The overall goal of this procedure is to administer and observe impulsive pressurization of cultured brain cells for traumatic brain injury study. This is accomplished by first inducing neuronal differentiation of cells cultured using retinoic acid. Next neuronal cells are placed in the ksky bar based cell pressurization device and are exposed to impulsive pressure at the magnitude and duration relevant to blast induced traumatic brain injury.
Lastly, the cells are subjected to molecular biology assays to evaluate cell damages and possible mechanisms of traumatic brain injury. The results from microscopy inspection show neuronal cell death and neurite axonal structural loss at impulsive pressurization of two megapascal magnitude and 0.7 millisecond duration. The main advantage of this technique over existing methods such as fluid percussion, is that there are no negative pressure sections and the time duration is well controlled.
This method can help answer key questions in the traumatic brain injury TBI field, such as the threshold for neuro cell death or injury as a function of impulsive pressure, magnitude, duration, and loading shape. Though this method has so far been used in our group to study blast induced TBI, but it can also be used for other types of traumatic brain injury such as impact induced TBI. The procedure will be demonstrated by Matthew, a graduate student from Professor Chen fang's Laboratory and by li ha, a technician from my laboratory.
This experiment utilizes a Ksky bar set up as a cell pressurization apparatus. The apparatus consists of two six meter aluminum alloy bars suspended by aligned brass bearings with an in vitro cell pressurization chamber. Sandwiched between the bars, the cell pressurization chamber is composed of a piston cylinder.
The cylinder has a small hole tapped into the base of the cavity, which during cell sample installation vents air and excess fluid. The piston is made from the same tube as the bar and is wrapped with two or three layers of Teflon tape. The end cap secures the glass cover slips with two small stainless steel screws.
This demonstration uses SH SY five Y human neuroblastoma cells. However, the protocol is applicable to many cell models culture. The cells on 18 millimeter diameter glass cover slips at a density of 3000 cells per square centimeter.
In supplemented DMEM while incubating the cells trigger their differentiation by supplementing the media with 10 micromolar retinoic acid for seven days With media changed every two days. On the seventh day, the cells show well-developed neurite axonal structure and are ready to be pressurized. Sterilize all the pressurization chamber parts using an autoclave and store them under UV light while assembling the chamber.
In a culture hood, make sure that the vent screw in the chamber is loosely engaged in the air vent. At the base of the cavity, fill the chamber with freshly made warm growth media. Then screw down a cell cultured glass slide on the cap of the piston with the cells facing outwardly, carefully load the piston into the cavity of the chamber.
Remove the vent screw, tilt the vent upward and push the piston into the chamber to a previously determined reference line. This leaves about six millimeters of fluid depth within the cell pressurization chamber. Avoid introducing bubbles, sanitize the vent screw and replace it to make the chamber watertight.
The chamber should not leak under the static load. Reset the Ksky bar system. Move the heavy mass back to its original position and replace the locking bolt.
The locking bolt holds a clamping force that prevents movement of the incident bar until the pins are broken. Engage the new locking bolt with the hydraulic jack to about 200 PS.I use the scissors jack to compress the preloading section of the incident bar to a pressure little higher than the desired value, and then back it off to engage the full friction of the JAK screw. Now set the data acquisition because pressurization are about a millisecond.
Set a sampling frequency of one megahertz using 2000 ohm strain gauges in a wheat stone bridge circuit. The voltage should be set to 45 volts at transmitted bar. Mount the cell chamber with the piston facing the transmitted bar.
Place the cell chamber in a V block supported by the small lab scissors jack and aligned with the two bars. Lightly grease each interface and eliminate any air between the budding surfaces by gently rubbing them together. Now the system is primed.
Arm the data acquisition and conduct the test by quickly pumping the hydraulic clamp driver. This forces the locking bolt to break, causing the clamp to separate and a pressure wave to be transmitted to the cells After the test. Unload the slide from the chamber in a culture hood and proceed with assessing cell behavior.
With experience. The apparatus can be used to pressurize about nine cell cultured slides per hour. When analyzing the readout, air bubble errors may be seen at the beginning of the transmitted bar signal, however they're not seen in this demonstration.
If the transmittance bar is long enough, then the most important data should begin with the first disturbance and terminate where the measurements show a negative pressure, this voltage reading is proportional to the stress experience by the cells through strain gauge analysis. The duration of the transmitted pulse may be shorter than the incident pulse. If a small bubble is trapped in the fluid within the cell pressurization chamber, the magnitude of the transmitted pulse may not exceed that of the incident pulse, but it should have a sufficiently long plateau before unloading.
Otherwise, either a large air bubble is trapped in the sealed chamber or the assembly and the bar are misaligned. After pressurization, the cells may be inspected immediately or further incubated for later examination. Compare the pressurized cells to the chamber control cells that were treated identically but not subjected to pressurization assess changes in neurite length with optical microscopy at various post incubation time points.
Actin immunofluorescent staining can be used to assess cytoskeletal changes. Also antibody for micro tubule associated protein two. MAP two can be used to visualize changes in one of the key neuronal markers.
To assess the effect of impulsive pressurization on neuronal and apoptotic gene expression use standard molecular biology techniques such as R-T-P-C-R if desired. Sufficient cell lysates for immuno blotting can be collected from several identically pressured cell samples. In this example, pressurization profile, the ksky bar apparatus successfully applied two megapascal level single pulse type impulsive pressure with duration of about 0.7 millisecond to the cultured neuronal cells as seen in the optical microscope images, SHSY five Y human neuronal cells, which were subjected to two megapascal impulsive pressure show distinct neurite axon breakdown relative to un depressurized control cells.
When attempting this procedure, you always wanna hold the fluid pressurization chamber like this and never like as it will subject the cells to negative pressure After its development. This technique paved the way for the researchers in the field of neuroscience to explore the cell mechanism of traumatic brain injury under controlled in prostate pressurization conditions. We thank the funding support from the Army UNL Center for Trauma Mechanics.
