25 / 162
animals. As further evidence of this immune activation, we see in-
creased numbers of neutrophils in acutely injured animals, while the
chronically injured animals show an increased number of T cells.
This data indicates that SCI causes acute and chronic phases of
injury in the testes, much like in the spinal cord itself. This research
is the first step in identifying a target for treatment designed to
improve or sustain BTB function after SCI in order to enhance
fertility; one of our most fundamental biological functions.
Keywords: Blood Testes Barrier
A2-18
DELAYED HYPOXIA FOLLOWING TRAUMATIC BRAIN
INJURY EXACERBATES AXONAL INJURY
Melissa Williams
1
, Umang Parikh
1
, Jodi Lapidus
1
, Jose Pineda
1
,
David Brody
2
,
Stuart Friess
1
1
Washington University at St. Louis, Pediatric Critical Care Medi-
cine, Saint Louis, USA
2
Washington University at St. Louis, Neurology, Saint Louis, USA
Hypoxia immediately following traumatic brain injury (TBI) has
been observed to exacerbate injury. In a retrospective cohort of 32
children admitted with TBI to an ICU, delayed hypoxemia (paO
2
<
60 mm Hg) was observed in 10/32 patients with 70% of the episodes
occurring in the first 48 hours after admission. It remains unclear
whether hypoxia beyond the immediate post injury period influences
axonal injury.
Five-week-old male mice (C57BL/6J), underwent controlled cor-
tical impact (CCI) followed by arterial catheterization on post-injury
day 1 with arterial blood gas sampling after 30 minutes of hypoxia
(8% FiO
2
and 4% CO
2
) as well as an assessment of tissue hypoxia
utilizing Hypoxyprobe. Twenty mice were subjected to either CCI
(N
=
12) or sham surgery (SHAM) (N
=
8). One day later awake ani-
mals were randomized to 30 minutes of hypoxia or normoxia. White
matter axonal injury was quantified 48 hrs post injury utilizing blinded
stereological methods on beta amyloid precursor protein (B-APP) and
NF-200 stained sections.
Twenty-four hours after CCI, 30 minutes of hypoxia in awake
spontaneously breathing mice revealed hypoxemia with normocarbia
(paO
2
50.1
–
1.9 mm Hg and paCO
2
40.7
–
2.0 mm Hg). Hypoxyprobe
immunohistochemistry demonstrated increased gray matter hypoxia in
SHAM
+
hypoxia and CCI
+
hypoxia compared with SHAM and CCI
respectively. However, pericontusional white matter hypoxia was only
observed in CCI
+
hypoxia. In the pericontusional corpus callosum
and external capsule there were increased axonal swellings in
CCI
+
hypoxia compared with CCI animals for both B-APP (37
–
6 vs.
21
–
6 10
3
axons/mm
3
, P
<
0.01) and NF-200 (34
–
4 vs. 24
–
3 10
3
axons/mm
3
, P
<
0.01). Minimal B-APP and NF-200 staining was ob-
served in SHAM and SHAM
+
hypoxia mice.
A clinically relevant model of delayed hypoxia following TBI re-
sulted in increased pericontusional axonal injury.
Keywords: hypoxia, axonal injury, traumatic brain injury, second-
ary injury, pediatric
A3 Poster Session I - Group A: Blood-Brain Barrier
A3-01
EVALUATING THE POTENTIAL FOR NANOPARTICLE
DELIVERY AFTER TRAUMATIC BRAIN INJURY
Vimala Bharadwaj
1
, Jonathan Lifshitz
2
, David Adelson
3
, Vikram D.
Kodibagkar
1
, Sarah E. Stabenfeldt
1
1
Arizona State University, Biomedical Engineering, Tempe, USA
2
Barrow Neurological Institute, Phoenix Children’s Hospital, Phoe-
nix, USA
3
University of Arizona, College of Medicine-Phoenix, Phoenix, USA
An estimated 1.7 million traumatic brain injuries (TBI) occur annually
and account for over 50,000 deaths in the U.S. TBI is initiated by a
mechanical insult that leads to a host of cellular and molecular al-
terations, including transient blood-brain-barrier (BBB) breakdown.
Nanoparticles (NP) have played an important role as diagnostic and
therapeutic (theranostic) agents in various diseases, but, limited per-
meability across BBB is a major obstacle for NP-based approaches for
neural disease/injury. Thus, the short-lived BBB permeability post-
injury may be effectively utilized to deliver NP-based theranostics for
TBI. Previous studies with pre-clinical TBI models demonstrated peak
permeability of small molecules (
*
5 nm) at 4–6h post-injury. Yet,
there is a critical gap in understanding the behavior of larger particle
delivery (
>
5 nm) after TBI. Therefore, the objective of this study was
to investigate the effect of NP’s size on extravasation after TBI.
Specifically, carboxylated polystyrene NPs of 20, 40, 100, and 500 nm
with unique fluorescent spectra were pegylated to both increase cir-
culation time and neutralize the surface charge of the nanoparticles.
Pegylated-NP cocktails were then intravenously injected in to mice
(n
=
6 retro-orbital injection) 5h post-injury (controlled cortical im-
pact) and allowed to circulate for 1h prior to sacrifice and perfusion.
The brains were frozen, sectioned, and imaged with fluorescent mi-
croscopy. Pegylation of NPs led to modest increase in hydrodynamic
diameter (
*
5–7 nm above baseline diameter) and reduced zeta-po-
tential (range:
-
9 mV to
-
29 mV). Histological analysis demonstrated
the presence of all pegylated-NPs exclusively within the injury pen-
umbra, indicating BBB breakdown and extravasation at 5–6h post-
injury. The presence of pegylated-NPs was significantly higher in
injured animals compared to control sham animals. In conclusion, we
have demonstrated the potential for NP extravasation acutely post-
TBI. Further time course characterization will provide insights as to
the full utility of NP-based theranostics agents for TBI.
Keywords: Nanoparticles, extravasation, theranostics, systemic
delivery
A3-02
ISG15 INTERACTS WITH JUNCTIONAL PROTEINS PRIOR
TO BBB DISRUPTION FOLLOWING TBI IN YOUNG MICE
Janet Rossi
1,2
, Tracey Todd
1
, Ludmilla Belayev
2
, Nicolas Bazan
2
1
LSUHSC/Children’s Hospital, Department of Pediatrics, New Or-
leans, USA
2
LSUHSC School of Medicine, Neuroscienc Center of Excellence,
New Orleans, USA
Introduction:
Recently we have shown that ISG15, an ubiquitin like
protein is upregulated prior to breakdown of the BBB following TBI
and colocalizes with MLCK, which plays a pivotal role in develop-
ment of cerebral edema. Here we show that ISG15 interactions with
junctional protein Claudin 5, and occurs prior to the disruption of the
BBB following TBI in young mice. ISG15 is increased following
focal ischemia and is neuroprotective. The significance of ISG15
following TBI is still unclear.
Methods:
PND21 and PND24 mice were anesthetized with avertin,
mechanically ventilated, physiologically regulated, and subjected to
lateral closed-skull injury model with impact depth of 2 or 2.25 mm
(bregma level - 0.10 mm). Mice were sacrificed at T0, T30m, T4h.
ISG15 and MLCK analyzed by western blot, immunohistochemistry;
BBB disruption with Fluorescein sodium salt and 4 Kda Dextran.
Results:
Protein expression
A-25