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Glutamate transporter expression and localization are potently reg-
ulated by protein kinases. We used a kinome array platform to de-
termine global changes in serine-threonine kinase activity after
lateral fluid percussion in prefrontal cortex (PHC) and hippocampus
(HPC). In PFC 25 substrates had changes in phosphorylation status
greater or less than 1.15 fold change from sham while in HPC 15
substrates exhibited a 1.15 fold change difference from sham. Using
publically available algorithms and databases we mapped protein
kinases predicted to target substrate sequences and performed ran-
dom sampling permutation analyses to identify kinases targeting the
reporter peptides at frequencies greater than what would be expected
by chance. From kinases identified by the permutation analyses we
constructed signaling network models based on known direct inter-
actions in the Ingenuity database. We performed inhibitor studies in
the prefrontal cortex using the kinome array platform to further in-
vestigate the role of several kinases implicated by our analyses and
in the literature in TBI pathology. In pooled prefrontal cortical
samples, we found differential regulation of protein kinase B (AKT)
in the presence of the AKT inhibitor. In TBI samples, kinase activity
increased on array substrates in the presence of AKT inhibitor, while
kinase activity on substrates of the sham sample was decreased.
Kinase activity in the presence of c-Jun kinase inhibitor or combine
protein kinase C (PKC) and mitogen activated protein kinase kinase
(MEK) showed much less divergence in activity profiles. Of the
kinases identified in our analyses, AKT, PKC and protein kinase A
(PKA) are known to actively regulate glutamate transporter ex-
pression, localization and transport activity and suggest dysfuntion
in the balance of pro- and anti- glutamate reuptake mechanisms after
brain injury.
Keywords: kinase, cell signaling, glutamate transport, AKT
T1-12
ADIPOSE STROMAL VASCULAR FRACTION CELL
TREATMENT MITIGATES INCREASED CERE-
BROVASCULAR PERMEABILITY AFTER TRAUMATIC
BRAIN INJURY
Nino Muradashvili
1
, Reeta Tyagi
1
, Jacob Dale
3
, Richard L. Benton
2,4
,
Suresh C. Tyagi
1
, James B. Hoying
1,3
, David Lominadze
1,4
1
University of Louisville, Physiology and Biophysics, Louisville, USA
2
University of Louisville, Anatomical Sciences and Neurobiology,
Louisville, USA
3
University of Louisville, Cardiovascular Innovation Institute,
Louisville, USA
4
University of Louisville, Kentucky Spinal Cord Injury Research
Center, Louisville, USA
Traumatic brain injury (TBI) is accompanied by a loss of memory that
can be attributed to the formation of un-degradable complexes of
proteins such as fibrinogen (Fg) and cellular prion protein (PrP
C
). This
complex can be formed as a result of increased cerebrovascular per-
meability resulting in deposition of Fg in the interstitium. We tested
the hypothesis that adipose-derived stromal vascular fraction (SVF)
cells, that are anti-inflammatory and reparative, can mitigate TBI-
induced hyper-permeability and, by decreasing Fg-PrP
C
complex
formation, reduce memory loss. SVF cells collected from syngeneic
mouse adipose tissue were intravenously injected into experimental
mice the next day after TBI. Mice in control group were injected with
vehicle alone. Ten days after TBI pial venular permeability was as-
sessed by measuring the extravascular accumulation of fluorescently-
labeled bovine serum albumin. Formation of Fg-PrP
C
complexes in
mouse brain vascular subendothelial matrix was assessed by immu-
nohistochemistry. The novel object recognition test (NORT) was used
to assess changes in short-term memory after TBI. Cerebrovascular
protein leakage was reduced in mice infused with SVF cells
(131
–
3%) as compared to that in mice infused with vehicle alone
(186
–
6%) after TBI. Accumulation of Fg and PrP
C
in the interstitium
was mitigated by SVF cell treatment. NORT results showed that there
was a tendency in reduction of memory loss in mice with SVF cells.
These results suggest that TBI-induced cerebrovascular hyper-
permeability to proteins can be ameliorated with SVF cells affecting
vascular endothelium and leading to reduction of Fg-PrP
C
complex
formation and short-term memory loss. Thus, our data indicate a novel
therapeutic role of SVF cells in treatment of vasculo-neuronal dys-
function after TBI.
NIH grants NS-084823 and P30 GM-103507
Keywords: Cerebrovascular permeability, Fibrinogen, Cellular
prion protein, Endothelial repair, Short-term memory
T1-13
NEURONAL PLASMALEMMAL PERMEABILITY/DENDRITIC
BEADING IN THE HIPPOCAMPUS FOLLOWING DIFFUSE
BRAIN INJURY IN SWINE
James Harris
1,2
, Kevin D. Browne
1,2
, John A. Wolf
1,2
, Douglas H.
Smith
2
, John E. Duda
1,3
, D. Kacy Cullen
1,2
1
Philadelphia Veterans Affairs Medical Center, Center for Neuro-
trauma, Neurodegeneration and Restoration, Philadelphia, USA
2
University of Pennsylvania, Center for Brain Injury and Repair,
Department of Neurosurgery, Philadelphia, USA
3
University of Pennsylvania, Department of Neurology, Philadelphia,
USA
Closed-head traumatic brain injury (TBI) is generally caused by
rapid angular acceleration/deceleration of the head, resulting in
strain fields throughout the brain. However, the neuroanatomical
distribution and loading thresholds of cells affected by these strain
fields remain poorly understood. Our objective was to characterize
the extent of immediate alterations in plasmalemmal permeability in
the hippocampus following inertial TBI in swine. Swine underwent a
closed-head rotational acceleration (single injury or two injuries
separated by 15 minutes or 7 days). To assess plasmalemmal com-
promise, Lucifer Yellow (LY), a small (457 Da) cell impermeant
dye, was administered into the lateral ventricles. Animals were
sacrificed within 15 minutes (LY injections), 8 hours, or 7 days post-
injury (n
=
29 total). We found acute plasmalemmal permeability,
predominantly neural cells. LY
+
cells were observed across different
hippocampal regions, including the hilar region, dentate granule
layer, and CA1 area. A decrease in NeuN immunoreactivity was
observed in a subset of LY
+
cells, suggesting altered NeuN expres-
sion could indicate stressed neurons. In addition to LY in somata and
neurites, morphological changes were observed via acute beading in
dendritic fields projecting from dentate granule neurons. The effect
was most pronounced following a single severe injury but also fol-
lowing repetitive mild injuries. Although mitochondrial dysfunction
is a potential cause of beading, mitochondrial fission labeling did not
co-localize with beading. Given the rapid onset of permeability and
beading, these are likely acute biophysical disruptions due to diffuse
strain fields. Ongoing analyses are assessing the neurophysiological,
inflammatory, and degenerative changes associated with these
structural responses. Understanding trauma-induced biophysical re-
sponses and pathophysiological progression is necessary to guide
development of therapeutics to ameliorate afflicted cell populations
following inertial TBI.
Keywords: traumatic brain injury, biomechanics, plasma mem-
brane, cell permeability
A-6