Page 44 - NNS 2014 Program & Abstract Book

OC2-03
A RABBIT MODEL OF PEDIATRIC TRAUMATIC BRAIN
INJURY
Zhang, Z., Saraswati, M., Robertson, C.L., Koehler, R.C., Kannan, S.
Johns Hopkins University, Department of Anesthesiology and Critical
Care, Baltimore, USA
Traumatic brain injury (TBI) is a common cause of disability in child-
hood, yet the mechanisms responsible for its complex pathologies re-
main largely unknown. A limitation of pediatric rodent models of TBI is
that they often do not demonstrate the spectrum of motor and cognitive
deficits seen in pediatric TBI patients. To address this, we developed a
model of pediatric TBI in New Zealand rabbits that mimics pediatric
brain development better. On postnatal day 5–7 (P5-7), rabbits were
injured with controlled cortical impact (CCI) (6mm impactor tip;
5.5m/s, 2mm depth, 50 msec duration). Rabbits from the same litter
served as control (no injury) and sham (craniotomy alone). Functional
abilities (cranial nerve, motor and sensory functions) and activity levels
(open field) were measured 24-h and 5-d after surgery. Maturation level
was monitored daily. The cognitive tests, spontaneous alternation in
T-maze and novel object recognition, were carried out during P14-26.
Animals were sacrificed 3, 7 and 21 days after TBI for evaluating lesion
volume and microglia (IBA1 staining). Significant decreases in the
overall motor functions, such as suck and swallow, head movements and
hops, was noted 24–48 hours after TBI. In addition, TBI kits showed a
delayed achievement of normal developmental milestones, such as eye
opening, and loss of cliff-avoidance. Significant cognitive deficits were
noted in the TBI kits with lower percentage of correct alternation rate in
the T-maze (n = 8-10/group; p < 0.001) and less discrimination between
novel and old objects ( p < 0.01) when compared with controls and
shams. Lesion volume increased from 10% at 3 days to 30% at 7 days
after injury, indicating ongoing secondary injury. Activated microglia
were noted at the injury site and also in white matter regions (peri-
ventricular region and internal capsule) of both the ipsilateral and con-
tralateral hemispheres. The short-term and long-term impairments of
this model are comparable to those reported clinically, providing a novel
platform for evaluating neuroprotective therapies in pediatric TBI.
Key words
developmental brain injury, microglia, pediatric
OC3-01
DISRUPTED AUTOPHAGY AFTER SPINAL CORD INJURY
IS ASSOCIATED WITH NEURONAL CELL DEATH
Liu, S.1, Sarkar, C.2, Koh, E.Y.1, Wu, J.2 , Lipinski, M.M.2
1University of Maryland School of Medicine, Department of Ortho-
paedics, Baltimore, USA
2University of Maryland, Department of Anesthesiology and the
Center for Shock, Trauma and Anesthesiology Research (STAR),
Baltimore, USA
Autophagy is a lysosome-dependent intracellular degradation pathway,
which plays a neuroprotective function in several neurodegenerative
diseases. Although elevated autophagic markers have been reported after
SCI, its mechanism, cell type specificity, and relationship with cell death
remain unknown. In a rat model of moderate contusive SCI, we found
increased levels of autophagy marker, LC3-II, by western blot and in-
creased numbers of cells accumulating LC3-positive autophagosomes by
immunohistochemistry (IHC), starting at 1 day after injury and con-
tinuing for up to 5 weeks. Initial accumulation of LC3 was accompanied
by pronounced elevation in the levels of the autophagy substrate, p62.
This indicates that the initial increase in markers of autophagy was due to
defective lysosomal clearance of autophagosomes and their cargo. Ac-
cumulation of p62 was resolved by day 7 after SCI, and was associated
with elevated levels of the lysosomal enzyme cathepsin D. Therefore,
increase in the size and activity of the lysosomal compartment may help
restore autophagy flux at that time. Furthermore, we used IHC to study
cell type specificity of autophagy after SCI. LC3 preferentially accu-
mulated in oligodendrocytes and microglia in the white matter, and co-
localized with the neuronal cell marker NeuN in the gray matter. LC3
was especially pronounced at day 1 after SCI in motor neurons in the
ventral horn. Since our data indicate that at that time autophagic
clearance is blocked, we hypothesize that it may contribute to neuronal
cell death. Consistently, we found that p62 labeled cells were also
positive for apoptotic cell death markers, cleaved caspase 3 and caspase
12, indicating association between disrupted autophagy and cell death.
Together, our data indicate that autophagic degradation is temporarily
blocked and may contribute to neuronal cell death after SCI.
Key words
apoptosis, autophagy flux, p62, SCI
OC3-02
PROTEASE ACTIVATED RECEPTOR-MEDIATED
MECHANISMS OF NEURAL INJURY
Yoon, H., Radulovic, M., Scarisbrick, I.A.
Mayo Clinic, Neurobiology of Disease Program, Department of
Physical Medicine and Rehabilitation, Rochester, MN, USA
Central nervous system injury, including that elicited by trauma, is-
chemia, infection, neurodegeneration or neoplasia, creates a complex
wound manifesting with a cascade of secondary cellular and molecular
responses that worsen the initial insult and limit repair and regeneration.
Among the deregulated neurotoxic factors are serine proteases of the
thrombolytic, fibrinolytic and kallikrein families, either as a result of
elevations in endogenous cells, secretion by infiltrating immune cells or
extravasation. Here we document changes in expression in kallikrein 6
and thrombin in a murine model of spinal cord contusion-compression
injury and document key pathophysiologic activities impacting the
development and progression of traumatic spinal cord injury (SCI),
including oligodendrogliopathy, neuron degeneration, axonopathy, and
astrogliosis. Importantly, we provide mechanistic evidence that the
neurotoxic effects of kallikrein 6 are mediated by activation of select
members of a G-protein coupled receptor family referred to as Protease
Activated Receptors (PARs). PAR activation is induced by proteolytic
cleavage within the extracellular N-terminus of the receptor revealing a
cryptic tethered ligand that binds intramolecularly to induce intracel-
lular signaling. Kallikrein 6 elicited PAR1-dependent dying back of
oligodendroglial processes and suppression of myelin gene expression.
PAR1, in addition to PAR2, were shown to play critical roles in kal-
likrein 6-mediated neuron injury, astrocyte stellation and secretion of
the pro-inflammatory cytokine IL-6. Moreover, genetic deletion of PAR
was associated with reductions in molecular signatures of injury in
murine SCI, including decreases in markers of apoptosis (BIM), as-
trogliosis (GFAP, Vimentin) and Th1 pro-inflammatory cytokines
(IL-6, IL1-b). These studies suggest that PARs or their protease ago-
nists represent new modalities to moderate pathophysiological re-
sponses that occur in association with traumatic SCI and to foster an
environment favorable to repair and regeneration.
Key words
demyelination, protease, spinal cord injury
A-12