This method can help answer key questions in the field of blast injurious traumatic brain injury and can be used to screen neuroprotective drugs before the use of more complex in Vivo models. The main advantage of this technique is that it uses a laboratory instrument to expose in Vitro mass brain tissue to a shockwave using a simple and high throughput protocol that allows the creation of a reproducible injury. First, insert sterile custom-made stainless steel rings into the wells of a six well plate.
Then add pre-warmed serum-free experimental medium with propidium iodide to the wells ensuring that the level of medium does not reach above the notch of the ring. Transfer the plate to the incubator for one hour to ensure that the medium is at 37 degrees Celsius before the tissue culture inserts are transferred. After an hour, transfer tissue culture inserts with organotypic slices from their growth dishes onto the rings in the six well plate.
Make a dot on the insert rim in the three o'clock position with a permanent marker pen to facilitate returning the inserts to its original position, then label each six well plate with a unique name and date and make a map of the wells of each plate, naming each well with a letter and each slice in the well with a number so that each slice has a unique identifier. Incubate at 37 degrees Celsius for one hour to ensure the slices are at 37 degrees Celsius immediately before imaging. One hour after transferring to experimental medium, assess slice health by quickly imaging each slice individually and sequentially under low power.
Use a fluorescence microscope fitted with an appropriate excitation and emission filter ideally in a dark room or a room with dim light. Keep the lid on the plate when imaging. Some condensation may build up on the inside of the lid.
If this happens, briefly use a hairdryer on the low setting on the outside of the lid. Ensure the imaging conditions are identical on different days and between experiments. At baseline, healthy slices show very little fluorescent staining.
Slices that exhibit areas of dense red staining indicate compromised viability and should be excluded from further analysis. For comparison, fluorescence from the blast injured slice at 72 hours is shown here. Immediately after imaging, remove one tissue culture insert from the six well plate and carefully transfer the insert to a sterile polyethylene bag containing 20 milliliters of pre-warmed experimental medium bubbled with 95%oxygen and 5%carbon dioxide.
Carefully remove any air bubbles and seal the sterile bag by twisting the top and applying a plastic clamp. Ensure that each sterile bag is correctly labeled with the plate and well identification. After transferring each tissue culture insert to an individual sterile bag, place the bags and the six well plates with experimental medium into the 37 degree Celsius incubator.
After one hour, carefully pack the sterile bags with the tissue culture inserts in plastic boxes inside a thermal regulated box filled with the ionized water at 37 degrees Celsius. The water should keep the organotypic slices at physiologic temperature throughout the shockwave exposure protocol. Wear steel-toed protective boots, a laboratory coat, and gloves during preparation of the shock tube and the shockwave exposure.
Use a blanking rod to bolt the sterile bag holder frame to the shock tube distal flange ensuring that the central hole is aligned with the shock tube outlet. Sensor one, a pressure transducer, is located in the middle part of the driven section, and sensor two is in the distal flange of the shock tube. Connect these pressure transducers to an oscilloscope through a current source power unit and turn on the oscilloscope.
Ensure the shock tube's solenoid valve and flow control are closed, then open the external compressed air line and charge the solenoid valve to 2.5 bar. Open the compressed air cylinder safety valve and slowly open the pressure regulator to increase the pressure to approximately five bar. Next, prepare diaphragms by cutting 23 micron thick polyester sheets into 10 by 10 centimeter squares.
Prepare handles from autoclave tape and stick them to the top and bottom of each diaphragm. Position one diaphragm in the breach and ensure they are centered. Next, clamp the diaphragm using four M24 bolts and nuts.
Fashion them sequentially in a diagonally symmetric way while ensuring the diaphragms are wrinkle free. Clamp a sterile bag in a vertical position on the holder frame ensuring that the surface of the tissue culture insert with the organotypic hippocampal slices is facing the shock tube outlet and the tissue culture insert is centered inside the sterile bag. For the double diaphragm configuration, the bursting pressure is dependent on the gas pressure differential between the driver and double breach chamber.
Therefore, for the diaphragms to burst in a controlled way, the double breach safety valve is opened manually once the target pressures are reached. Put on ear defenders and safety spectacles if not already worn. Close the solenoid valve.
Switch on the current source power unit to acquire the shockwave data. Manipulate the flow control knob on the shock tube control panel to slowly pressurize the driver volume section of the shock tube for single diaphragm configuration or both the driver volume section and the double breach section of the shock tube for double diaphragm configuration. As soon as the diaphragm ruptures, quickly closed the compressed air flow using the flow knob and open the solenoid valve.
The ideal combination of shockwave parameters should be enough to cause tissue injury but not so high that it causes tissue culture insert or sterile bag distortion or rupture. After exposing each slice to a single shock tube wave, return it immediately to the thermal-regulated box. Then take the next sterile bag from the box and clamp it on the holder frame.
Perform the switch smoothly and swiftly to prevent cooling of the experimental medium as temperatures below 37 may interfere with injury development. Once all tissue culture inserts have been exposed to a shockwave or sham protocol, return the tissue culture inserts to their wells in thee original six well plate and return to the incubator until further imaging. This image is a representative example of a shockwave obtained using 23 micron thick polyester film with 55 kilopascal peak overpressure.
The shockwave velocity was 440 meters per second. Both 50 and 55 kilopascal peak overpressure shockwaves caused significant injury that developed throughout the 72 hour protocol when compared with the sham group. The injury resulting from a 55 kilopascal peak overpressure wave exposure was significantly higher than after 50 kilopascal at 48 hours and 72 hours, demonstrating the development of the injury is proportional to the intensity of the shockwave.
This image shows a slice 72 hours after a 50 kilopascal blast. A high level of diffuse injury is visible. Injury is more pronounced after a 55 kilopascal peak overpressure blast.
This propidium iodide fluorescence image shows an organotypic slice from a sham experiment. The sham slice shows low levels of fluorescence. While attempting this procedure, it is important to remember to maintain an aseptic technique throughout to avoid contamination of the tissue and to manipulate tissue cultures rapidly but very gently to avoid unintended cell damage.
Following this procedure, other methods like immunofluorescence staining can be performed in order to answer additional questions like what are the mechanisms of cell death involved in blast induced traumatic brain injury? This technique allows a high throughput assay for researchers in the field of neuroprotection to explore the potential for drugs to prevent the spread of cell death and brain tissue after exposure to blast shockwaves.
