53 / 198
Blast-induced traumatic brain injury (bTBI) is a significant cause of
morbidity in US soldiers. Understanding the underlying mechanisms
of cellular and molecular neuropathology in bTBI is essential for the
development of effective treatments. This study characterized acute
effects of blast overpressure in rat organotypic hippocampal slice
cultures (OHCs). OHCs were exposed to a single blast overpressure
of 150 or 280 kPa generated by an open-ended helium-driven shock
tube. Cell death was visualized by the fluorescent cell death marker
propidium iodide (PI). Co-labeling with the neuronal marker (neuronal
class III
b
-tubulin; Tuj1), the microglial marker (ionized calcium-
binding adapter molecule 1; Iba1), or the astrocyte marker (glial
fibrillary acidic protein; GFAP) revealed the phenotype of cells sus-
ceptible to blast injury. At 2 h, following exposure to either blast
overpressure, dead neurons and glial cells were observed. Additionally,
in both blast groups we detected the presence of phagocytic microglia
cells, suggesting their role in dead cell clearance. In previous studies,
we demonstrated at 72 h post-injury the majority of dead cells were
neurons while only few dead glial cells were observed at this later time
point. Together, our results suggest the progression of blast-evoked cell
death depends on the cell type. Specifically, microglia and astrocytes
appear susceptible to blast overpressure in the acute phase, yet we do
not observe the progression of cell death at 72 h post-injury. In contrast,
neuronal loss progresses over time following blast exposure. Future
studies will reveal potential correlations between the acute effects of
blast on glial cells and delayed neuronal loss.
Supported by Department of Neurosurgery, MCW and VA Research
Keywords: blast injury, hippocampus, organotypic slice culture,
traumatic brain injury
A1-04
INHIBITION OF CA
2
+
-INDEPENDENT PLA2
c
EXACERBATED
THE MECHANICAL STRETCH INJURY IN PRIMARY COR-
TICAL NEURONS
Honglu Chao
1
, Xiupeng Xu
1
, Zheng Li
1
, Yinglong Liu
1
, Huimei
Chen
2
, Ning Liu
1
, Jing Ji
1
1
Nanjing Medical University, Department of Neurosurgery, Nanjing,
China
2
Nanjing University School of Medicine, Department of Medical
Genetics, Nanjing, China
Ca
2
+
-independent PLA
2
c
(iPLA
2
c
), known as Group VIA PLA
2
,
hydrolyze phospholipids at the sn-2 position, thereby releasing free
fatty acids and a lysophospholipids. In some studies found iPLA
2
c
not
only like other PLA
2
releasing FFAs to regulate inflammation, but
also have key role in membrane phospholipid metabolism and re-
modeling especially under oxidative stress. And the location of
iPLA
2
c
is on endoplasmic reticulum and mitochondria. This implies
that iPLA
2
c
may participate the membrane phospholipid injury and
repair when mitochondria under oxidative stress. And previously, we
found that selective peroxidation of mitochondria membrane phos-
pholipid cardiolipin as an important pathogenic mechanism for TBI.
So we use (R)-BEL, a specific inhibitor of iPLA
2
c
, to clarify the role
of iPLA
2
c
when under mitochondrial oxidative stress damage after
stretch in primary cortical neurons. In the current report, we found that
iPLA
2
c
inhibitor deteriorate the mechanical stretch injury. Cyto-
chrome c is released into the cytoplasm and caspase3 apoptosis
pathway is activated. And we found mechanical stretch damage the
mitochondrial membrane potential, and iPLA
2
c
inhibitor aggravated
the damage. The inhibition of iPLA
2
c
may increase the mitochondrial
apoptosis and injury. Furthermore, MDA,4-HNE, products of lipid
peroxidation, increase after stretch, and iPLA
2
c
inhibitor exacerbated
the lipid peroxidation. And iPLA
2
c
inhibitor also increased stretch-
induced ROS production by DCFH-DA staining. Interestingly after
stretch iPLA
2
c
expression level did not change significantly, but
iPLA
2
c
activity significant increased. Our result suggested that
iPLA
2
c
is neuroprotective and iPLA
2
c
can be up-regulated to relieve
the mitochondrial injuries. And this protective effect may via mito-
chondrial membrane phospholipid remodeling pathway. Further de-
tailed mechanism about the iPLA
2
c
after stretch needs to be clarified.
Keywords: Ca
2
+
-independent PLA2
c
, mitochondria, oxidative
stress, lipid peroxidation
A1-05
A NOVEL METHOD FOR TRAUMATIC INJURY OF ORGA-
NOTYPIC HIPPOCAMPAL SLICE CULTURES
David Shellington
1,2
, Mimi Yao
1
, Hang Yao
1
, Dan Zhou
1
, Gabriel
Haddad
1,2
1
University of California, San Diego, Department of Pediatrics, San
Diego, CA, USA
2
Rady Children’s Hospital of San Diego, Department of Pediatrics,
San Diego, CA, USA
Background:
Organotypic hippocampal slice cultures (OHSC) can be
used to model molecular changes after traumatic brain injury. Pre-
vious OHSC models of traumatic injury have used proprietary
equipment. We report a novel method of inducing mechanical injury
in OHSC using a commercially available device.
Methods:
Briefly, 400-micrometer slices were prepared from hip-
pocampi from P5-7 C57Bl6 mice and placed on Bioflex plates
(Flexcell Int) coated with polyornithine and laminin. Slices were in-
cubated on a rocker until DIV10. Culture medium was changed to
artificial cerebrospinal fluid with propidium iodide (PI). Images were
obtrained from a Zeiss Axiovert microscope with a custom-built stage
adapter and rhodamine filter. The Cell Injury Controller II (Custom
Design and Fabrication, VA) stretched OHSC with pulse duration
80 msec and varied injury pressures (3.3 to 7.6 PSI). Images were
recorded at 2, 6, 12, and 24 hours post-injury. Slices were treated with
sucrose overnight to determine maximum PI uptake. Fluorescence
intensity was measured (Image J). Fractional PI uptake was calculated
to quantify cell death (1.0
=
maximum cell death).
Results:
Control slices demonstrated low baseline PI uptake after
24h (CA1 0.11
–
0.03, mean
–
SD). Fractional PI uptake was altered
for CA1, CA3, and DG regions after stretch injury at 24h (p
<
0.001,
ANOVA). PI staining at low levels of stretch was not significantly
different than control OHSC (3.5 PSI:0.11
–
0.03; 4.1 PSI:0.14
–
01;
4.4 PSI:0.17
–
0.04; p NS). Maximum PI uptake occurred at injuries
above 5.2 PSI (0.31
–
0.05). Additional PI uptake did not occur with
higher pressures (6.4 PSI: 0.32
–
0.04, 7.4 PSI: 0.29
–
0.04). In CA1,
control values differed from 5.2 PSI, 6.4 PSI, and 7.4 PSI (p
<
0.01 for
all, Dunn’s). Mild stretch injury (3.3 PSI, 4.4 PSI) differed from se-
vere stretch injuries (5.2 PSI, 6.4 PSI, p
<
0.01). Other hippocampal
regions (CA3, DG) showed similar patterns of cell death.
Conclusions:
Our methods yielded consistent, reproducible injury
to neuronal regions in OHSC. This model can be used to study effects
of varying ACSF constituents or screening therapeutic agents in iso-
lated brain tissue.
Keywords: Tissue Culture, Hippocampus, Artificial Cerebrospinal
Fluid
A-17