Our protocol demonstrates how to maintain the LFPI device properly. It highlights the importance of routine cleaning and monitoring to avoid malfunctioning of the device in inconsistent injuries. So LFPI is an injury model used to assess TBI in vivo.
It closely reproduces pathologies that are associated with human TBI, thus making it one of the most well-defined methods of experimental TBI. Given that the LFPI is used to reproduce human TBI symptoms, it allows us to set up experiments that investigate different aspects of traumatic brain injuries in the hope of finding therapeutic measures. Air bubbles in the fill port or transducer are particularly difficult to remove due to the limited visibility in those areas.
When filling those areas, be patient, slow, and careful not to introduce air. To begin, carefully detach the syringes attached to the transducer housing and fill port and the cable connected to the pressure transducer. Unscrew the hand knobs at the back of the device from the cylinder clamps to free the cylinder.
Remove the piston at the end of the cylinder, transducer, transducer housing, and plunger O-rings. Drain fluid out of the cylinder, add a mild detergent such as dishwashing detergent to the cylinder, and scrub lightly using a dish or bottle brush. To ensure that all the detergent is rinsed off completely, fill the cylinder with water and rinse thoroughly.
Apply a thin layer of petroleum jelly to the piston plunger and attach the piston plunger with the plunger protruding approximately 32 millimeters from the cylinder. Apply a thin layer of petroleum jelly to the other O-rings as well and attach them to the cylinder except for the O-ring on the fill port. Wrap Teflon tape twice around the transducer's threads.
Connect a 10-milliliter syringe filled with degassed fluid and free of air bubbles to the Luer lock hub on the transducer housing. Hold the transducer with the threaded end pointing upward and completely fill the well inside the threaded region of the transducer with the degassed fluid. The goal here is to fill the transducer well without introducing any air bubbles.
Be careful not to damage the delicate membrane at the bottom of the well. With the cylinder placed at an angle to prevent air from reentering the transducer housing, attach the transducer housing to the cylinder and use a wrench to tighten it snugly. Remove the cap from the fill port and cylinder once the degassed fluid reaches approximately two-thirds of the cylinder capacity.
Place the cylinder horizontally and finish filling the cylinder with degassed fluid. Replace the cap at the fill port and close all stopcocks. Manipulate the cylinder to work any air bubbles to the fill port.
Open the stopcock on the fill port and inject fluid using the syringe on the transducer housing to force any air bubbles out of the port. Inspect the entire device and ensure that there are no air bubbles. Add a 10-milliliter syringe filled with degassed fluid to the Luer lock hub on the fill cap and reattach the cylinder to the base using the hand screws.
Ensure that the cylinder is horizontal and lined up with the center of the weighted hammer on the pendulum. The image of the pressure waveform produced by a properly cleaned and functioning LFPI device is shown in this figure. When performing an injury or testing the LFPI device, the pressure waveform should show a single sharp peak.
The presence of air bubbles in the device will result in a waveform with several short peaks. The oscilloscope outputs for the noncontaminated, 5 milliliter of air injection, 10 milliliter of air injection, and 15 milliliter of air injection are shown here. The techniques shown in this video reduce the likelihood of gases being introduced into the device and help remove any small pockets of gas that may be contaminating the fluid.
Therefore, removing air bubbles is the most crucial step to remember when using the LFPI device. So after this procedure, our lab is able to induce injuries in mice and use live brain slices in order to record neuronal activity. You can actually find one of our videos on this on JoVE's website.
This device has been a game changer in the world of TBI. It is currently the most widely used method to induce injury in animal models and has paved the way to find better therapeutics for human TBI.
