Page 125 - NNS 2014 Program & Abstract Book

Traumatic axonal injury is a major contributor to morbidity after se-
vere traumatic brain injury (TBI). Reduction and avoidance of in-
creases in intracranial pressure (ICP) continues to be the mainstays of
treatment. It remains unclear whether elevations in ICP influence
axonal injury.
Six week old male mice (C57BL/6J) were subjected to either con-
trolled cortical impact (CCI) (N = 48) or sham surgery (SHAM, N = 12).
Immediately after CCI, injured animals were randomized to a loose
fitting plastic cap (OPEN) or replacement of the previously removed
bone flap (CLOSED). Animals were sacrificed at 1 day, 7 days and 4
weeks post injury. Brain parenchymal ICP measurements were taken
via a contralateral burr hole. Severity of white matter axonal injury was
quantified utilizing stereological methods of beta amyloid precursor
protein (B-APP) stained sections at 1 day and 7 days post injury.
Elevated ICP was observed in CLOSED animals 15 minutes and 1
day after injury compared to OPEN and SHAM (15 min 21.4 – 4.2 vs.
12.3 – 2.9 and 8.8 – 1.8 mm Hg, P < 0.0001; 1 day 17.8 – 3.7 vs.
10.6 – 2.0 and 8.9 – 1.9 mm Hg, P < 0.0001). Stereologic quantification
of B-APP staining in the corpus callosum and ipsilateral external
capsule revealed increased axonal swellings and bulbs in CLOSED
compared to OPEN animals at 1 day (136 – 24 vs. 94 – 29 103 axons/
mm3, P < 0.01) and 7 days (99 – 29 vs. 58 – 15 103 axons/mm 3,
P < 0.001) post injury. At 4 weeks post injury, CLOSED animals had
increased white matter atrophy compared to OPEN and SHAM, re-
sulting in smaller corpus callosum and external capsule volume
(1.2 – 0.1 vs. 1.5 – 0.2 and 2.0 – 0.1 mm3 , P < 0.0001).
Following controlled cortical impact, even moderate elevations in
intracranial pressure were associated with increased axonal injury and
white matter atrophy. Therapeutic interventions that ameliorate in-
tracranial hypertension may influence white matter injury severity.
Key words
axonal injury, intracranial pressure, mouse, seconday injury
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IMPAIRED SYNAPTIC VESICLE DOCKING IS A NOVEL
CONTRIBUTOR TO REDUCED NEUROTRANSMISSION
AFTER TRAUMATIC BRAIN INJURY
Carlson, S.W., Yan, H., Dixon, C.E.
Neurosurgery, Safar Center for Resuscitation Research, and Veteran’s
Affairs, University of Pittsburgh, Pittsburgh, USA
Traumatic brain injury (TBI) impairs neuronal function and can cul-
minate in lasting cognitive impairment. While impaired acetylcholine
release has been well established after experimental TBI, little is
known about the mechanisms underlying this consequence. We hy-
pothesized that alterations in synaptic vesicle distribution and reduced
vesicular docking at the pre-synaptic membrane contribute to im-
paired neurotransmission. To examine the ultrastructural distribution
of synaptic vesicles, Sprague-Dawley rats received 2.7 mm controlled
cortical impact (CCI) or sham injury (n = 6/group) and the brains were
processed for transmission electron microscopy at 1 week post-injury.
In each animal, 20 randomly selected synaptic nerve terminals from
the molecular layer of the hippocampus were imaged at 100 k mag-
nification. Synaptic vesicle distribution was assessed by measuring the
distance of each vesicle from the active zone for all terminals. CCI
resulted in a significant reduction in vesicle frequency within 200 nm
of the active zone (p < 0.01 compared to sham, repeated measures one-
way ANOVA). Recent reports highlight that reduced vesicular density
within 100nm of the active zone impairs vesicular docking and blunts
neurotransmitter release. In a normal synapse, vesicular docking and
neurotransmitter release requires formation of the SNARE complex. To
examine the effect of TBI on the SNARE complex, rats received CCI or
sham injury and were sacrificed at 6 hr, 1 d, 1, 2, or 4 weeks post-injury
(n = 6/injury/time). Immunoblotting of unboiled hippocampal homog-
enates showed that SNARE complex formation, identified by SNAP-25
and syntaxin immunoreactivity, was reduced by at least 48% at 1 week
(p < 0.05) and 2 weeks (p < 0.01) after CCI. Neurotransmitter release
deficits have been well characterized at 1 and 2 weeks post-injury,
suggesting that changes in synaptic vesicle docking contributes to im-
paired neurotransmission. In this study, we provide the first evidence
that TBI alters synaptic vesicle distribution using quantitative ultra-
structural analysis of electron micrographs. Our findings suggest that
reductions in the standing pool of readily releasable vesicles and im-
paired SNARE complex formation are two novel mechanisms that
contribute to the impaired neurotransmission after TBI.
Key words
electron microscopy, neurotransmission, synapse, vesicle
C3-13
SECONDARY MEMBRANE DAMAGE AND THE POTEN-
TIAL FOR MEMBRANE-TARGETED NEUROPROTECTION
Dastgheyb, R.M.1, Gallo, G.2, Barbee, K.A.1
1Drexel University, Philadelphia, US
2Temple University, Shriners Hospitals Pediatric Research Center,
Philadelphia, United States
Traumatic Brain Injuries (TBI) result in primary and secondary
damage. The extended timescale of secondary injuries provides a
larger window of opportunity for therapeutic interventions, but un-
fortunately there are currently no clinically successful pharmaceutical
treatments for TBI. This indicates a need for a better understanding of
the mechanisms and pathways of cellular degeneration and dysfunc-
tion after injury in order to better develop effective therapeutic in-
terventions for patients with TBI. Plasma membrane damage, calcium
influx, mitochondrial damage, and increased oxidative stress have all
been identified as key players in the TBI pathway. Membrane damage
has been hypothesized to be an initiating factor in the secondary
damage pathway and previous studies have shown that sealing the
damage using Poloxamer 188(P188) is neuroprotective after me-
chanical shear stress injury. However, the therapeutic potential of
P188 and targeted membrane protection is limited if membrane
damage is only occurring at the beginning of the secondary damage
pathway. The results here make the case that secondary membrane
damage is occurring and that it can be targeted using P188. Aspects of
the injury pathway downstream of initial membrane damage were
targeted and induced using Calcium Ionophore A23187 to increase
intracellular calcium without general membrane damage, CCCP to
damage mitochondria, and the hydroperoxide donor tert-butyl hy-
droperoxide to increase oxidative stress. These treatments were used
to create isolated perturbations of pieces of the TBI pathway in cul-
tured chick forebrain neurons. Axonal injury was quantified by nor-
malizing the number of focal swellings (beads) by the length of the
axon. P188 used in combination with each of these resulted in a
statistically significant reduction in beading. Membrane permeability
was quantified by measuring the loss of intracellular Calcein Red/
Orange. Together, these results provide evidence for the presence of
secondary membrane damage and the possibility of targeted mem-
brane sealing as a therapeutic tool for treating TBI.
Key words
calcium, membrane damage, membrane protection, mitochondria,
oxidative stress, poloxamer 188
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