Our research focuses on the long-term effects of repeated low level blast exposure in mice. Our goal is to determine the long-term effects of this exposure during military training. For animal research, the effects of low level blast are subtle, and research teams will often use techniques like electron microscopy to detect differences between blast and sham mice.
Our work suggests increased blast repetition makes these differences easier to detect using more accessible microscopy techniques. There's currently very limited literature on both low level blast exposures, which are also called low intensity blasts, and the models that use these exposures over multiple days. We designed our protocol to include multiple low level blasts over many days to address these gaps.
Begin by performing a safety check, ensuring the helium gas supply and master power are off. To prepare the membranes for low intensity blasts, cut one sheet of plastic cling wrap into a 5.5 by 5.5 inch square. Also, cut one sheet of standard 8.5 by 11 inch copier paper to size 5.5 by 11 inch and fold this sheet of paper in half to form a square.
Obtain one sheet of 500 G Mylar membrane with a thickness of 125 micrometers. Then take a square of cling wrap and a square of folded paper and set them out on a flat surface. Place the folded paper on top of the cling wrap and align the two with each other.
Roll the Mylar membrane into a small tube and insert it between the driver and the spool. Let go of the membrane to allow it to enroll against the rubber seal, separating the driver from the spool. Push the spool toward the driver to secure the Mylar sheet in place.
This will unseal the spool from the driven section of the shock tube. Place the fingers under the top half of the cling wrap and carefully roll both the cling wrap and paper together. Insert the membrane stack between the spool and the driven sections of the shock tube.
Allow the membrane stack to unroll so that the plastic seal faces the spool and the paper faces the driven section of the tube. Close the spool assembly and tighten the bolts to secure the driver's spools shock tube assembly. Pressurize the shock tube to 500 pounds per square inch.
Begin by check-Ear punch the mouse for identification and place its nose in the nose cone for sustained anesthesia. Use laboratory tape to restrain the mouse's limbs against the gurney. Place a wire twist tie around each limb and twist tightly, securing the mouse to the gurney at the wrists and angles.
Place a larger twist tie around the chest, tying it loosely. Lift the tail of the mouse and place it under the left foot to prevent it from getting pinched. Then, open the animal exposure section of the shock tube and orient the mouse so that it faces the oncoming blast wave.
Secure the gurney in the animal exposure section. Then, close the door tightly, ensuring that the door does not pinch the anesthetic flow tube. Reduce the anesthesia to 2.5 to 3%isoflurane with one liter per minute of oxygen.
Next, power on the system. Connect to the supply line for the compressed helium gas. Leave the blast room to access the blast control tube console in an adjoining room.
From the console, turn on the acquisition software to record the blast event. Disengage any safety lock, close both gas vents, and passively pressurize the spool. Continue to fill until the membrane ruptures at the target pressure.
Turn off the fill mechanism, then record the peak pressure, positive phase duration, and impulse at the animal location. Return to the shock tube. Disconnect the helium feed line, and turn off the power supply to the blast control circuit.
To conduct repeated LLB exposures on the same animal, open the spool, remove and replace the spool membrane stack. After completing the LLB exposures, remove the animal from the shock tube, leaving the anesthesia on. Untie the animal while under anesthesia and place it on its back on the heated water pad.
Record the time until the mouse flips onto its ventral side as the righting time. Upon recovery, return the mouse to its home cage. Experiments to study the effects of explosive blasts on mice were evaluated using pressure versus time analysis, with successful experiments featuring a controlled wave pattern with a sharp pressure rise and a predictable decline.
No overt injuries were observed in low level blast experiments, even after multiple blasts. Analysis of righting time indicated that repetitive LLB exposure led to faster recovery in blast mice than controls, indicating neurobehavioral arousal changes. Further, correlating pressure time profiles with biological responses helped establish causal relationships, supporting longitudinal studies without animal loss for up to six months post-exposure.
