We thank all the members of the Christie Laboratory at the University of Victoria, past and present, for their contributions to the development of this protocol. This project was supported with funds from the Canadian Institutes for Health Research (CIHR: FRN 175042) and NSERC (RGPIN-06104-2019). The Figure 1 skull graphic was created with BioRender.

The authors have no conflicts of interest to disclose.

Most preclinical research has utilized models of mTBI that do not recapitulate the biomechanical forces seen in the clinical population. Here, it is shown how the ACHI model can be used to induce r-mTBIs in juvenile rats. This closed-headed model of r-mTBI has significant advantages over more invasive procedures. First, the ACHI does not normally cause skull fractures, brain bleeds, or fatalities, all of which would be contraindications of a &#34;mild&#34; TBI in clinical populations61. Second, the ACHI does not require the use of craniotomies, which is significant because they are known to cause inflammatory responses that can exacerbate symptomologies and neuropathology67. Third, the ACHI does not require the use of anesthesia. This is also significant, as anesthesia can have neuroprotective properties and can impair synaptic plasticity, in addition to learning and memory performance48,49,50,51,68. Finally, the ACHI can produce subtle transient changes in neurological function that can be assessed immediately post injury.
As the ACHI does not normally induce loss of consciousness or apnea, this model mimics mTBI in a significant proportion of the clinical population69,70,71. Despite this, the ACHI model produced a significant reduction in the NAP scores. This reduction persisted with repeated administrations of the ACHI procedure but did not exacerbate sensorimotor impairments within the r-mTBI group. This indicates that the ACHI model induces a mild injury analogous to that observed following concussive or sub-concussive head impacts in clinical populations72,73. A primary advantage of the NAP is the detection of subtle behavioral deficits seen in the acute timeframe following r-mTBI. This quick examination may allow researchers to categorize rats based on their behavioral responses. However, the use of more robust behavioral tests at subacute and chronic time points may be necessary to detect motor, cognitive, and affective symptomologies74,75,76. It is important to note that while there were no differences in NAP scores over the eight injuries, rodent behavior can be influenced by changes in environment and familiarity with the experimenter77,78. Rats should be allowed to acclimate to the procedure room prior to administering r-mTBI or sham injuries. In addition, it is important for one individual to be responsible for administering the impacts to help ensure consistency.
Despite the previously mentioned benefits of the ACHI model, it is not without limitations. First, the paradigm was designed to mimic the cumulation of impacts in a single session and not repetitive injuries following a recovery period. Following injury, the brain resides in a window of cerebral vulnerability that extends from 1 to 5 days post injury in rodents15,79,80. Receiving eight injuries on a singular day does not allow for acute and subacute injury cascades to develop. Therefore, depending on the research question of interest, the injury paradigm may need to be adjusted within the window of vulnerability. Second, while it is beneficial to limit the use of anesthetic, an unintended consequence of the ACHI model is subjecting the rats to restraint stress. It has been shown that exposure to acute and chronic stressors may initiate an inflammatory response, influence a variety of behaviors, and alter synaptic plasticity in the hippocampus81,82,83.
The protocol described above provides a clear-cut method to produce high-quality transverse hippocampal slices from r-mTBI-administered animals with the ACHI model. Additionally, the protocol allows for stable electrophysiological recordings and shows that the hippocampus is still capable of exhibiting synaptic plasticity following r-mTBI, although there may be transient disruptions. With any electrophysiological recordings, slice health is paramount for the ability to record suitable fEPSPs. To preserve brain tissue, prior to slicing, it is imperative that the brain remains ice-cold in carbogenated aCSF. The brain&#39;s removal and slicing should be done quickly, but not if this comes at the expense of care. This protocol on juvenile animals utilizes aCSF as cutting solution, but depending on the age of the animal, protective cutting solutions (such as choline-, sucrose-, NMDG-, or glycerol-based solutions) may be required84,85,86.
Field electrophysiological recordings allow researchers to gauge hippocampal synaptic plasticity. However, there are a number of limitations to the technique. The process of slicing the brain has shown to cause changes in spine numbers87, which could affect synaptic plasticity. The use of in vivo recordings would preserve pathways and allow for measurement of synaptic plasticity in anesthetized or live animals88. Additionally, the use of field recordings probes the properties of groups of neurons but does not inform about changes in individual neurons. The use of whole-cell patch-clamp recordings can give temporally detailed information about neuronal properties in response to pharmacological or optogenetic manipulations89. Additionally, the combination of electrophysiological recordings with complementary techniques, such as calcium imaging, Western blotting, immunohistochemistry, or electron microscopy, would allow researchers to gain insight into the mechanisms of action.
Cognitive deficits are commonly reported following r-mTBI, and the current protocol can help investigate some of the underlying physiological processes associated with these deficits. In particular, the mild nature of the ACHI procedure opens up the possibility of examining changes in synaptic physiology across the lifespan of animals that have incurred r-mTBI. The ACHI model appears to be an ecologically valid model of mTBI than can be used to study r-mTBI. Preliminary studies utilizing the ACHI model have showed acute neurological impairment without overt structural damage, administering one, four, and eight repeated injury paradigms61,90. Future studies will examine how r-mTBI can impact synaptic plasticity during developmental periods and in the aging brain. By better understanding the pathophysiology of mTBI and r-mTBI for synaptic function, the hope is to better direct potential therapeutic interventions to help reduce cognitive function.

Traumatic brain injury (TBI) is a significant health issue, with ~2 million cases in Canada and the United States every year1,2. TBI affects all age groups and genders and has an incidence rate greater than any other disease, notably including breast cancer, AIDS, Parkinson&#39;s disease, and multiple sclerosis3. Despite the prevalence of TBI, its pathophysiology remains poorly understood, and treatment options are limited. In part, this is because 85% of all TBIs are classified as mild (mTBI), and mTBI has previously been thought to produce only limited and transient behavioral changes with no long-term neuropsychiatric consequences4,5. It is now recognized that mTBI recovery can take weeks to years5,6, precipitate more serious neurological conditions4, and that even repeated &#34;sub-concussive&#34; impacts affect the brain7. This is alarming as athletes in sports such as hockey/football have &#62;10 head sub-concussive impacts per game/practice session7,8,9,10.
Adolescents have the highest incidence of mTBI,&#160;and in Canada, roughly one in 10 teens will seek medical care for a sport-related concussion annually11,12. In reality, any sub-concussive head impact or mTBI can cause diffuse damage to the brain, and this could also create a more vulnerable state for subsequent injuries and/or more serious neurological conditions13,14,15,16,17. In Canada, it is recognized legally via Rowan&#39;s law that prior injury can increase the vulnerability of the brain to further injury18, but mechanistic understanding of r-mTBI remains woefully inadequate. It is clear, however, that single and r-mTBI can impact learning capacity during school years19,20, have sex-specific outcomes21,22,23,24, and impair cognitive capacity later in life16,25,26. Indeed, cohort analyses strongly associate r-mTBI early in life with dementia later on27,28. r-mTBI is also potentially associated with chronic traumatic encephalopathy (CTE), which is characterized by the accumulation of hyperphosphorylated tau protein and progressive cortical atrophy and precipitated by significant inflammation27,29,30,31. Although the links between r-mTBI and CTE are currently controversial32, this model will allow them to be explored in greater detail in a preclinical setting.
An mTBI is often described as an &#34;unseen injury,&#34; as it occurs within a closed skull and is difficult to detect even with modern imaging techniques33,34. An accurate experimental model of mTBI should adhere to two tenets. First, it should recapitulate the biomechanical forces normally observed in the clinical population35. Second, the model should induce heterogeneous behavioral outcomes, something that is also highly prevalent in clinical populations36,37,38. Currently, the majority of preclinical models tend to be more severe, involving craniotomy, stereotaxic head restraint, anesthesia, and controlled cortical impacts (CCI) that produce significant structural damage and more extensive behavioral deficits than normally observed clinically33. Another concern with many preclinical models of concussion that involve craniotomies is that this procedure itself creates inflammation in the brain, and this can exacerbate mTBI symptoms and neuropathology from any subsequent injury39,40. Anesthesia also introduces several complex confounds, including reducing inflammation41,42,43, modulating microglial function44, glutamate release45, Ca2+ entry through NMDA receptors46, intracranial pressure, and cerebral metabolism47. Anesthesia further introduces confounds by increasing blood-brain barrier (BBB) permeability, tau hyperphosphorylation, and corticosteroid levels, while reducing cognitive function48,49,50,51. Additionally, diffuse, closed-headed injuries represent the vast majority of clinical mTBIs52. They also allow one to better study the multitude of factors that can influence behavioral outcomes, including sex21, age53, inter-injury-interval15, severity54, and the number of injuries23.
The direction of the accelerative/decelerative forces (vertical or horizontal) is also an important consideration for behavioral and molecular outcomes. Research from Mychasiuk and colleagues have compared two models of diffuse closed-headed mTBI: weight-drop (vertical forces) and lateral impact (horizontal forces)55. Both the behavioral and molecular analyses revealed heterogeneous model- and sex-dependent outcomes following mTBI. Thus, animal models that help avoid surgical procedures, while incorporating linear and rotational forces, are more representative of the physiological conditions under which these injuries normally occur33,56. The ACHI model was created in response to this need, allowing for the rapid and reproducible induction of mTBI in rats while avoiding procedures (i.e., anesthesia) that are known to bias sex differences57.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D-printed helment&#160;</td>
			<td></td>
			<td></td>
			<td>Designed and constructed by Christie laboratory (See Specifications in Christie et al. (2019), Current Protocols in Neuroscience)&#160;</td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>Fisher Scientific (BioReagents)</td>
			<td>BP160500</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia chamber</td>
			<td>Home Made</td>
			<td>N/A</td>
			<td>Plexiglass Container</td>
		</tr>
		<tr>
			<td>Automatic Heater Controller</td>
			<td>Warner Electric</td>
			<td>TC-324B</td>
			<td></td>
		</tr>
		<tr>
			<td>Axon Digidata</td>
			<td>Molecular Devices</td>
			<td>1440A</td>
			<td>Low-noise Data Acquisition System</td>
		</tr>
		<tr>
			<td>Balance beam&#160;</td>
			<td></td>
			<td></td>
			<td>Can be constructed or purchased (100 cm long x 2 cm wide x 0.75 cm thick)</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Bio Basic Canada Inc.&#160;</td>
			<td>CD0050</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Dage MTI</td>
			<td>NC-70</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen tank</td>
			<td>Praxair</td>
			<td>MM OXCD5C-K</td>
			<td>Carbon Dioxide 5%, Oxygen 95%</td>
		</tr>
		<tr>
			<td>Clampex Software</td>
			<td>Molecular Devices</td>
			<td>Clampex 10.5 Version</td>
			<td></td>
		</tr>
		<tr>
			<td>Compresstome Vibrating Microtome</td>
			<td>Precisionary</td>
			<td>VF 310-0Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Concentric Bipolar Electrode</td>
			<td>FHC Inc.</td>
			<td>CBAPC75</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose (D-Glucose)</td>
			<td>Fisher Scientific (Chemical)</td>
			<td>D16-3</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>Digital Stimulus Isolation Amplifier&#160;&#160;</td>
			<td>Getting Instruments, Inc.&#160;</td>
			<td>Model 4D</td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium Phosphate</td>
			<td>Fisher Scientific (Chemical)</td>
			<td>S373-500</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Dissection Tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Feather Double Edge Blade</td>
			<td>Electron Microscopy Sciences</td>
			<td>72002-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Paper</td>
			<td>Whatman 1</td>
			<td>1001-055</td>
			<td></td>
		</tr>
		<tr>
			<td>Flaming/Brown Micropipette Puller</td>
			<td>Sutter Instrument</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair Claw Clip</td>
			<td></td>
			<td></td>
			<td>Can be obtained from any department store</td>
		</tr>
		<tr>
			<td>Home and Recovery Cages</td>
			<td></td>
			<td></td>
			<td>Normal rat cages from animal care unit.</td>
		</tr>
		<tr>
			<td>Hum Bug Noise Eliminator</td>
			<td>Quest Scientific&#160;</td>
			<td>726300</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane USP</td>
			<td>Fresenius Kabi</td>
			<td>CP0406V2</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp 215 Digital Water Bath</td>
			<td>Fisher Scientific&#160;</td>
			<td>15-462-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Impact One CCI unit</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td>Tip is modified to hold 7mm rubber impact tip</td>
		</tr>
		<tr>
			<td>Long-Evans rats, male</td>
			<td colspan="3">Charles River Laboratories (St. Constant, PQ)</td>
		</tr>
		<tr>
			<td>Low-Density Foam Pad</td>
			<td></td>
			<td></td>
			<td>3&#34; polyurethane foam sheet&#160;</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Fisher Scientific (Chemical)</td>
			<td>M33-500</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>Male Long Evans Rats</td>
			<td>Charles River Laboratories</td>
			<td></td>
			<td>Animals ordered from Charles River Laboratories, or pups bred at the University of Victoria</td>
		</tr>
		<tr>
			<td>MultiClamp 700B Amplifier</td>
			<td>Molecular Devices</td>
			<td>Model 700B</td>
			<td></td>
		</tr>
		<tr>
			<td>pH Test Strips</td>
			<td>VWR Chemicals BDH</td>
			<td>BDH83931.601</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific (Chemical)</td>
			<td>P217-500</td>
			<td>aCSF, PBS</td>
		</tr>
		<tr>
			<td>Potassium Phosphate</td>
			<td>Sigma</td>
			<td>P9791-500G</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Push Button Controller</td>
			<td>Siskiyou Corporation&#160;</td>
			<td>MC1000e</td>
			<td>Four-axis Closed Loop Controller Push-Button</td>
		</tr>
		<tr>
			<td>Sample Discs</td>
			<td>ELITechGroup</td>
			<td>SS-033</td>
			<td>For use with Vapor Pressure Osmometer</td>
		</tr>
		<tr>
			<td>Small towel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Fisher Scientific (Chemical)</td>
			<td>S233-500</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific (Chemical)</td>
			<td>S271-3</td>
			<td>For aCSF, PBS</td>
		</tr>
		<tr>
			<td>Sodium Phosphate</td>
			<td>Fisher Scientific (Chemical)</td>
			<td>S369-500</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>Soft Plastic Restraint Cones</td>
			<td>Braintree Scientific</td>
			<td>model DC-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Stopwatch</td>
			<td></td>
			<td></td>
			<td>Many lab members use their iPhone for this</td>
		</tr>
		<tr>
			<td>Table or large cart with raised edges&#160;</td>
			<td></td>
			<td></td>
			<td>For NAP and ACHI</td>
		</tr>
		<tr>
			<td>Thin Wall Borosilicate Glass (with Filament)</td>
			<td>Sutter Instrument</td>
			<td>BF150-110-10</td>
			<td>Outside diameter: 1.5 mm; Inside diameter: 1.10 mm; Length: 10 cm</td>
		</tr>
		<tr>
			<td>Upright Microscope</td>
			<td>Olympus</td>
			<td>Olympus BX5OWI</td>
			<td>5x MPlan 0.10 NA Objective lens</td>
		</tr>
		<tr>
			<td>Vapor Pressure Osmometer</td>
			<td>Vapro</td>
			<td>Model 5600</td>
			<td>aCSF should be 300-310 mOSM</td>
		</tr>
		<tr>
			<td>Vetbond Tissue Adhesive</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibraplane Vibration Isolation Table</td>
			<td>Kinetic Systems</td>
			<td>9101-01-45</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The awake closed-head injury model is a viable method of inducing r-mTBI in juvenile rats. Rats exposed to r-mTBI with the ACHI model did not show overt behavioral deficits. Subjects in these experiments did not exhibit latency to right or apnea at any point during the r-mTBI procedure, indicating that this was indeed a mild TBI procedure. Subtle behavioral differences did emerge in the NAP; as described above, the rats were scored on four sensorimotor tasks (startle response, limb extension, beam walk, and rotating beam) on a scale from 0 to 3, with 3 representing no impairment with the task. Thus, the lower the NAP score, the more impaired the animal was. At baseline, there were no differences in the NAP scores between sham and r-mTBI rats. Following all ACHI sessions, the r-mTBI rats showed significant impairments within the NAP tasks when compared to shams (Figure 4). However, as reported previously for impacts delivered over multiple days (i.e., 2 or 4 days), the subsequent addition of injuries over the course of the day did not compound or produce additional behavioral deficits. Thus, the ACHI model of r-mTBI produces subtle, yet significant, behavioral deficits during these acute post injury time points.
Following the injury protocol, evoked field responses and synaptic plasticity were examined in the MPP input to the DG of the hippocampus on post injury day 1 (PID1), and PID7. Slice health was examined using fEPSPs in response to an ascending series of pulse widths in each slice. As is shown in Figure 3C, there was no difference in the input-output curves generated in slices obtained from sham and r-mTBI rats. To examine presynaptic transmitter release, a series of paired pulses (50 ms interpulse interval) were administered, and the ratio of the size of the second fEPSP was calculated relative to the first fEPSP. The paired-pulse ratios did not differ between sham and r-mTBI rats (Figure 3D). Thus, these data indicate that r-mTBI did not alter basic synaptic physiology in the MPP input to the DG. To examine LTD, a 10 Hz LTD protocol was administered to induce an LTD dependent on endocannabinoids64. On PID1, there was a significant decrease in the capacity of the MPP input to the DG to sustain LTD (Figure 3E). This reduction in LTD was transient, however, and by PID7, slices from sham and r-mTBI animals displayed equivalent LTD (Figure 3F), although there was an indication of a slight trend for slices from r-mTBI animals to exhibit an increase in LTD.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64592/64592fig01.jpg" /> 
Figure 1: The ACHI procedure setup used to model r-mTBI. (A) A modified controlled cortical impactor was used to rapidly displace the animal&#39;s head 10 mm at a velocity of 6.0 m/s. (B,C) Custom 3D printed helmet with a left parietal cortex target site. (D) Subjects were placed in a plastic restraint bag on a foam platform, with the helmet placed around the restraint cone and positioned so the target site is directly under the impactor tip. Abbreviations: ACHI = awake closed-head injury; r-mTBI = repeated mild traumatic brain injury. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64592/64592fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64592/64592fig02.jpg" /> 
Figure 2: Materials and setup required for slice preparation. (A) Tools used for brain extraction, mounting, slicing, and incubation: (a) culture dish with filter paper; (b) various dissection tools, including standard scissors, dissecting scissors, forceps, a rongeur, and spatulas; (c) tissue adhesive; (d) Compresstome piston and specimen tube; (e) feather blade and blade holder; (f) chilling block; (g) slice incubation chamber. (B)&#160;Compresstome tissue slicer. (C)&#160;Slices incubating in a bath containing artificial cerebrospinal fluid that is continuously oxygenated with 95% O2/5% CO2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64592/64592fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64592/64592fig03.jpg" /> 
Figure 3: Acute impairments in synaptic plasticity in juvenile male rats due to r-mTBI using the ACHI model. (A)&#160;The major hippocampal pathways. The medial perforant pathway is comprised of the input from the entorhinal cortex into the dentate gyrus (blue). The medial perforant path inputs synapse onto granule cells in the dentate gyrus (purple). (B) Brightfield photomicrograph of a hippocampal brain slice (4x magnification), showing the actual placement of a bipolar stimulating electrode (left) and a glass recording electrode pipette (right) in the medial performant path of the dentate gyrus. (C)&#160;Input-output plot (fEPSP slope) for different simulation intensities (10-300 &#181;s) on PID1 and PID7 for sham and r-mTBI rats. (D)&#160;Paired-pulse ratios for sham and r-mTBI rats (50 ms interpulse interval). (E)&#160;Time course of fEPSP changes prior to, and following, administration of an LTD induction paradigm in hippocampal slices obtained from sham and r-mTBI rats at PID1. (F) Time course of fEPSP changes prior to, and following, administration of an LTD induction paradigm in hippocampal slices obtained from sham and r-mTBI rats at PID7. Abbreviations: ACHI = awake closed-head injury; r-mTBI = repeated mild traumatic brain injury; PID = post injury day; fEPSP = field excitatory postsynaptic potential. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64592/64592fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64592/64592fig04.jpg" /> 
Figure 4: Acute neurological impairment in juvenile male rats due to r-mTBI using the ACHI model. The rats underwent eight ACHI procedures at 2 h intervals over 1 day, with a neurological assessment protocol conducted at baseline and after each injury. The NAP consisted of four tasks: startle response, limb extension, beam walk, and rotating beam. Each task was scored out of 3, giving a total possible score of 12 for each session. Data presented as mean &#177; SEM. (*) indicates p &#60; 0.05. Abbreviations: ACHI = awake closed-head injury; r-mTBI = repeated mild traumatic brain injury; NAP = neurological assessment protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64592/64592fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Table S1: ACHI procedure animal and impact information. Abbreviation: ACHI = awake closed-head injury. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64592/64592_Supp Table S1.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Table S2: Restraint scoring for awake mTBI. Abbreviation: mTBI = mild traumatic brain injury. &#34;Turn around in restraint&#34; refers to the researcher placing the animal in the restraint, before closing the bag around the tail. After the bag is closed, the animal should not be able to turn around. Vocalization and squirming should be scored after the bag is closed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64592/64592_Supp Table S2.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary File 1: Cage-side monitoring checklist. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64592/Supp 1_Cage-side monitoring checklist.docx" target="_blank">Please click here to download this File.</a>
Supplementary File 2: Pain scale and advanced monitoring checklist. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64592/Supp 2_Pain scale and advanced monitoring checklist.docx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Assessing Changes in Synaptic Plasticity Using an Awake Closed-Head Injury Model of Mild Traumatic Brain Injury at JoVE.com

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

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 ...

assessing-changes-synaptic-plasticity-using-an-awake-closed-head

Nanyang Technological University

Research

JoVE Journal

Neuroscience

2.2K Views.  University of Victoria. 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.

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

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 ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

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 ...

Pentylenetetrazole-Induced Kindling Mouse Model

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high dose, whereas sequential injections of a subconvulsive dose have been used for the development of chemical kindling, an epilepsy model. A single low-dose injection of PTZ induces a mild seizure without convulsion. However, repetitive low-dose injections of PTZ decrease the threshold to evoke a convulsive seizure. Finally, continuous low-dose administration of PTZ induces a severe tonic-clonic seizure. This method is simple and widely applicable to investigate the pathophysiology of epilepsy, which is defined as a chronic disease that involves repetitive seizures. This chemical kindling protocol causes repetitive seizures in animals. With this method, vulnerability to PTZ-mediated seizures or the degree of aggravation of epileptic seizures was estimated. These advantages have led to the use of this method for screening anti-epileptic drugs and epilepsy-related genes. In addition, this method has been used to investigate neuronal damage after epileptic seizures because the histological changes observed in the brains of epileptic patients also appear in the brains of chemical-kindled animals. Thus, this protocol is useful for conveniently producing animal models of epilepsy.

This protocol describes a method of chemical kindling with pentylenetetrazole and provides a mouse model of epilepsy. This protocol can also be used to investigate vulnerability to seizure induction and pathogenesis after epileptic seizures in mice.

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high ...

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.

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 ...

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 ...