-electrically stimulated, to allow later subtraction of non-stimulated background. Record resting light intensity (fluorescence reading with shutter closed) prior to opening shutter. Each VSD trial will be an x pixels by y pixels by time "movie" of fluorescence readings. Record 10 - 12 VSD trials in each test condition.</li>
 <li>All measurements are performed on the intra-pixel pre-electrical-stimulus normalized fluorescence change, dF/F, which is calculated as follows: 
 Subtract the resting light intensity from the VSD raw acquired trial. For each pixel in each trial, normalize according to the pre-electrical-stimulus mean value for that trial, and subtracting unity. Do this for both the electrically stimulated and non-electrically stimulated trials. Create an average "background fluorescence" trial from the non-electrically stimulated trials, and subtract this background trial from the electrically stimulated trials. Further analysis is typically performed on the average of 10 - 12 such trials, which are also usually filtered in space and time to improve the signal to noise ratio.</li>
</ol>
7. Regional Dissections for Biochemical Analyses
<ol>
 <li>Remove the brain from the mouse as previously described.</li>
 <li>Prepare slices using tissue chopper.</li>
 <li>Lay section flat, microdissect area CA1 and DG.</li>
 <li>Immediately immerse in lysis buffer containing protease inhibitor. Freeze in liquid nitrogen and store at -80 &deg;C.</li>
</ol>
8. Representative Results
Our experiments typically begin with behavioral data to confirm the expected cognitive deficits that exist in brain-injured mice. We employ contextual conditioned fear response testing as it is a hippocampal dependent behavior that is reliable and requires only one training session and one testing session. The data depicted in Figure 2 represents behavioral testing to measure anterograde memory; however, the test can also be used to measure retrograde memory if the training session is performed before the injury.
Field excitatory postsynaptic potentials (fEPSPs) are measured to determine the net synaptic efficacy of a large population of cells (Figure 3A). We commonly employ three different types of stimulation patterns, each affording its own results and conclusions. First, by increasing the stimulation intensity through a series of steps, we create an input/output curve (Figure 3B/C).
Next, by delivering two stimulations of the same intensity separated by a brief delay (usually 50 msec) we investigate potential alterations in synaptic vesicle release probability.; Additionally, we often perform long-term potentiation (LTP) experiments. After establishing a baseline response, brief high frequency stimulation (usually 100Hz) at the same intensity is delivered, causing an intracellular cascade that leads to a potentiated synaptic response when normal stimulation is resumed.
Electrical activity from whole-cell patch-clamped cells can be recorded in two modes. In voltage clamp mode, the experimenter controls the membrane voltage of the patch-clamped cell in reference to the bath, via computer software associated with the amplifier. In this case currents mediating postsynaptic events are recorded, providing information about presynaptic release frequency, number of postsynaptic receptors activated and vesicular neurotransmitter concentration (Figure 4A). In current clamp mode the experimenter manipulates injected current and measures the voltage response. This can be useful to determine characteristics of the action potential, such as action potential threshold and half-width. These characteristics allow for the functional categorization of neurons as excitatory or inhibitory based on their action potential firing patterns (Figure 4B). In order to verify the cellular identity we recommend filling cells with Lucifer Yellow after recording and visually confirming identity by the presence or absence of dendritic spines. (Figure 4C, D).
An example of results from an experiment using voltage sensitive dye to measure changes in membrane voltage is depicted in Figure 5. In this case, a stimulating electrode has been placed in the Schaffer collateral pathway and the resulting neuronal activity in area CA1 is analyzed. Voltage sensitive dye does not report absolute values of membrane voltage, but rather the change in voltage from a baseline condition without stimulation. However, analyses comparing two conditions (e.g. brain-injured vs. sham-operated) can be used to determine the spatiotemporal parameters of physiological alterations that cannot be measured using fEPSPs or whole-cell patch-clamp recording.
<img alt="Figure 1" src="/files/ftp_upload/4411/4411fig1.jpg" /> 
Figure 1. Hippocampal Circuit Diagram. A horizontal section through the hippocampus. The major pathways through the hippocampus are shown in yellow. The axons from neurons in the entorhinal cortex project via the perforant pathway into the dentate gyrus forming synapses with the dendrites of dentate gyrus granule cells. Granule cell axons project via the mossy fiber pathway to CA3 where they form synapses with the dendrites of CA3 neurons. The axons of CA3 neurons project via the Schaffer collateral pathway onto the dendrites of CA1 pyramidal cells. The axons of CA1 pyramidal cells project out of the hippocampus through the subiculum. Note: Also depicted are CA3 pyramidal cell axon collaterals projecting to the contralateral hippocampus via the fimbria. (CA1: Cornu Ammonis 1, CA2: Cornu Ammonis 2, CA3: Cornu Ammonis 3, DG: Dentate Gyrus, dashed line indicates cell body layer, solid line indicates structural boundaries.)
<img alt="Figure 2" src="/files/ftp_upload/4411/4411fig2.jpg" /> 
Figure 2. Representative Behavioral Data. A: Behavioral data depicting the difference in freezing rates between brain-injured mice (FPI) and sham-operated controls (sham) in the conditioned fear response paradigm. Training occurred on the 6th day following injury, with the test period occurring 24 hr later. (** denotes p&lt;.01.)
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/4411/4411fig3highres.jpg" src="/files/ftp_upload/4411/4411fig3.jpg" /> 
Figure 3. Representative Extracellular Recording Data. A: An example of a field excitatory post-synaptic potential (fEPSP) recording in area CA1. The first downward deflection is the stimulus artifact, followed by the presynaptic fiber volley and finally the fEPSP. B: Input/output curves depicting a decrease in net synaptic efficacy in CA1 following fluid percussion injury (FPI). C: Input/output curves depicting an increase in net synaptic efficacy in dentate gyrus (DG) following FPI. (* denotes p&lt;.05.) <a href="https://www.jove.com/files/ftp_upload/4411/4411fig3large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 4" src="/files/ftp_upload/4411/4411fig4.jpg" /> 
Figure 4. Representative Whole-cell Patch-clamp Data. A: An example spontaneous excitatory postsynaptic current (sEPSC) from a CA1 pyramidal cell. B: Examples of action potential trains from a CA1 pyramidal cell (upper trace) and a fast spiking CA1 interneuron (lower trace). C: Example of a Lucifer Yellow filled CA1 inhibitory interneuron. Note the absence of dendritic spines (upper panel). D: Example of Lucifer Yellow filled CA1 pyramidal neuron. Note the presence of dendritic spines (upper panel). <a href="https://www.jove.com/files/ftp_upload/4411/4411fig4large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 5" src="/files/ftp_upload/4411/4411fig5.jpg" /> 
Figure 5. Representative Voltage Sensitive Dye Imaging Data. A: Mouse coronal hippocampal slice displayed to show anatomy. (DG: dentate gyrus, CA1: Cornu Ammonis 1) B: Yellow to red pixels representing excitatory activity in area CA1 14 msec after afferent Schaffer collateral stimulation. Red indicates more depolarization while yellow indicates a lower, but still significant depolarization. C: Blue pixels representing sites of inhibitory activity in area CA1 56 msec after the same afferent Schaffer collateral stimulation shown in B. Darker blue indicates more hyperpolarization.

<ol>
	<li>Faul, M., Xu, L., Wald, M. M., Coronado, V. G. <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+the+United+States:+Emergency+Department+Visits+Hospitalizations+and+Deaths+2002-2006.">Traumatic Brain Injury in the United States: Emergency Department Visits Hospitalizations and Deaths 2002-2006.</a> Centers for Disease Control and Prevention, National Center for Injury Prevention and Control. , (2010).</li><li>McAllister, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropsychiatric+sequelae+of+head+injuries.">Neuropsychiatric sequelae of head injuries.</a> Psychiatr. Clin. North Am. 15, 395-413 (1992).</li><li>Pierce, J. E., Smith, D. H., Trojanowski, J. Q., McIntosh, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enduring+cognitive,+neurobehavioral+and+histopathological+changes+persist+for+up+to+one+year+following+severe+experimental+brain+injury+in+rats.">Enduring cognitive, neurobehavioral and histopathological changes persist for up to one year following severe experimental brain injury in rats.</a> NSC. 87, 359-369 (1998).</li><li>Dixon, C. 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=A+fluid+percussion+model+of+experimental+brain+injury+in+the+rat.">A fluid percussion model of experimental brain injury in the rat.</a> J. Neurosurg. 67, 110-119 (1987).</li><li>McIntosh, T. K., 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+the+rat:+characterization+of+a+lateral+fluid-percussion+model.">Traumatic brain injury in the rat: characterization of a lateral fluid-percussion model.</a> Neuroscience. 28, 233-244 (1989).</li><li>Carbonell, W. S., Grady, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+and+temporal+characterization+of+neuronal,+glial,+and+axonal+response+after+traumatic+brain+injury+in+the+mouse.">Regional and temporal characterization of neuronal, glial, and axonal response after traumatic brain injury in the mouse.</a> Acta Neuropathol. 98, 396-406 (1999).</li><li>Toth, Z., Hollrigel, G. S., Gorcs, T., Soltesz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instantaneous+perturbation+of+dentate+interneuronal+networks+by+a+pressure+wave-transient+delivered+to+the+neocortex.">Instantaneous perturbation of dentate interneuronal networks by a pressure wave-transient delivered to the neocortex.</a> J. Neurosci. 17, 8106-8117 (1997).</li><li>D'Ambrosio, R., Maris, D. O., Grady, M. S., Winn, H. R., Janigro, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+loss+of+hippocampal+long-term+potentiation,+but+not+depression,+following+fluid+percussion+injury.">Selective loss of hippocampal long-term potentiation, but not depression, following fluid percussion injury.</a> Brain Res. 786, 64-79 (1998).</li><li>Witgen, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+hippocampal+alteration+associated+with+cognitive+deficit+following+experimental+brain+injury:+A+systems,+network+and+cellular+evaluation.">Regional hippocampal alteration associated with cognitive deficit following experimental brain injury: A systems, network and cellular evaluation.</a> Neuroscience. 133, 1-15 (2005).</li><li>Schwarzbach, E., Bonislawski, D. P., Xiong, G., Cohen, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+underlying+the+inability+to+induce+area+CA1+LTP+in+the+mouse+after+traumatic+brain+injury.">Mechanisms underlying the inability to induce area CA1 LTP in the mouse after traumatic brain injury.</a> Hippocampus. 16, 541-550 (2006).</li><li>Cole, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+branched+chain+amino+acids+ameliorate+injury-induced+cognitive+impairment.">Dietary branched chain amino acids ameliorate injury-induced cognitive impairment.</a> Proceedings of the National Academy of Sciences. 107, 366-371 (2010).</li></ol>

No conflicts of interest declared.

Each technique outlined above contributes to the greater understanding of the underlying mechanism causing the observed behavioral deficit. By combining the unique information gained from each method we are able to examine the biological mechanisms with more precision.
	Measuring fEPSPs is useful for quantifying the net synaptic efficacy of large, spatially defined regions of neurons. It also can provide information about the potential of a group of cells to undergo synaptic plasticity, a ne...

<table border="1">
	<tbody>
 <tr>
 <td>Name of the equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Axopatch 200B amplifier</td>
 <td>Molecular Devices</td>
 <td>AXOPATCH 200B</td>
 <td>Patch-clamp rig</td>
 </tr>
 <tr>
 <td>Digidata 1322A digitizer</td>
 <td>Molecular Devices</td>
 <td></td>
 <td>Patch-clamp rig</td>
 </tr>
 <tr>
 <td>MP-225 micromanipulator</td>
 <td>Sutter</td>
 <td>MP-225 </td>
 <td>Patch-clamp rig</td>
 </tr>
 <tr>
 <td>DMLFSA microscope</td>
 <td>Leica</td>
 <td></td>
 <td>Patch-clamp rig</td>
 </tr>
 <tr>
 <td>Multiclamp 700B amplifier</td>
 <td>Molecular Devices</td>
 <td>MULTICLAMP 700B</td>
 <td>Multipurpose (field) rig</td>
 </tr>
 <tr>
 <td>Digidata 1440 digitizer</td>
 <td>Molecular Devices</td>
 <td></td>
 <td>Multipurpos (field) rig</td>
 </tr>
 <tr>
 <td>MPC-200 micromanipulator</td>
 <td>Sutter</td>
 <td>MPC-200</td>
 <td>Multipurpose (field) rig</td>
 </tr>
 <tr>
 <td>BX51WI microscope </td>
 <td>Olympus</td>
 <td>BX51WI</td>
 <td>Multipurpose (field) rig</td>
 </tr>
 <tr>
 <td>Axoclamp 900A amplifier</td>
 <td>Molecular Devices</td>
 <td>AXOCLAMP 900A</td>
 <td>VSD rig</td>
 </tr>
 <tr>
 <td>Digidata 1322 digitizer</td>
 <td>Molecular Devices</td>
 <td></td>
 <td>VSD rig</td>
 </tr>
 <tr>
 <td>Redshirt CCD-SMQ camera</td>
 <td>Redshirt</td>
 <td>NCS01</td>
 <td>VSD rig</td>
 </tr>
 <tr>
 <td>VT 1200S Vibratome</td>
 <td>Leica</td>
 <td>14048142066 </td>
 <td></td>
 </tr>
 <tr>
 <td>P-30 Electrode puller</td>
 <td>Sutter</td>
 <td>P-30/P </td>
 <td></td>
 </tr>
 <tr>
 <td>cOmplete protease inhibitor</td>
 <td>Roche</td>
 <td>11697498001 </td>
 <td></td>
 </tr>
	 </tbody>
 </table>

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.

Watch this Scientific Journal Video about Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury at JoVE.com

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

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 ...

investigations-on-alterations-hippocampal-circuit-function-following

Research

JoVE Journal

Neuroscience

15.2K Views.  Children's Hospital of Philadelphia. 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.

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

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 ...

Low-intensity Blast Wave Model for Preclinical Assessment of Closed-head Mild Traumatic Brain Injury in Rodents

Traumatic brain injury (TBI) is a large-scale public health problem. Mild TBI is the most prevalent form of neurotrauma and accounts for a large number of medical visits in the United States. There are currently no FDA-approved treatments available for TBI. The increased incidence of military-related, blast-induced TBI further accentuates the urgent need for effective TBI treatments. Therefore, new preclinical TBI animal models that recapitulate aspects of human blast-related TBI will greatly advance the research efforts into the neurobiological and pathophysiological processes underlying mild to moderate TBI as well as the development of novel therapeutic strategies for TBI.
Here we present a reliable, reproducible model for the investigation of the molecular, cellular, and behavioral effects of mild to moderate blast-induced TBI. We describe a step-by-step protocol for closed-head, blast-induced mild TBI in rodents using a bench-top setup consisting of a gas-driven shock tube equipped with piezoelectric pressure sensors to ensure consistent test conditions. The benefits of the setup that we have established are its relative low-cost, ease of installation, ease of use and high-throughput capacity. Further advantages of this non-invasive TBI model include the scalability of the blast peak overpressure and the generation of controlled reproducible outcomes. The reproducibility and relevance of this TBI model has been evaluated in a number of downstream applications, including neurobiological, neuropathological, neurophysiological and behavioral analyses, supporting the use of this model for the characterization of processes underlying the etiology of mild to moderate TBI.

We present here a protocol of a blast wave model for rodents to investigate neurobiological and pathophysiological effects of mild to moderate traumatic brain injury. We established a gas-driven, bench-top setup equipped with pressure sensors allowing for reliable and reproducible generation of blast-induced mild to moderate traumatic brain injury.

Traumatic brain injury (TBI) is a large-scale public health problem. Mild TBI is the most prevalent form of neurotrauma and accounts for a large number of ...

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 ...

Assessing Changes in Synaptic Plasticity Using an Awake Closed-Head Injury Model of Mild Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of closed-head mTBI in the preclinical setting to increase translatability to the clinical population. The awake closed-headed injury (ACHI) model uses a modified controlled cortical impactor to deliver closed-headed injury, inducing clinically relevant behavioral deficits without the need for a craniotomy or the use of an anesthetic.
This technique does not normally induce fatalities, skull fractures, or brain bleeds, and is more consistent with being a mild injury. Indeed, the mild nature of the ACHI procedure makes it ideal for studies investigating repetitive mTBI (r-mTBI). Growing evidence indicates that r-mTBI can result in a cumulative injury that produces behavioral symptoms, neuropathological changes, and neurodegeneration. r-mTBI is common in youths playing sports, and these injuries occur during a period of robust synaptic reorganization and myelination, making the younger population particularly vulnerable to the long-term influences of r-mTBI.
Further, r-mTBI occurs in cases of intimate partner violence, a condition for which there are few objective screening measures. In these experiments, synaptic function was assessed in the hippocampus in juvenile rats that had experienced r-mTBI using the ACHI model. Following the injuries, a tissue slicer was utilized to make hippocampal slices to evaluate bidirectional synaptic plasticity in the hippocampus at either 1 or 7 days following the r-mTBI. Overall, the ACHI model provides researchers with an ecologically valid model to study changes in synaptic plasticity following mTBI and r-mTBI.

Here, it is demonstrated how an awake closed-head injury model can be used for examining the effects of repeated mild traumatic brain injury (r-mTBI) on synaptic plasticity in the hippocampus. The model replicates important features of r-mTBI in patients and is used in conjunction with in vitro electrophysiology.

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of ...

Validating Injury Response and Behavioral Assessments in an mTBI Rat Model

Rat Model of Closed-Head Mild Traumatic Injury and its Validation

Animal models are crucial for advancing our understanding of mild traumatic brain injury (mTBI) and guiding clinical research. To achieve meaningful insights, developing a stable and reproducible animal model is essential. In this study, we report a detailed description of a closed-head mTBI model and a representative validation method using Sprague-Dawley rats to verify the modeling effect. The model involves dropping a 550 g mass weight from a height of 100 cm directly onto the head of a rat on a destructible surface, followed by a 180-degree turn. To assess the injury, rats underwent a series of neurobehavioral assessments 10 min post-injury, including time of loss of consciousness, first seeking-behavior time, escape ability, and beam balance ability test. During the acute and subacute stages following the injury, behavioral tests were conducted to assess motor coordination ability (Beam task), anxiety (Open Field test), and learning and memory abilities (Morris Water Maze test). The closed-head mTBI model produced a consistent injury response with minimal mortality and replicated real-life situations. The validation method effectively verified the model development and ensured the stability and consistency of the model.

Author Spotlight: Advancing Traumatic Brain Injury Research - A Closed-Head Model for Accurate Replication and Rapid Assessment 

Here, we present a closed-head mild traumatic brain injury (mTBI) rat model and its validation exhibiting remarkable similarity to human mTBI concerning behavioral manifestations during the acute and subacute stages.

Animal models are crucial for advancing our understanding of mild traumatic brain injury (mTBI) and guiding clinical research. To achieve meaningful ...

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 ...

Fluorescent Dye-Based Visualization of Blood-Brain Barrier Breakdown in Mice

Evaluation of Blood-Brain Barrier Breakdown in a Mouse Model of Mild Traumatic Brain Injury

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is a complex process influenced by several factors, such as the concentration of dyes in the blood, permeability of brain vessels, duration of dye extravasation, and reduction in dye concentration in the tissue due to degradation and diffusion. In a mild traumatic brain injury model, exposure to blast-induced shock waves (BSWs) triggers BBB breakdown within a limited time window. To determine the precise sequence of BBB breakdown, Evans blue, and fluorescein isothiocyanate-dextran were injected intravascularly and intracardially into mice at various time points relative to BSW exposure. The distribution of dye fluorescence in brain slices was then recorded. Differences in the distribution and intensity between the two dyes revealed the spatiotemporal sequence of BBB breakdown. Immunostaining of the brain slices showed that astrocytic and microglial responses correlated with the sites of BBB breakdown. This protocol has broad potential for application in studies involving different BBB breakdown models.

A method was developed to visualize dye extravasation due to blood-brain barrier (BBB) breakdown by administering two fluorescent dyes to mice at different time points. The use of glycerol as a cryoprotectant facilitated immunohistochemistry on the same sample.

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is ...