delivery of a traumatic brain injury to a mouse brain

Delivery of a Traumatic Brain Injury to a Mouse Brain

To begin, apply ophthalmic ointment to the anesthetized mouse&#39;s eyes. Remove the hair from the top of the head by trimming with regular scissors. After establishing a 12-to-15-millimeter-long midline incision, excise the skin over the left and right hemispheres of the skull using curved-tipped spring scissors.
Once this hole is exposed, remove the periosteum by gently rubbing it with a sterile cotton-tipped applicator, and flushing it with sterile saline. Once the school area is dried, mark the Traumatic Brain Injury, or TBI impact site at the coordinates 2.5-millimeter posterior to the bregma, and two millimeters lateral from the sagittal suture to the right.
Quickly remove the mouse from the stereotactic frame, and place the head on the buffer cushion under the TBI device. Align the impactor tip with the marked impact site. Lift the metal column by pulling the tethered nylon string 15 centimeters above the mouse head, and then, release it allowing the weight to fall freely onto the transducer rod, which is in contact with the skull top at the TBI site.

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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. Administration of a repetitive traumatic brain injury (TBI) induction
NOTE: The TBI parameters are adjusted from previous reports, in which the TBI impact was delivered once. The protocol here applies the same parameter, except increasing the total impact number to 10.
<ol>
	<li>TBI equipment
	<ol>
		<li>Use a custom-built portable device to administer a closed-skull TBI (Figure 1A) to the mouse head on the right side, as indicated in Figure 1B.</li>
	</ol>
	</li>
	<li>Preparation before surgery
	<ol>
		<li>Autoclave surgery equipment, including regular scissors, a surgical caliper, surgical spring scissors, surgical forceps, mosquito forceps, headpost pieces, gauze, and cotton-tipped applicators.</li>
		<li>Weigh and record the mouse&#39;s body weight 2 h before surgery. To minimize brain edema during cranial window implantation, inject dexamethasone sodium phosphate at a dose of 4.8 mg/kg [2 mg/mL, usually ~70 &#181;l (need to inject at two sites)] into the quadriceps muscle using an insulin syringe. After the injection of dexamethasone, but prior to starting TBI surgery, prepare the glass windows as indicated in step 2.1 for later use.</li>
		<li>Disinfect the surgical stage and TBI equipment using 75% ethanol. Expand the disposable sterile surgical drape to cover the surgery stage on the stereotaxic platform. Turn on the heating apparatus and the temperature monitor, setting the target temperature to 37 &#176;C.</li>
		<li>Open the oxygen tank valve and adjust the oxygen flow to 1.5 L/min. Open the anesthesia machine and set the isoflurane level value to three. 
		&#8203;NOTE: 100% pure oxygen can help the animal to survive the TBI impacts.</li>
		<li>Place the mouse in the induction chamber and let it stay for 5 minutes to achieve full anesthesia.</li>
		<li>Once the mouse is fully anesthetized (i.e., slow but steady breathing rates at approximately one cycle per 2 s, coupled with the absence of a tail-pinch reflex), remove the mouse from the induction chamber and place the mouse head in the stereotaxic frame.</li>
		<li>Place the anesthesia tubing nose cone over the mouse&#39;s snout.</li>
		<li>Maintain anesthesia using 1.5% isoflurane mixed with 100% pure oxygen at a 1 L/min flow rate until the end of surgery. Periodically check the breath frequency and deliver a tail or toe pinch at least every 15 min throughout surgery to assess appropriate anesthesia. Adjust isoflurane concentration as appropriate to maintain the desired depth of anesthesia.</li>
	</ol>
	</li>
	<li>TBI surgery
	<ol>
		<li>Administer buprenorphine (1 mg/kg, subcutaneously) using disposable insulin syringes and apply ophthalmic ointment to the mouse&#39;s eyes.</li>
		<li>Remove the hair from the top of the head by trimming it with scissors 1 and then applying a depilatory agent for 1 min. 
		&#8203;CAUTION: Do not apply the depilatory agent product for more than 3 minutes to the scalp, as it is a skin irritant. Also, avoid getting any depilatory agent into the mouse&#39;s eyes.</li>
		<li>Clean the scalp skin by applying gauze and cotton-tipped applicators. Then, disinfect the skin, using 75% ethanol first and then betadine. Repeat this three times (i.e., ethanol followed by betadine), leaving the final application of betadine on the skin.</li>
		<li>Re-check the anesthetic depth to ensure that the mouse is fully anesthetized (i.e., slow but steady breathing rates at approximately one cycle per 2 s, coupled with the absence of a tail-pinch reflex). Tent the skin with surgical forceps 3 and make a 12-15 mm long midline incision starting approximately 3 mm posterior from the eyes.</li>
		<li>Excise the skin over the left and right hemisphere of the skull using spring scissors 2.</li>
		<li>Once the skull is exposed, remove the periosteum by gently rubbing it with a sterile cotton-tipped applicator and flushing it with sterile saline.</li>
		<li>Visually inspect the condition of the skull to ensure that it is intact, except for the two small holes, which were made for the previous virus injection surgery.</li>
		<li>Dry the skull area and mark the TBI impact site at the following coordinates: 2.5 mm posterior to the Bregma and 2 mm lateral from the sagittal suture to the right (Figure 1B).</li>
		<li>Quickly remove the mouse from the stereotaxic frame and place the head on the buffer cushion under the TBI device.</li>
		<li>Align the impactor tip with the marked impact site.</li>
		<li>Lift the metal column by pulling the tethered nylon string 15 cm above the mouse head and then release it, allowing the weight to fall freely onto the transducer rod, which is in contact with the skull top at the TBI site. Do not touch the mouse head when delivering the TBI impact.</li>
		<li>Move the mouse onto the heating blanket and place it on its back while monitoring the breathing status.</li>
		<li>Once the mouse rights itself from a supine to a prone position, place it in the isoflurane induction chamber for ~5 min with 3% isoflurane mixed with 100% pure oxygen at a 1.5 L/min flow rate.</li>
		<li>Once the mouse is fully sedated (i.e., slow but steady breathing rates at approximately one cycle per 2 s, coupled with the absence of a tail-pinch reflex), repeat steps 1.3.9-1.3.14 to achieve a total of 10 impacts. 
		&#8203;NOTE: Ten TBI impacts have been found to induce robust phenotype with low mouse mortality in our study (data not published yet). The parameters may be adjusted to achieve a different severity of brain injury by increasing or decreasing the number of impacts and/or the height from which the weight is released. All parameters are subject to approval by local IACUC.</li>
		<li>Check the skull under a surgical microscope and remove the mouse from the study if a skull fracture has occurred.</li>
		<li>For sham surgery, follow the same procedures as described above, including placing the animal under the impactor but without delivering the TBI impacts.</li>
		<li>After the mouse rights itself from a supine to a prone position following the 10th TBI impact, place it in the isoflurane induction chamber for ~5 min. Then, follow steps 1.2.6-1.2.8 to place the mouse head onto the stereotaxic frame for the cranial window implantation surgery described below.</li>
	</ol>
	</li>
</ol>

Source: Zhong, J., et al. <a href="https://app.jove.com/v/64701">Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope.</a> J. Vis. Exp. (2023)
This video demonstrates the delivery of Traumatic Brain Injury (TBI) in mice. After exposing the skull, the TBI site is marked. The mouse is then positioned under the TBI device. The impact is delivered by lifting the metal rod and allowing it to drop freely onto the impactor, which is in contact with the skull.

<img alt="Figure 1" src="/files/ftp_upload/22967/22967_Figure1.jpg" /> 
Figure 1: Diagrams for the TBI device, TBI impact site, and window preparation. (A) Equipment for the weight-drop TBI device. 1: pedestal, 2: bracket, 3: adjustable clamp, 4: nylon string, 5: weight dropping tunnel, 6: metal weight column (50 g), 7: impactor, 8: buffer cushion. (B) A schematic of the virus injection and TBI impact site, with the coordinates 2.5 mm posterior to the Bregma and 2 mm...

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 anesthetized mouse with its head secured in a stereotaxic apparatus.
Apply ointment to prevent eye dryness.
Remove the hair from the head and disinfect the area.
Make a midline incision on the scalp and remove the connective tissue to expose the skull.
Clean the area with saline and allow the skull to dry.
Using the point where the skull bones join and the bone boundary as a reference, mark the desired area for the traumatic brain injury or TBI.
Position the mouse under the TBI device.
The TBI device consists of an impactor and a metal column within a tunnel.
Align the impactor tip with the marked area on the skull.
Lift the metal column to a desired height and release it freely to deliver an impact on the skull, inducing a TBI at the specified location.

12 Views. Delivery of a Traumatic Brain Injury to a Mouse Brain

Delivery of a Traumatic Brain Injury to a Mouse Brain

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