The overall goal of this procedure is to establish an animal model of PTSD. This is accomplished by physically stressing the animal and then characterizing its feeding behavior and startle response. Also demonstrated is a procedure for measuring circulating court levels.
Ultimately, biomarkers presented by white blood cells and traumatized animals can be correlated with expression of the same biomarkers in the brain tissue. Thus, this model opens new inroads For the study of PTSD, We use animal model that has PTSD like syndrome. Using this animal model allow us to explore the neurobiological mechanisms of PTSD neurobiological markers revealed in this animal model has been shown to correlate it with syndrome or PTSD.
Perhaps one in three soldiers exposed to the trauma of combat will develop excessive fearfulness aggressiveness, or other distress symptoms. For reasons we don't fully understand, we need to identify methods to identify these most vulnerable soldiers as early as possible, even before they enter combat so we can do more to support them. One of the key components of post-traumatic stress disorder is hyper arousal and fear response, sometimes called startle.
Understanding that particular response, both as it occurs in humans and in our animal models, will aid us in advancing our understanding of this disorder. Dr.Lee's laboratory is focused on understanding animal models of this exaggerated fear response. My work is with the PTSD biomarkers.
These PTSD biomarkers are taken from peripheral white blood cells, joined from patients and from postmortem brain tissue. The biomarkers must be correlated with the living brains. This correlation can only be done in an animal model of PTSD.
Emotion is subsumed in different ways by different regions of the brain. Having an animal model of PTSD makes it possible to excise those different regions of the brain. For independent discovery of biomarkers, Select 32 male albinos bra dolly rats weighing between 150 and 200 grams.
Each animal must be housed individually with strict attention paid to maintaining consistent environmental conditions for the three days before the trial, three days during the trial, and three days after the trial Record each animal's body weight as well as each animal's food and water consumption, acclimate all the animals to the animal facility and to an acoustic startle chamber. For the three days prior to the trial, allow each animal to spend five minutes per day in the startle chamber with no additional stimuli on the day before the trial begins. A baseline measurement of acoustic startle response is made.
Then baseline response and weight are used to divide the 32 animals into two normalized groups that will be controlled and experimental during the three testing days. The experimental group is stressed. Daily stress is delivered by restraint and tail shocks during the morning hours.
Then couple an electrode to the tail of the rat with electrode gel and attach the electrode clip onto the tail such that it does not affect the blood circulation of the tail to titrate a current that is sensational but not painful. Dr.Lee originally placed the electrode on his own finger, which is more sensitive than the rat's tail and adjusted the current to be aversive, but neither painful nor damaging. On the seventh, 14th, and 21st day after the three day stress test.
Acoustic startle response is measured using a calibrated system of weight sensitive platforms in a sound attenuated chamber. When the system is first set up, the transducer, which is a strain gauge, requires calibration switch the coupler to DC mode, so the coupler's output directly follows the input from the platform and calibrate the output to static weights on the platform. During testing, the transducer is switched to the AC coupled mode.
So the only rapid change in force, which indicates startle response is output. The test consists of six types of stimuli delivered eight times in a random order. These stimuli include a 100 decibel tone with or without a pre pulses, a 110 decibel tone with or without a prese.
The pre pulses alone and a zero decibel control the inter trial intervals ranges are randomly assigned between 15 and 25 seconds. The animal's movements are recorded as the maximum response occurring within 200 milliseconds of the onset of the startle eliciting stimulus from each of the eight trials. The maximum value for each of the six stimuli is recorded.
The startle response data is divided by the animal's body weight measured on the same day. Consumption data and body weight are therefore measures on seven different days. After stress testing on days 1, 14, 21 and 30 from the first day after stress, blood is withdrawn from the tail vein under light isof fluorine anesthesia into heparinized tubes and frozen until ready For analysis, use an active RAC core Eliza to analyze plasma court levels.
Prepare the assay by marking the micro titration strips and mixing the rat core enzyme conjugate solution. Once prepared, begin the assay by pipetting 20 microliters of standards, controls and unknowns into wells of the ELI A plate. Second, add 100 microliters of enzyme conjugate solution to each well for just five to 10 seconds.
Gently tap the plate to mix the solutions. Third, add 100 microliters of rat core enzyme conjugate to each well and incubate the plate for one hour at room temperature with shaking. After the incubation, use an automatic microplate washer to aspirate and wash each well five times with wash solution.
Then blot the plate dry by inverting it on an absorbent material. Next, 100 microliters of the TMB chromogen solution is added to each well using a semi-automatic dispenser. Finally at 100 microliters of stopping solution to each well and shake the plate by hand for five to 10 seconds.
Within 30 minutes, proceed to collect the data after the stress test, food and water consumption between the control and experimental groups were recorded. Body weight measurements following stress revealed reduced weight gain in the experimental group compared to controls. Plasma samples revealed elevated court levels in animals.
Given the stress test, the startle response of the experimental animals differed significantly from the control group. Biomarker screening revealed unique correlations between brain tissue and white blood cell expression in stressed animals not seen in controls. These markers will serve as candidates for following experiments Together with clinical evaluation.
Neurobiological measurement will identify the pathophysiological process or PTSD more objectively in the future. Discover the biomarker from this animal model will be diagnostic. In addition, this will lead to a more effective pharmacotherapies for the patient with PTSD.
