The Quantification of Activity Regulated Cytoskeletal (ARC) Gene in Rat Hippocampus After Diffuse Traumatic Brain Injury

1. THE QUANTIFICATION OF ACTIVITY REGULATED CYTOSKELETAL (ARC) GENE IN RAT
HIPPOCAMPUS AFTER DIFFUSE TRAUMATIC BRAIN INJURY
1BASIS Chandler High School, Chandler, AZ, 2BARROW Neurological Institute at Phoenix Children’s Hospital- Phoenix, AZ, 3Department of Child Health, University of Arizona College of Medicine –
Phoenix, AZ, 4Phoenix VA Healthcare System- Phoenix, AZ, 5Department of Neuroscience, University of Strasbourg, France, 6Neuroscience Program, Arizona State University, Tempe, AZ,
References
Background
• Diffuse traumatic brain injury (dTBI) is caused by rapid
acceleration-deceleration of the brain inside the skull resulting in
diffuse axonal injury.
• dTBI incites secondary cascades of molecular processes,
resulting in circuit reorganization that likely leads to neurological
impairment, including sensory sensitivity.
• The whisker nuisance task (WNT) demonstrates late-onset,
persistent sensory sensitivity to whisker stimulation manifesting
by 28 days post-diffuse TBI in rats (McNamara et al., 2010).
• During the development of sensory sensitivity, we have
documented pathological and functional changes supporting
circuit reorganization in the whisker barrel circuit, but not the
hippocampus.
• The efficacy of therapeutic approaches on maintaining in vivo
circuit integrity after dTBI could be carried out using an immediate
early gene as a molecular marker of circuit activation after circuit-
specific stimulation.
• Activity-regulated cytoskeleton-associated (ARC) gene is
considered to be an immediate early gene that is tightly coupled
to behavioral encoding of information in neuronal circuits in vivo.
• Here, we identified the time course of ARC gene expression after
exploration of a novel environment and determined whether the
time course of ARC gene expression changed as a function of TBI
at 28 days post-injury in the hippocampus.
• McNamara et al., (2010) J Neurotrauma 27(4): 695-706.
• Centers for Disease Control and Prevention.—. MMWR 2011;
60(39):1337–1342.
• Khodadad et al. (2015) Behavioral Brain Research;284:249-56
• Moser et al(1998). J Neuroscience 18(18): 7535-7542.
Hypothesis
Diffuse traumatic brain injury leads to changes in the
time course of ARC gene expression in the
hippocampus after exploration of novel environment.
Methods
Adult male Sprague-Dawley rats (300-350g) were subjected to moderate midline
fluid percussion injury ( FPI; n=67; 1.9 atm; 6-10 min righting reflex time) or a sham
surgery (Fig. 4). At 28 days post-injury, the rats explored a novel environment for 20
minutes, activating their spatial memory (Fig. 5). After 20 minutes of exploring the
novel environment, animals were housed individually for 15, 30, 60 or 90 minutes.
At the specific time point, rats were perfused with iced cold phosphate buffered
saline PBS, the hippocampus was dissected out and stored in RNAlater® at -20°C
until mRNA extraction. Life Technology’s RNA extraction kit was used to extract
mRNA from the dissected tissue and converted mRNA to CDNA for q-PCR.
Quantitative PCR (q-PCR): The TaqMan® Gene Expression Assay for ARC was
optimized to run under universal thermal cycling conditions, with amplification
efficiencies of 100%. Within each animal, relative gene expression was normalized
to the 18s endogenous control and the expression level in the naive group using the
2-ΔΔCT.
Methods-Continued
Statistical analyses: For the both time courses, relative gene expression was normalized to naïve rats
(unstimulated/uninjured). ARC gene expression was compared to naïve over time using a one-way ANOVA
with a Tukey’s post-hoc analysis (“naïve” in Fig 6). After dTBI, relative gene expression was compared to
sham and “naïve” animals after exploration of the novel environment as a function of time post-stimulation
and injury using a two-way ANOVA with Tukey’s post-hoc analysis. The data are represented as the mean ±
standard error of the mean (SEM) *, p<0.05. All statistical analyses were performed using Prism®
(GraphPad, CA).
Conclusions
• Although there were no significant changes in ARC gene expression with respect to sham,
there were significant changes in ARC expression within the injury group.
• However, we did not see ARC expression increase significantly at 30 minutes exploration in
the sham group, which confounds the interpretation of the data.
• The lack of ARC expression at 30 minutes post-stimulation in sham can be due to insufficient
exploration of the novel environment by sham animals.
• Based on this data, ARC gene expression in the hippocampus may not be as valuable as
other areas of the brain for assessing therapeutic efficacy on restoring circuit function in
experimental diffuse TBI models.
Scientific Value
Where previous data from other areas of the brain support ARC as a valuable tool for
assessing circuit re-organization after TBI, these data do not indicate that signaling in the
hippocampus follows similar re-organization patterns.
Acknowledgements
The authors wish to thank Daniel Griffiths and Megan Evilsizor for performing
rodent surgeries and the Translational Neurotrauma Research Program for
valuable feedback throughout my internship. These experiments are partly
supported by, ADHS14-00003606, NIH R03 NS-077098, NIH R01 NS-065052
and Phoenix Children’s Hospital Mission Support Funds.
Fig 6. Exploration of the novel environment results
in a significant increase of ARC gene expression in
the hippocampus in animals with no surgical
preparation. The figure shows the time course for ARC
rats explored a novel environment for 20 minutes. These
animals were normalized to the naïve group which was
euthanized without being given time to explore the box.
There is a significant 2.5 fold increase of ARC
expression at 15 minutes relative to naïve. ARC
expression peaked at 30 minutes and then decreased to
baseline levels in the following time points.
Fig 5. Exploration of novel
environment The novel group was
introduced to the behavioral box lined
with an absorbent pad and allowed to
habituate for 5 minutes. For the next 15
minutes they were free to explore their
environment, voluntarily using their
whiskers.
Fig 4. FPI device. A cranial hub is
implanted and attached to the injury
device where an adjustable pendulum
falls and strikes the plunger, generating
a fluid pulse that impacts the dural
surface, resulting in diffuse axonal
injury without cavitation or contusion.
Hippocampus and Spatial Memory
Fig 3. Exploring a novel environment requires
spatial memory, which activates the hippocampus.
When a rat interacts with a novel environment, it
encodes its surroundings in spatial memory, activating
hippocampal circuitry (Moser et al., 1998).
Researchers lesioned parts of the hippocampus in rats
and tested their ability to complete a water maze. They
found that while the rats were still able to encode their
surroundings as spatial memory, they were unable to
retrieve the memories from the hippocampus. ARC is
an immediate early gene which responds to the
encoding of spatial memories in the hippocampus. Our
study will focus on the time course of ARC activation in
the hippocampus after exploration of a novel
environment after dTBI.
Traumatic Brain Injury
• There are over 1.7 million cases of traumatic brain injury per
year in the United States. In reality, the actual number is higher
due to undocumented cases of TBI.
• From 2001-2009, the rate of emergency department visits for
sports and recreation-related injuries with a diagnosis of
concussion or TBI, alone or in combination with other injuries,
rose 57% among children (age 19 or younger).
• TBI causes long-lasting debilitating neurological deficits (Fig 2).
• Currently, there is no cure for persisting TBI-induced neurological
deficits; only treatment of specific physical, cognitive, emotional
and sleep-related symptoms as they arise.
Fig 1. Leading causes of TBI in the United
States. Falls disproportionally affect the chart in
that more than 50% of children (ages 0-14) and
more than 80% of seniors (ages 65+) receive
TBI via a fall. Blunt trauma (dTBI) is the highest
cause of TBI in adolescents and adults (ages 15-
64), often occurring during sports and motor
vehicle accidents (CDC 2011).
Fig 2. Debilitating neurological
deficits of TBI. The onset of these
morbidities can occur from hours to
months after the TBI. Neurological
deficits can be partially or fully
alleviated through medication;
however, the cognitive and
emotional deficits can significantly
impact a person’s career and family
life (CDC 2011).
Fig 7. Diffuse TBI does not cause a significant
change in ARC expression in the hippocampus. The
figure shows the relative gene expression of ARC in the
injured group of rats across the selected time points.
ARC expression significantly increases at 30 minutes
post-stimulation in the injured group. ARC then
decreased to baseline levels in the following time
points. The change in relative gene expression of ARC
was not significant at any time point in the injured group
with respect to sham.
Khodadad et al. (2015)
Moser et al., 1998
Results
Arjun Ganesh1,2,3, Aida Khodadad3,5, Jonathan Lifshitz2-4,6, Theresa Currier Thomas2-4