Generating a Closed-Head Mild Traumatic Brain Injury in a Murine Model

Protokoll

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

1. Preparation of experimental setup and induction of mild traumatic brain injury (TBI) in rodents

NOTE: Transfer rodents to the holding area 30 min to 1 h prior to the start of traumatic brain injury (TBI) experiments to acclimatize. Select a holding area that is minimally affected by the noise of the procedure.

1. Prepare all materials required for the experiment and check setup for proper installation (e.g., adjust parameters according to aim of study) (~5 – 10 minutes).
   NOTE: Injury severity can be adjusted by selecting the thickness of the polyester membrane. Based on our studies, a membrane thickness of 25.4 to 102 μm is utilized for mild to moderate TBI in mice. We have previously utilized membranes with a thickness of 76.2 to 127 μm to produce mild to moderate TBI in rats.
   1. Carefully cut the polyester membrane, insert it between the driven and driver sections, and secure it by tightening the connecting bolts.
   2. Connect the gas tank to the shock tube through the use of quick-release fittings. Ensure that the membrane is tightly fixed between driver and driven sections.
   3. Place three pressure sensors at the exit of the shock tube, 120° apart, to monitor the blast wave properties during the TBI induction.  
   4. Ensure distance from end of shock tube apparatus is correct for each respective subject using the installed micrometer. Keep the positioning of the rodent’s head (i.e., position, distance) constant within studies to allow for consistent injury evaluation.
      NOTE: Different types of injury can be induced by selecting the direction in which the blast wave impacts the head. For the procedure to induce mild to moderate TBI described here, the body is placed perpendicular to the shock tube where the blast wave impacts the side of the head. In this setting, the head is allowed free mobility and hence is exposed to the blast wave and rapid rotational forces allowing the generation of coup and contrecoup effects.
   5. Initiate the recording from the pressure sensors using the graphical user interface (GUI) of the software.

2.  Anesthesia and positioning of rodents in setup

1. Transfer rodents from the holding room and induce anesthesia with 4% isoflurane in oxygen and maintain with 2% isoflurane in oxygen to reduce distress and pain.
   NOTE: Make sure the animal is non-responsive to toe or tail pinch prior to proceeding. Make sure the induction of anesthesia is consistent for all experimental animals, including sham controls. This procedure requires a low-level and short duration of anesthesia.
2. Place the fully anesthetized rodent in the polyvinyl chloride (PVC) pipe shield with cushioning to protect peripheral organs from the blast wave.
   NOTE: Control subjects are anesthetized and placed in proximity to the setup, but are not directly subjected to the blast wave. Ensure that the controls are subjected to the noise generated by the shock tube.
3. Place the head of the rodent within the head placement area and support it from below, either by support built directly into the shielding apparatus or a gauze pad. Determine the head alignment according to each rodent’s anatomy, with the occipital condyle aligned with the edge of the protective shielding.
   NOTE: Avoid directing the pressure wave directly towards the brainstem to decrease mortality. Injury to the respiratory center of the brainstem and the cervical spinal cord is known to contribute to breathing abnormalities and even death in rodent models of TBI.

3.  Exposure of rodents to blast wave

1. Rapidly open the main valve of the compressed gas tank to produce a pressure spike that ruptures the membrane and produces a loud explosion that confirms the generation of a pressure wave. The membrane will be visually ruptured when removed after the experiment.
   NOTE: A high-speed camera can be used to capture the coup and contrecoup effects of the rotational acceleration experienced by the rodent for further analysis.
2. Turn off the gas flow immediately after hearing the explosion.

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