Name: assessing changes in synaptic plasticity using an awake closed-head injury model of mild traumatic brain injury
Uploaded: 2023-01-20T12:55:00
Description: 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

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.

Approval for all animal procedures was provided by the University of Victoria Animal Care Committee in compliance with Canadian Council on Animal Care (CCAC) standards. All male Long-Evans rats were bred in-house or purchased (see the Table of Materials).
1. Housing and breeding conditions
<ol>
	<li>Allow the animals to acclimate to their housing environment for 1 week before weaning at postnatal day (PND) 21.</li>
	<li>Maintain the rats in standard cage hou...

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, th...

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...

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 ...

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