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

Summary

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.

Abstract

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.

Introduction

Traumatic brain injury (TBI) is a significant health issue, with ~2 million cases in Canada and the United States every year^1,2. TBI affects all age groups and genders and has an incidence rate greater than any other disease, notably including breast cancer, AIDS, Parkinson'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....

Protocol

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

1. Allow the animals to acclimate to their housing environment for 1 week before weaning at postnatal day (PND) 21.
2. Maintain the rats in standard cage housing at 22.5 °C ± 2.5 °C, with ad libitum access to food and water, on a 12 h light/dark cycle.
3. Group and house the ....

Results

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.......

Discussion

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 "mild" TBI in clinical populations⁶¹. Second, th.......

Materials

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