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The Centers for Disease control (CDC) reports that
approximately 1.5 million people per year in the United States sustain
head injury. Of those injured 230,000 require medical attention
for traumatic brain injury. Those who survive TBI face years of
cognitive and physical rehabilitation with 25% those patients remaining
permanently disabled. The cost of treatment and long-term care for
one patient with TBI can be as high as $4 million. Currently there
is no effective pharmacological treatment for TBI that promotes
functional recovery in either the acute or chronic stages of injury.
In this research experiment we the researchers primarily focused
on the pharmacology of neuroregeneration synaptic repair and neuroplasticity
after brain injury. The researchers began to determine the effect
of experience and timing of DHEAS treatment on recovery of function
by evaluating the importance of combining experience with DHEAS
treatment to the extent of recovery of function. We measured the
levels of proteins related to neuroplasticity using quantitative
methods (e.g. Western blots) to determine if there are changes in
neuronal growth and repair which would parallel our behavioral results.
We hypothesized that DHEAS could significantly influence the post-injury
milieu by acting at each of these levels to enhance reparative mechanisms.
This research investigated the most effective way to systemically
administer DHEAS in the promotion of functional recovery after bilateral
controlled cortical impact injury to the medial frontal cortex in
rats. The results of this research showed significant differences
between injury with administration of DHEAS and injury with administration
of the vehicle. The injury plus DHEAS was significantly similar
to the sham. These results were seen through Morris Water Maze testing.
Further research will look closely at the molecular markers for
neuroplasitiy that were seen in the significant behavioral testing.
Approximately 1.5 million people per year are affected
by traumatic brain injury (TBI) and 5.3 million people are living
with its affects. Majority of current research concentrates on neuroprotection
while less attention has been given to compounds that enhance neuroplasticity
and repair after TBI Recent research demonstrates that progesterone
and its metabolite allopregnanolone are neuroprotective when administered
after injury. Progesterone and allopregnanolone are inhibitory neurosteroids
that reduce excitotoxicity and inflammation. Conversely we have
now shown that excitatory neurosteroids such as dehydroepiandrosterone
sulfate (DHEAS) have been shown to enhance plasticity and recovery
of function after TBI. Our previous results using DNA Microarrays
indicated that several genes related to neuroplasticity were increased
in expression in injured animals treated with DHEAS as compared
to injured-controls. Subsequent protein analyses confirmed that
MAP2 and GluR3 were significantly increased in brains of injured
rats given DHEAS as compared to their injured-controls. Our goal
in this current study was to further confirm our DNA Microarray
results by using Western blot analyses.
Rat Model of TBI
- 11 male Sprague-Dawley rats weighing approximately 350g
- Controlled cortical impact of medial frontal cortex
- Velocity = 2.25 m/s depth=2mm; duration=150ms; angle=0°
DHEAS Treatment
- DHEAS (Sigma; 10mg/kg) or equal volume of vehicle (15% 2-Hydroxypropyl-b-cyclodextrin
in sterile water)
- Subcutaneous injections 1 hour prior to MWM testing starting
on the 7th day post-injury
Behavioral Testing
- MWM: 5 days of testing beginning on the 7th day post-injury
Each rat received two 90-s trials per day with 20-s on the platform
and a 30-s ITI
Western Blots
- Brains were homogenized in T-Per™ (Pierce) and assayed
for protein concentration via commassie staining
- Samples were loaded on Tris-HCl acrylamide gels (Biorad) then
transferred to a PVDF membrane
- Blots were developed and scanned using HRP-reactive substrate
and quantified using densitometry
Experimental Timeline
Protocol
- CCI Injury
- MWM Testing and DHEAS Treatment
- Brain Extraction
- DNA Microarry
- Used Gene Expression to Choose Antibodies
- Western Blot
Morris Water Maze
Figure 2. Mean latency (seconds) to reach platform
in the Morris Water Maze. (A) Analysis over 5 days of testing (*:
different from shams; p<0.05 and ** different from shams and
injured+10 mg/kg DHEAS; p<0.05) and (B) the overall mean of all
5 days of testing (*: significantly different from and ** significantly
different from shams and injured+10 mg/kg DHEAS).
DNA MicroArray
Figure 3. The results of the DNA microarray assay
of the brain tissue indicated that DHEAS-treated rats compared to
injured+vehicle showed a consistent modulation of genes which been
to neuroplasticity. The enzyme responsible for the metabolism of
DHEA was also upregulated indicating that DHEAS was most likely
entering the CNS.
Western Blot Analysis
Figure 4. (A) Representative blots for each treatment
group with TUC-4 antibody. (B) The analysis of the optical density
of the blots indicates that injury increases the level of TUC-4
(p<0.05). *: significantly different from shams.
This study supports initial findings that delayed chronic treatment
with DHEAS (10 mg/kg) improves MWM performance after TBI. DHEAS
treatment increases expression of genes associated with neuroplasticity.
From this analysis of TUC-4 protein levels we can conclude that
TBI significantly increases the expression of this protein. However
due to low number of samples and normal variability it is too early
to determine if DHEAS treatment has any effect on the levels of
this protein after TBI. Ongoing analyses will continue to investigate
genes from our DNA microarray assay to determine if changes in gene
expression are accompanied by similar changes in protein synthesis.
This material is based upon work supported by Howard Hughes Medical
Institute Grant No. 52003727 and by NIH grant 5R03HD040295.
The authors would like to sincerely thank Sarah Cutler for her technical
assistance on this study, Emory University Emergency Medicine Department,
and the Summer Undergraduate Research Program advisors.
My research was done in the Emergency Medicine Department
of Emory University in Don Stein's Lab. The lab deals with tramatic
brain injury which refers to direct impact to the brain for example
in a car/motorcycle accident. In tramatic brain injury what happens
is the neurons (cells in the brain) begin to die and release excess
toxic chemicals in the brain. This later disrupts the blood brain
barrier which causes things to happen such as oxidative stress (less
oxygen to brain) and cerebral edema (extra fluid in the brain).
All of the things that happen to the brain after impact which were
just previously listed are known as the secondary injury cascade.
My research for the summer looked at this phenonmena of the secondary
injury cascade by administration of DHEAS. DHEAS is a neurosteriod
that acts to excite the neurons around the damage area and produce
new neurons. This is known as neuroplasicity in the brain. My research
this summer was dealing with protein markers for neuroplasicity
after adminstration of DHEAS. We as researchers prformed controlled
medial contusions to the frontal cortex of the brain of rats to
create a model that is similar to a tramatic injury in humans. After
this was complete we did several behavioral tests on the rats. There
were 3 groups of rats: One group of rats were not injured another
group of rats were injured and given DHEAS as a pharmalogical drug
to regrow neurons and the last group was injured but not given any
pharmalogical drug. The results showed that with administration
of DHEAS the rats were able to learn and regrow neurons better than
the rats that were given no drug but were injured!
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