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ISG15 down-regulation: PND21 normalized to actin T0 Sham:
14.83 2.00 mm:9.83, 2.25 mm: 3.83 and T4h Sham: 4.5 2.00 mm: 5.5,
2.25 mm: 6.5.
ISG15 up-regulation: PND24 T0 Sham: 5.167, 2.00 mm: 9.167,
2.25 mm: 14.17 and T4 Sham: 4.5, 2.00 mm: 9.5, 2.25 mm: 14.
ISG15 and Claudin develop protein-protein interactions on co-im-
munoprecipitation increased in TBI compared to shams.
Blood Brain Barrier Disruption
Fluorescein and Dextran ng/grams of brain: Fluorescein- PND21
T0 Sham: 2, 2.00 mm: 3.5, 2.25 mm: 4.25. T4h T0 Sham: 2.25,
2.00 mm: 6.33, 2.25 mm: 13. PND24 T0 Sham: 2.25, 2.00 mm: 3,
2.25 mm 4. T4h Sham: 2, 2.00 mm 5.33, 2.25 mm 9.33.
Dextran-PND21 T0 Sham: 6.8, 2.00 mm 8.6 2.25 mm: 9.3. T4h
Sham: 10 2.00 mm: 20, 2.25 mm: 40. PND24 T0 Sham: 2.25,
2.00 mm: 3.25, 2.25 mm: 3.25. T4h Sham: 2, 2.00 mm: 5, 2.25 mm:
10.5. 13.223, 4h 2.00 mm: 9.333, 2.25 mm: 15.50
Conclusion:
Delay in upregulation of ISG15 in PND21 mice
compared to PND24 mice may play a role in worse outcomes in
younger children following TBI.
Keywords: pediatrics, mouse model, development, proteins
A3-03
ENDOTHELIAL TARGETED ANTIOXIDANT ENZYME
THERAPY TO COMBAT SECONDARY INJURY AND PRE-
SERVE BBB INTEGRITY FOLLOWING EXPERIMENTAL TBI
Evan Lutton
1
, Steven Merkel
1
, Allison Andrews
1
, Roshanak
Razmpour
1
, Vladimir Shuvaev
2
, Vladimir Muzykantov
2
, Servio
Ramirez
1
1
Temple University School of Medicine, Pathology & Laboratory
Medicine, Philadelphia, USA
2
University of Pennsylvania, Pharmacology, Philadelphia, USA
An estimated 1.7 million traumatic brain injuries (TBI) occur each
year, and TBI is a contributing factor to one third of all injury related
deaths in the United States alone. Current treatment for TBI is sup-
portive, and the pathophysiology is not fully understood; however,
evidence suggests high-energy oxidants and oxidative stress as me-
diators of secondary damage in TBI, including blood brain barrier
(BBB) hyperpermeability. A novel endothelial targeted antioxidant
enzyme approach to TBI therapy is hypothesized to quench reactive
oxygen species (ROS) at their source to protect BBB function. While
constitutively expressed molecules can be targeted for prophylactic
and therapeutic drug delivery, determinants expressed or upregulated
in pathological states are ideal for localized therapeutic intervention.
Here, we propose a strategy that targets the activated endothelium
following TBI. Conjugates of anti-PECAM-1 and anti-ICAM-1 anti-
bodies to catalase were prepared through amino chemistry for ad-
ministration to C57BL/6 mice after controlled cortical impact (CCI)
model of moderate TBI (impact parameters: 3.5 m/s, 2 mm tip, 1 mm
depth, 0.5 s dwell time). Previously, the anti-PECAM-1/catalase
conjugate was found to alleviate vascular oxidative stress in lung
ischemia/reperfusion injury. The use of antibody/antioxidant enzyme
conjugates has not been investigated in the context of TBI. Pre-
liminary data from isolated cerebral microvasculature suggests an
increase in expression of cell adhesion molecules, PECAM-1 and
ICAM-1, after TBI. Furthermore, we have demonstrated increased in
situ dihydroethidium (DHE) fluorescence detection of ROS in the
brain, thus supporting the proposed therapy as a potential means to
ameliorate secondary mechanisms of injury and to maintain BBB
homeostasis in neurotrauma.
Keywords: Antioxidant enzymes, Traumatic brain injury, Con-
trolled cortical impact, Endothelial biology
A3-04
PROFILE OF BLOOD BRAIN BARRIER DISRUPTION FOL-
LOWING SINGLE AND REPEATED CLOSED HEAD IMPACT
CONCUSSION IN RATS
Jenny Browning
, Ying Deng-Bryant, Weihong Yang, Xiaofang
Yang, Frank Tortella, Deborah Shear, Lai Yee Leung
Walter Reed, BTNN, Silver Spring, USA
Blood brain barrier (BBB) disruption is a pathologic hallmark of
severe traumatic brain injury (TBI) that is associated with neuroin-
flammatory events contributing to cell death and edema. However,
the extent to which the BBB may be compromised following con-
cussion or mild TBI (mTBI) are not well understood. In the current
study the WRAIR projectile concussive impact (PCI) injury model
was used to assess the effect of concussion on the expression of BBB
proteins. Rats were anesthetized, fitted with a helmet designed to
prevent skull fracture and a projectile was launched at their head to
induce a closed-head concussive impact injury resulting in mTBI.
Groups consisted of animals exposed to sham surgery (anesthesia
only), a single concussion and multiple concussions (4 impacts
spaced 1 hour apart). Animals were sacrificed at 6h, 24h, or 72h
post-injury. Immunohistochemistry was performed to quantify the
expression of proteins associated with the BBB structure/functions
including claudin-5 (CL5), zona occulden-1 (ZO1), and aquaporin-4
(AQ4). Positive-stained areas were quantified in the parietal cerebral
cortex. Significant reductions in CL5 expression were detected at
72h post-injury following a single concussion (p
<
0.05 vs. sham).
However, repeated concussion produced significant decrements in
CL5 expression that were apparent at 6h post-injury and sustained
out to 72h post-injury (p
<
0.05 vs. sham). Likewise, significant re-
ductions in Z01 protein expression were detected at 72h post-injury,
but only in animals that received multiple concussions (p
<
0.05 vs.
sham). No significant effects were detected in AQ4 expression at any
post-injury time point following single or repeated concussion.
Overall, these results indicate initial evidence of BBB disruption
following a single concussion that is worsened following multiple
concussions in the PCI model. Notably, the pattern of delayed deg-
radation of CL5 and Z01 protein expression following single con-
cussion or repeated concussion suggests a progressive gradient in
BBB disruption that is likely mediated by secondary causes, such as
neuroinflammation and oxidative stress.
Keywords: Concussion, mTBI
A4 Poster Session I - Group A: Pediatric
A4-01
PHASE I TRIAL OF N-ACETYLCYSTEINE IN COMBINA-
TION WITH PROBENECID IN CHILDREN AFTER SEVERE
TRAUMATIC BRAIN INJURY
Robert Clark
1,2
, Philip Empey
3
, Samuel Poloyac
3
, Hulya Bayir
1,2
,
Bedda Rosario-Rivera
4
, Patrick Kochanek
2
, Thomas Nolin
3
, Stephen
Wisniewski
4
, Michael Bell
1,2
1
Children’s Hospital of Pittsburgh, Pediatrics, Pittsburgh, USA
2
Safar Center, CCM, Pittsburgh, USA
3
School of Pharmacy, P&T, Pittsburgh, USA
4
School of Public Health, Epidemiology, Pittsburgh, USA
N-acetylcysteine (NAC) is being evaluated in clinical trials targeting
multiple neurological diseases including TBI; yet existing data
suggest it has limited if any penetration into normal brain. NAC
serves as a cysteine donor for synthesis of the prominent intracellular
A-26