Page 94 - NNS 2014 Program & Abstract Book

B4-08
REPETITIVE MILD TRAUMATIC BRAIN INJURY IN THE
IMMATURE BRAIN
Fidan, E.G.1, Lewis, J.1, Kline, A.E.1, Garman, R.1, Alexander, H.1,
Dezfulian, C.1, Clark, R.S.1 , Kochanek, P.M.1, Bayır, H.1,2
Safar Center for Resuscitation Research, Pittsburgh, USA
Repetitive traumatic brain injury (TBI) occurs in unique pediatric
conditions, such as abusive head trauma (AHT) and sports concussion.
While accumulating evidence suggests that repeated mild TBI
(rmTBI) may cause long-term cognitive dysfunction in adults, it is
unknown whether rmTBI causes similar deficits in the immature
brain. We studied the effect of rmTBI in immature brain on acute and
chronic histological and neurocognitive outcome. 18d-old rats were
divided into two groups. The 1st (n = 3/grp) received either two closed
head impacts (CHI, 9.5mm rubber tip impactor, 4m/s velocity, 1mm
depth), one CHI paired with sham (S) or two sham (S/S) insults 24 h
apart. Histology at 7d included silver staining to detect axonal injury,
Iba-1 immunohistochemistry to assess microglial activation, H&E to
evaluate cell survival. The 2nd group received three CHI (n = 18) or
three sham insults (n = 12) 24 h apart and evaluated for motor [beam
balance (BB); inclined plane (IP) tests, d1-5] and cognitive [Morris
water maze (MWM; d11-15, 60); Novel Object Recognition (NOR;
d18); Elevated Plus Maze (EPM; d90); and Fear Conditioning (FC;
d92)] dysfunction. Silver staining revealed argyrophilia and axonal
staining in the ipsilateral external capsule of CHI/S and CHI/CHI
groups. H&E staining showed no overt neuronal loss. Second impact
increased axonal staining in ipsilateral external capsule. Increased
Iba1 positivity with morphological appearance of activated microglia
was observed in bilateral amygdala after CHI/S and CHI/CHI. There
were no differences in BB, IP, MWM and EPM performance between
groups. However, rmTBI rats were impaired in the NOR (P < 0.05,
66 – 2.4 vs 75 – 4%) and froze less than sham to a context (P < 0.05,
89 – 3.4 vs 99 – 0.3%) or a discrete auditory cue (P < 0.05, 26.4 – 5.9
vs 44.9 – 8.4%). In conclusion, rmTBI amplifies the mTBI response
producing diffuse axonal injury, microglial activation and memory
deficits. This model could be useful in therapy development for AHT
or sports concussion in children. Support: NS061817, U19AIO68021,
NS076511
Key words
abusive head trauma, immature brain, repetetive mild traumatic brain
injury, sport concussions
B4-09
NORMAL BACKGROUND OF APOPTOSIS IN JUVENILE
RATS USED AS BASIS TO DETERMINE INDUCED DEGEN-
ERATION BY THE NEUROTOXIN MK-801
Switzer, R.C.1, Parker, R.M.2, Thake, D.C.3
1NeuroScience Associates, Knoxville, USA
2Huntingdon Life Sciences, East Millstone, US
3ToxPath Sciences, Chesterfield, USA
Degeneration/apoptosis in juvenile brains from PND 0 to PND 24 is
minimal to abundant in different brain regions as a function of time.
This ‘backdrop’ of degeneration (DEG)/apoptosis (APO) must be
taken into account when assessing possible induction of degeneration
by chemical or physical insults. This study sought to determine the
relationship of normal changes and the effects of MK-801 (a non-
competitive NMDA antagonist with known neurotoxic properties) on
the development of juvenile Sprague-Dawley rat brains. The MK-801
group received a single dose (3 mg/kg, IP) on PNDs 7, 8, 9, 11 13, 16,
23, 39, 69 or 111). Distilled water was the control article. Thirty or
twenty rats per sex per time-point had brains perfused/harvested on
PNDs 8, 9, 10 and 12, or on PNDs 14, 17, 24, 40, 71 and 113,
respectively. The brains were MultiBrain embedded, coronally sec-
tioned at 40l and 1/8 th section stained with the amino cupric silver
method (DEG changes) and activated caspase 9 immunohistochem-
istry staining (APO). APO and DEG were prevalent in numerous brain
regions of younger control animals (primarily from PND 8 through
PND 24). Increased DEG and APO were present in MK-801 animals
at all time-points. Increased DEG and APO in MK-801 rats were
present in a large number of brain sites in the earlier PNDs with
severities ranging from minimal to marked while these changes at
later PNDs were substantially diminished. Females had more brain
sites involved than did males especially at the later PNDs. By PND
40, males had only 3 or 4 sites at which DEG was observed. APO that
was present in MK-801 rats was distinct and unequivocal when
compared with the control animals. Based on this data, isolated
minimal or mild occurrences of DEG and APO should not be con-
sidered treatment related events in juvenile Sprague-Dawley rats.
Key words
apoptosis, degeneration, juvenile, neonate, trauma
B4-10
EXAMINING D-CYCLOSERINE ADMINISTRATION PRO-
TOCOLS IN DEVELOPING RATS FOLLOWING LFPI AND
REDUCING VARIABILITY IN PCAMKII LEVELS
Segal, A.J., Biswas, C., Cai, Y., Giza, C.C., Hovda, D.A.
University of California, Los Angeles, Los Angeles, United States
This study examined the effects of different doses of D-cycloserine
(DCS) administration on NMDA signaling in the subacute injury
phase following severe lateral fluid percussive injury (LFPI) on
postnatal day 19 (P19) rats. DCS is a partial agonist at the NMDAR
glycine-binding site, and has been investigated as a potential thera-
peutic approach to cognitive dysfunction following TBI in the adult,
but not the juvenile, rat. Additionally, this study investigated exposing
P19 rats to an enriched environment prior to sacrifice in an attempt to
decrease the variability of pCaMKII levels in hippocampus through
functional stimulation. P19 rats underwent LFPI and received either a
single DCS dose (30 mg/kg; 0.25 ml/kg) 24 hours post-injury, 5 DCS
doses every 12 hours starting 24 hours post-injury, or saline. We
found a significant increase in pCaMKII levels in the 5 injection
protocol group when compared to the vehicle (p = 0.017), while no
significant increase occurred between the single injection and vehicle
(p = 0.188). Thus, a protocol with multiple injections is more appro-
priate for further investigation into the therapeutic benefits of DCS
following TBI in the juvenile rat. We additionally hypothesized that
20 minutes of novel environment exploration would reduce variability
of pCaMKII levels in the hippocampus via activation of NMDA
signaling. Four days following craniotomy, subjects were either
housed in a new cage with novel objects or returned to homecage
for 20 minutes, followed by sacrifice, micro-dissection of hippo-
campus, and Western analysis. Although no significant difference
in pCaMKII levels (p = 0.772) was observed between the two
groups, the variance was reduced in the group exposed to an en-
riched environment prior to sacrifice compared to the control
group, suggesting a more consistent activation of pCaMKII prior to
euthanization. This decreased variability potentially allows for
smaller animal groups in future experiments.
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