Page 122 - NNS 2014 Program & Abstract Book

5Safar Center for Resuscitation Research, Pittsburgh,
6Center for Neuroscience, University of Pittsburgh, Pittsburgh,
7RS Dow Neurobiology Labs, Legacy Research Institute, Portland
Post-traumatic epilepsy (PTE) is a common complication following
traumatic brain injury (TBI). Cell surface A1 receptor activation by
extracellular adenosine is a powerful endogenous anticonvulsant
mechanism. We hypothesized that genetic variation within receptors,
transporters or enzymes influencing the extracellular adenosine/A1
receptor system would predict PTE risk. Previous work impli-
cates genetic variation within the adenosine A1-receptor gene with
PTE risk. Adenosine kinase (ADK) is up-regulated in astrocytes
chronically after TBI and converts the endogenous intracellular
anticonvulsant adenosine to inactive 5¢-AMP. Ecto-5 ¢-nucleotidase
[(CD73); metabolizes extracellular 5¢-AMP to adenosine, thereby
increasing extracellular adenosine and lowering 5¢ -AMP)] and
equilibrative nucleoside transporter type-1 [(ENT-1); allows equili-
bration of intracellular/extracellular adenosine concentrations]. 203
adult Caucasians without premorbid seizures and with moderate/
severe TBI were recruited, and nine ADK tagging SNPs, three CD73
SNPs, and two ENT-1 SNPs genotyped. PTE was defined as seizures
first occurring > 1wk post-TBI. Survival analyses were used to in-
vestigate time to first seizure and PTE risk while adjusting for time
to mortality. Kaplan-Meier analysis revealed that TT (rs946185),
AA (rs11001109), and GG (rs11001111) homozygosity within the
ADK gene was associated with a shorter time to first seizure. Mul-
tivariate Cox Proportional Hazard analyses showed these genotypes
were individually associated with increased PTE risk up to 3 yrs
post-TBI, and a cumulative effect of each ADK variant on PTE risk
(Hazard Ratio = 5.236; p = 0.012) was significant using an ADK gene
risk score. This is the first clinical investigation on genetic vari-
ability within the ADK gene and epilepsy risk in any population.
These results suggest that extracellular adenosine and/or intracellu-
lar 5-AMP may modulate biochemical mechanisms facilitating epi-
leptogenesis and leading to PTE.
Support
NIH-R01HD048162; DODW81XWH-071-0701; NIHR01NR013342;
NIH5P01NS030318
Key words
adenosine kinase, biomarker, genetic association studies, PTE, TBI
C3-04
DIFFERENTIAL REGULATION OF THE AKT SIGNALING
PATHWAY IN RAT BRAIN AFTER PRIMARY BLAST IN-
DUCED TRAUMATIC BRAIN INJURY
Wang, Y., Sawyer, T.W., Weiss, M.T., Nelson, P., Hennes, G.,
Barnes, J., Vair, C., Josey, T.
DRDC Suffield Research Centre, Medicine Hat, Canada
Traumatic brain injury (TBI) has been a leading cause of morbidity
and mortality in recent conflicts in Iraq and Afghanistan. However,
the mechanisms of blast-induced TBI are not known. Akt, also
known as Protein Kinase B (PKB), is a serine/threonine-specific
protein kinase that plays a key role in neuroprotection and survival
in the CNS. In the present study, the effect of a simulated single
pulse primary blast wave on the levels of Akt and its downstream
effector kinase, glycogen synthase kinase (GSKb), in rat hip-
pocampus and frontal cortex were investigated. Male Sprague-
Dawley (SD) rats (350 – 400 g) were anaethetized in 3% isoflurane
and stablized in a plastic sleeve for 8 min. The sleeve was then
placed in the shock tube with the rat head positioned in the test area
for shock wave exposure (25 psi). This system has been developed
so that simulated single pulse ‘‘primary blast’’ exposure is ac-
complished with only minimal concussive and whiplash effects.
After exposure, rats were closely observed for either 1 day or 7
days before being sacrificed. Phosphorylation of Akt and GSK was
detected using their respective phosphorylated antibodies. Results
showed that Akt and GSK phosphorylation were decreased or little
changed 1 day after blast in the hippocampus and front cortex.
However, p-Akt and p-GSK levels were dramatically increased 7
days after blast on the ipsilateral hippocampus, while p-GSK was
also significantly increased on the contralateral hippocampus.
Furthermore, p-Akt was increased on the contralateral cortex while
p-GSK was increased on both sides of the frontal cortex. No sig-
nificant changes in total protein levels of Akt and GSK were ob-
served in both the hippocampus and front cortex. Because both Akt
and GSK phosphorylation have been indicated in neuro-protection
and neuro-damage, changes in the levels of Akt and GSK phos-
phorylation may contribute to neuropathology observed after pri-
mary blast exposure. Therapies targeting Akt and GSK
phosphorylation pathways may help to protect the brain against
blast-induced TBI.
Key words
Akt, GSK, primary blast, TBI
C3-05
ENDOGENOUS ELEVATION OF ACROLEIN FOLLOWING
ACUTE TRAUMATIC BRAIN INJURY IN RATS
Marquis, A.M., Shi, R., Duerstock, B.S.
Purdue University, West Lafayette, USA
Under healthy conditions, levels of reactive oxygen species are
maintained to prevent the accumulation of damage by an endogenous
antioxidant system, notably glutathione and vitamin E. In addition to
the generation of free radicals, Acrolein is one of several reactive
unsaturated aldehydes that are produced as a byproduct of lipid per-
oxidation during neuronal membrane damage. These aldehydes cause
further bystander damage to biological macromolecules, phospholip-
ids, and crosslink proteins, and result in the depletion of antioxidant
reserves. Due to the relatively long activation of acrolein after lipid
peroxidation, a therapeutic window exists to ameliorate cell damage
caused by aldehydes. In this experiment hydralazine, a well-known
scavenger of acrolein, was applied after traumatic brain injury (TBI)
to reduce anatomical damage.
Sprague-Dawley rats were administered closed skull, weight drop
TBIs. The treatment group received 5mg/kg hydralazine in saline
injected intra-parenterally daily post-injury. A sham group received
identical surgery injury group only omitting the weight drop.
The functional behavior of the rats were tested using a roto-rod and
open field activity box after TBI. Forty-eight hours post-injury the
brains were frozen, every tenth section collected for immunohisto-
chemistry. Brain sections were for acrolein-lys protein adducts.
Acrolein fluorescence of injured rat brains was increased 80% over
sham injuries and 40% more than hydralazine-treated animals. The
relative luminosities of the acrolein-lys signal between the injury
group (1.80) and the injury and treatment group (1.44) was statisti-
cally significant. We also examined specific brain regions for partic-
ular increases in acrolein staining.
Treatment with hydralazine at the dose used in this experiment did not
significantly affect the blood pressure of the animals. The short post-
injury window did not allow us to observe differences in the groups in the
roto-rod and activity box tests. Future work will correct this.
A-90