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Methods:
Primary cortical astrocytes were isolated from neonatal
Sprague-Dawley pups. We exposed astrocytes to thrombin or specific
PAR-1 agonist, and measured ROCK pathway activation and MLC
phosphorylation. To determine the role for ROCK in MLC signaling
we examined the effect of a rho kinase inhibitor on thrombin-induced
MLC phosphorylation.
Results:
Thrombin induced phosphorylation of MLC in a time-
dependent manner. Rho kinase protein level was increased by treat-
ment with thrombin. Treatment with the ROCK inhibitor Y27632 at-
tenuated thrombin-mediated MLC phosphorylation without affecting
rho kinase protein level. Treatment with the thrombin agonist peptide
which acts via the PAR-1 receptor also caused an increase in phospho-
MLC levels.
Conclusion:
Thrombin activates the ROCK pathway to phosphor-
ylate MLC, likely via the PAR-1 receptor. These results suggest an-
other mechanism by which thrombin may affect BBB integrity
through its effects on astrocyte cytoskeletal properties.
Keywords: thrombin, rho kinase, myosin light chain
D7-02
TRAUMATICALLY INJURED ASTROCYTES RELEASE A
PROTEOMIC SIGNATURE MODULATED BY STAT3 DE-
PENDENT CELL SURVIVAL
Jaclynn Levine
1
, Kwon Eunice
1
, Pablo Paez
2
, Weihong Yan
3
, Gregg
Czerwieniec
3
, Joseph Loo
3
, Michael Sofroniew
4
, Ina-Beate Wanner
1
1
University of California Los Angeles, Semel Institute for Neu-
roscience, Los Angeles, USA
2
SUNY University at Buffalo, Hunter James Kelly Research Institute,
Buffalo, USA
3
University of California Los Angeles, Department of Biological
Chemistry, Los Angeles, USA
4
University of California Los Angeles, Department of Neurobiology,
Los Angeles, USA
Molecular markers associated with CNS injury are of diagnostic
interest. Mechanical trauma generates cellular deformation with
membrane damage and poration. We used an
in vitro
model of
stretch-injury and proteomic analysis to determine protein changes
in murine astrocytes and their surrounding fluids. Abrupt pressure-
pulse stretching resulted in rapid release of over 75 astrocytic pro-
teins with release profiles reflecting membrane permeability and cell
lysis. This acute astrocyte leak-associated ‘‘traumatome’’ was
overrepresented with metabolic and catabolic proteins compared to
their uninjured cellular proteome, bearing implications for post-
traumatic metabolic depression. Stretched astrocytes deficient in
signal transducer and activator of transcription 3 (STAT3-CKO)
released more protein complexes, nuclear proteins, cytoskeletal and
transport proteins, consistent with a larger population of lysed cells
compared to non-transgenic astrocytes. STAT3-CKO astrocytes also
had reduced basal expression of GFAP, lactate dehydrogenase B
(LDHB), aldolase C (ALDOC) and astrocytic phosphoprotein 15
(PEA15), and elevated levels of tropomyosin 4 (TPM4) and a-actinin
4 (ACTN4), as well as reduced stretch-injury tolerance and elevated
necrosis. Stretching led to cellular depletion of PEA15 and GFAP,
and caused GFAP filament disassembly in distinct subpopulations of
injured astrocytes that varied between genotypes. PEA15 and AL-
DOC signals were reduced in injured astrocytes at 6 hours after
mouse crush spinal cord injury (SCI) and were elevated in reactive
astrocytes 1–3 days post-injury. In contrast, a-crystallin (CRYAB)
was elevated in acutely injured astrocytes, and absent from uninjured
and reactive astrocytes, demonstrating marker heterogeneity. To-
gether these findings reveal a proteomic signature of traumatically-
injured astrocytes reflecting STAT3-dependent cellular survival,
with potential diagnostic value.
Keywords: traumatome, proteomic, GFAP, PEA 15, TBI, Bio-
marker
D8 Poster Session VIII - Group D: Neuroprotection
D8-01
NECK STRENGTH IS ASSOCIATED WITH HISTORY OF
CONCUSSION IN AMATEUR ADULT SOCCER PLAYERS
Eva Catenaccio
1
, Weiya Mu
1
, Namhee Kim
1
, Tamar Glattstein
1
,
Malka Zughaft
1
, Oren Jaspan
1
, Atira Kaplan
2
, Walter Stewart
3
,
Richard Lipton
4
, Michael Lipton
1,2
1
Albert Einstein College of Medicine, GMRRC, Bronx, USA
2
Montefiore Medical Center, Radiology, Bronx, USA
3
Sutter Healthcare, R&D, Sacramento, USA
4
Montefiore Medical Center, Neurology, Bronx, USA
Cervical musculature is essential for maintenance of posture and
stabilizing the head. Greater neck strength (NS) has been associated
with reduced risk of concussion in high school athletes. Soccer players
are at risk of concussion both from collisions and from heading the
ball. Our objective was to assess the association of measures of NS
and anthropometric measurements as predictors of risk of concussive
injury in soccer players. 57 players (41 men, 16 women, ages 18–51)
were drawn from an ongoing longitudinal study of sub-concussive and
concussive mild TBI in amateur soccer players. Number of prior
concussions was reported by each subject. Subjects underwent NS
measurement in isometric extension (EXT), forward flexion (FF), and
right (RLF) and left lateral flexion (LLF) using the microFET2 digital
dynamometer (Hoggan Scientific) mounted on a custom wall frame.
Height, weight, neck length, neck circumference, and head circum-
ference were measured and body mass index (BMI) was computed.
Players without a history of concussion (n
=
36) had significantly
greater NS in FF than players with a history of concussion (n
=
21)
(p
=
0.011). Number of prior concussions was negatively correlated
with NS in FF (p
=
0.003) and EXT (p
=
0.019). NS was correlated
with weight in all 4 directions (p
<
0.05) and with neck circumference
in FF, EXT, and LLF (p
<
0.01). However, neck circumference and
weight were not correlated with number of prior concussions. Results
were corrected for multiple comparisons using false discovery rate
correction (alpha
=
0.05). In soccer players increased NS is associated
with decreased risk of concussion. Anthropometric measurements are
correlated with NS, but not with history of concussion and may not
provide a reliable means for risk assessment. Further study is war-
ranted toward developing interventions to mitigate concussion risk.
Keywords: sports-related concussion, mild traumatic brain injury,
neck strength, anthropometrics
D8-02
NEUROPROTECTION WITH PEG-HYDROPHILIC CARBON
CLUSTERS IN MILD TRAUMATIC BRAIN INJURY COM-
PLICATED BY HYPOTENSION IN RODENTS
Roberto Garcia
1
, Lamin Mbye
1
, Thomas Kent
1
, James Tour
2
, Claudia
Robertson
1
1
Baylor College of Medicine, Neurosurgery, Houston, USA
2
Rice University, Chemistry, Houston, USA
A-113