generating a traumatic brain injury mouse model using an impactor probe

Generating a Traumatic Brain Injury Mouse Model Using an Impactor Probe

To perform a unilateral craniectomy, confirm a lack of response to toe pinch in the anesthetized mouse, and secure the mouse in a stereotaxic frame. Apply antibiotic ophthalmic ointment to the eyes with a sterile cotton swab, and iodine-based solution to the shaved scalp area. Remove the iodine with 70% ethanol, and cover the animal with a fenestrated surgical drape.
Make a 1.5 to 2-centimeter midline incision on the scalp, and use sterile cotton swabs to clean the wound and to clear the fascia left of the midline at bregma. Use an impactor probe to identify the craniectomy site, and set the x equals 0, y equals 0 stereotaxic reference point to bregma. Adjust the probe laterally to two millimeters left of the midline. Use a fine tip surgically safe marker to landmark a five-millimeter diameter circle around the presumptive impact area.
Raise and rotate the impactor out of position and complete the 5-millimeter circle. Briefly return the impactor into position to verify accuracy of the 5-millimeter circle. Then, raise and rotate the impactor out of position, and use a high-speed rotary micromotor kit hand tool equipped with a round tip of 0.6 or 0.8-millimeter bird drill bit to make an open hole in the skull at 70% to 80% maximum speed. Apply light pressure to the skull while drilling along the 5-millimeter circumferential outline to thin this border, and use forceps to lift the resulting bone flap.
Drilling through suture lines may present additional difficulty as the bone is interrupted and requires additional pressure. Blood vessels may be in close association, and therefore bleeding events are likely.
To induce a mild controlled cortical impact injury, clean the impactor probe with a sterile alcohol prep pad, and move the impactor probe back into position over the exposed cortex. Lower the probe until it touches the dura mater surface, and mark this position as z equals 0. Withdraw the piston, and move to z equals minus 1 millimeter, and discharge the piston to impact the cortex. Immediately raise the piston and move the arm out of position, and irrigate the cortex with a generous volume of saline, and use simple interrupted stitches and 5-0 silk suture to close the scalp incision.

generating-traumatic-brain-injury-mouse-model-using-an-impactor

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Trauma

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Craniectomy and controlled cortical impact
<ol>
	<li>Preparation of the controlled cortical impact device and surgical supplies.
	<ol>
		<li>Load a 1 mL slip-tip syringe with 0.5 mL of sterile saline for wound irrigation. Attach a 25 G needle to the syringe to control irrigation.</li>
		<li>Prepare a dilute solution of cyclosporine A (CsA) in dimethyl sulfoxide (DMSO) to a final concentration of 1 mg/mL. Load a second 1 mL slip-tip syringe with 0.5 mL of CsA solution for immunosuppression. Attach a 25 G needle or larger to the CsA syringe.</li>
		<li>Attach the controlled cortical impact piston to an arm on a stereotaxic frame and set it to a 15 &#176; angle. Attach a 3 mm impactor probe to the piston.</li>
		<li>Set the velocity of impact to 1.5 m/s and the impact dwell time to 0.1 s to produce a mild cortical injury.</li>
	</ol>
	</li>
	<li>Perform a unilateral craniectomy
	<ol>
		<li>Place the mouse in an anesthesia induction chamber connected to an isoflurane vaporizer with a compressed oxygen source. Induce anesthesia with ~3% isoflurane at ~0.7 L/min oxygen. Check for the depth of anesthesia by the lack of response to the toe pinch.</li>
		<li>Shave the scalp using electric clippers and wipe away any loose fur.</li>
		<li>Place the mouse in a stereotaxic frame with an attached anesthetic delivery nose cone.
		<ol>
			<li>Place a 37 &#176;C warming pad on the stereotaxic frame under the mouse to maintain body temperature under anesthesia. Fix the head in place with ear bars and a bite bar and orient the head so that the skull frontal bone is horizontal. Maintain anesthesia at ~1.5%-2% isoflurane for the duration of the surgery.</li>
		</ol>
		</li>
		<li>To perform preoperative care and aseptic surgical preparation, apply antibiotic ophthalmic ointment to the eyes using a sterile cotton swab. Apply an iodine-based solution to the shaved scalp area. Remove this with 70% ethanol. Cover the animal with a fenestrated surgical drape so that the top of the head is visible, but the eyes are covered.</li>
		<li>Make a midline incision (1.5-2 cm) on the scalp using a scalpel or scissors. Use sterile cotton swabs to clean the wound and to clear the fascia left of the midline at bregma.</li>
		<li>Use the impactor probe to identify the craniectomy site.
		<ol>
			<li>Set the stereotaxic reference point (X = 0, Y = 0) to bregma. Adjust the probe laterally to 2 mm left of the midline. Outline a 5 mm diameter circle around the probe using a fine-tip surgically safe marker. Raise and rotate the impactor out of position.</li>
		</ol>
		</li>
		<li>Use the high-speed rotary micromotor kit hand tool to make an open hole in the skull using a round-tip 0.6 mm or 0.8 mm burr drill bit at ~70%-80% maximum speed. Apply light pressure to the skull while drilling along the 5 mm circumferential outline to thin this border.
		<ol>
			<li>Do not apply excess pressure while drilling. Vibration, compression, or accidental penetration can cause cortical injury. Instead, allow the speed of the drill bit to do the work.</li>
			<li>Do not drill in any given spot for too long to avoid excessive friction heating of the skull. Irrigate the craniectomy occasionally with sterile saline to remove debris and reduce the heating from the rotary tool.</li>
			<li>Pay close attention while drilling over the coronal suture line as these points are vulnerable to hemorrhage.</li>
		</ol>
		</li>
		<li>Use a pair of fine tweezers to remove the skull flap when the craniectomy outline is sufficiently thinned. Grasp the flap medially, and gently lift and pull laterally with a radial motion.
		<ol>
			<li>Do not damage the dura mater when lifting the flap; this can cause severe injury and hemorrhaging.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Perform a mild controlled cortical impact injury
	<ol>
		<li>Clean the impactor probe with a sterile alcohol prep pad. Move the impactor probe back into position over the exposed cortex. Lower the probe until it touches the dura mater surface. Mark this position as Z = 0.</li>
		<li>Withdraw the piston and move to Z = -1.0 mm. Discharge the piston to impact the cortex.&#160;</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 ml syringes</td>
			<td>Becton Dickinson (BD)</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>25G Precision Glide syringe needles</td>
			<td>Becton Dickinson (BD)</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>anesthetic vaporizer</td>
			<td>Vetland</td>
			<td>0521-11-22</td>
			<td></td>
		</tr>
		<tr>
			<td>animal handling cloth</td>
			<td></td>
			<td></td>
			<td>Purchase from department store</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Purdue Products</td>
			<td>NDC-67618-151-32</td>
			<td></td>
		</tr>
		<tr>
			<td>compressed oxygen</td>
			<td></td>
			<td></td>
			<td>Product of choice; varies by region</td>
		</tr>
		<tr>
			<td>cyclosporine A</td>
			<td>Sigma-Aldrich</td>
			<td>30024-100mg</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8418-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen (ThermoFisher)</td>
			<td>A14430-01</td>
			<td></td>
		</tr>
		<tr>
			<td>fine tweezers</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>Fine Science Tools</td>
			<td>91106-12</td>
			<td></td>
		</tr>
		<tr>
			<td>High Speed Rotary Micromotor Kit</td>
			<td>Foredom Electric Co.</td>
			<td>K.1070 - K.107018</td>
			<td></td>
		</tr>
		<tr>
			<td>Ideal Micro Drill Burr Set Of 5</td>
			<td>Cell Point Scientific</td>
			<td>60-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Impact One Stereotaxic Impactor for CCI</td>
			<td>Leica Biosystems</td>
			<td>39463920</td>
			<td></td>
		</tr>
		<tr>
			<td>ophthalmic ointment</td>
			<td>Falcon Pharmaceuticals</td>
			<td>NDC-61314-631-36</td>
			<td></td>
		</tr>
		<tr>
			<td>small rodent stereotaxic frame</td>
			<td>Stoelting</td>
			<td>51925</td>
			<td></td>
		</tr>
		<tr>
			<td>small scissors</td>
			<td>Fine Science Tools</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile alcohol prep pads</td>
			<td>Fisherbrand</td>
			<td>06-669-62</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile cotton swabs/Kendall Q-tips</td>
			<td>Tyco Healthcare</td>
			<td>540500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile saline</td>
			<td>Hospira</td>
			<td>NDC-0409-1966-07</td>
			<td></td>
		</tr>
		<tr>
			<td>sutures - 5.0 silk with curved needle</td>
			<td>Oasis</td>
			<td>MV-682</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Furmanski, O., et al. <a href="https://app.jove.com/v/59561">Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells.</a> J. Vis. Exp. (2019).
This video demonstrates the procedure for inducing an open-skull traumatic brain injury in a mouse by making a midline skull incision, drilling through the skull and removing the bone flap to expose the brain cortex, and applying high pressure to induce trauma. The wound is then sutured to prepare the mouse model for subsequent experiments.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin with an immobilized, anesthetized mouse with a shaved head.
Apply an eye ointment and disinfect the scalp with iodine.
Remove the excess iodine, and cover the mouse with a surgical drape.
Make a midline incision to expose the skull and clean the wound.
Mark a circle on one side of the skull, targeting the brain cortex.
The cortex contains motor and sensory neurons crucial for signal transmission between the brain and the body.&#160;
Drill through the skull bone over the marking in a circular fashion, then remove the bone flap to expose the cortex.
Position a sterilized impactor probe over the exposed area. Hit the probe over the tissue to deliver high pressure.
This pressure induces trauma in the brain cortex, disrupting the motor and sensory neurons and impairing signal transmission.
Clear the wound and suture it. The mouse model with traumatic brain injury is ready for further experiments.

37 次观看. 使用撞击器探针生成创伤性脑损伤小鼠模型

视频: 使用撞击器探针生成创伤性脑损伤小鼠模型 - 实验

Assessing Dominant-Submissive Behavior in Adult Rats Following Traumatic Brain Injury

Competition over resources such as food, territory, and mates significantly influences relationships within animal species and is mediated through social hierarchies that are often based on dominant-submissive relationships. The dominant-submissive relationship is a normal behavioral pattern among the individuals of a species. Traumatic brain injury is a frequent cause of social interaction impairment and the reorganization of dominant-submissive relationships in animal pairs. This protocol describes submissive behavior in adult male Sprague-Dawley rats after the induction of traumatic brain injury using a fluid-percussion model compared to naive rats through a series of dominant-submissive tests performed between 29 days and 33 days after induction. The dominant-submissive behavior test shows how brain injury can induce submissive behavior in animals competing for food. After traumatic brain injury, the rodents were more submissive, as indicated by them spending less time at the feeder and being less likely to arrive first at the trough compared to the control animals. According to this protocol, submissive behavior develops after traumatic brain injury in adult male rats.

The present protocol describes a rat model of fluid percussion-induced traumatic brain injury followed by a series of behavioral tests to understand the development of dominant and submissive behavior. Using this model of traumatic brain injury in conjunction with specific behavioral tests enables the study of social impairments following brain injury.

评估创伤性脑损伤后成年大鼠的显性顺从行为

Competition over resources such as food, territory, and mates significantly influences relationships within animal species and is mediated through social ...

科研

JoVE Journal

行为学

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.

研究小鼠创伤性脑损伤后凯库姆微生物群的改动

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also ...

神经科学

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

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

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

创伤性脑损伤诱导受控皮质冲击的母模型

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

Generating a Traumatic Brain Injury Mouse Model Using an Impactor Probe

成績單

Generating a Traumatic Brain Injury Mouse Model Using an Impactor Probe