Page 121 - NNS 2014 Program & Abstract Book

1

University of Iowa, Iowa City, USA

2

UT Southwestern Medical Center, Dallas, USA

3

Iowa City Veterans Affair Hospital, Iowa City, USA

The prevalence of soldiers suffering from blast-mediated traumatic

brain injury (TBI) has increased due to use of improvised explosive

devices by enemy combatants. TBI results in progressive neuronal

damage associated with chronic cognitive and neurological symp-

toms. Currently, there are no available pharmacotherapeutic inter-

ventions to prevent TBI induced neurological damage. Previously, we

have demonstrated that P7C3-A20, a neuroprotective aminopropyl

carbazole, protects rats from neurological deficit after fluid percussion

injury. We have now evaluated the efficacy of P7C3-243, a more

recently developed analog in the P7C3-series of neuroprotective

compounds, after blast-mediated TBI in mice. When daily adminis-

tration of P7C3-243 is initiated within 36 hr after TBI, mice are

protected from deficits in the hippocampal-dependent Barnes maze

task of learning and memory. This is associated with similar protec-

tion in electrophysiologic measures of synaptic transmission in the

hippocampus (long term potentiation and paired pulse facilitation).

We further reveal that blast-mediated TBI precipitates axonal de-

generation in the absence of frank neuronal cell loss, and that P7C3-

243 blocks this axonal loss. Our hope is that the chemical scaffold

represented by P7C3-243 will provide a basis for developing new

pharmacologic agents for patients with TBI.

Key word

therapeutic

C3-01

DELAYED MICROBLEEDS AND WHITE MATTER DAMAGE

AFTER EXPERIMENTAL TRAUMATIC BRAIN INJURY

Glushakova, O.Y.

, Johnson, D., Hayes, R.L.

Banyan Biomarkers, Inc., Alachua, USA

This study evaluates microvascular abnormalities observed at acute

and chronic stages following TBI in rats and examines pathological

processes associated with these abnormalities. TBI in adult rats was

induced by controlled cortical impact (CCI) of two magnitudes. Brain

pathology was assessed in white matter of the corpus callosum for 24

h to 3 months following injury using immunohistochemistry (IHC).

TBI resulted in focal microbleeds that were related to the magnitude

of injury. At the lower magnitude of injury, microbleeds gradually

increased over the 3 month duration of the study. IHC revealed TBI-

induced focal abnormalities including brain barrier (BBB) damage

(IgG), endothelial damage [Intercellular Adhesion Molecule 1

(ICAM-1)], activation of reactive microglia [Ionized calcium binding

adaptor molecule 1 (Iba1)], gliosis [Glial Fibrillary Acidic Protein

(GFAP)] and macrophage mediated inflammation [Cluster of Differ-

entiation 68 (CD68)], all showing different temporal profiles. At

chronic stages (up to 3 months), apparent myelin loss (Luxol fast blue)

and scattered deposition of microbleeds were observed. Microbleeds

were surrounded by glial scars and colocalized with CD68 and IgG

puncta stainings, suggesting that localized BBB breakdown and in-

flammation were associated with vascular damage. Our results indi-

cate that evolving white matter degeneration following experimental

TBI is associated with significantly delayed microvascular damage

and focal microbleeds that are temporally and regionally associated

with development of punctate BBB breakdown and progressive in-

flammatory responses. Increased understanding of mechanisms un-

derlying delayed microvascular damage following TBI could provide

novel insights into chronic pathological responses to TBI and poten-

tial common mechanisms underlying TBI and neurodegenerative

diseases.

Key words

BBB, CD 68, chronic TBI, GFAP, ICAM-1, microbleeds, microglia

C3-02

TRAUMATIC BRAIN INJURY-INDUCED MICRORNAS SUP-

PRESS PROSURVIVAL GENE EXPRESSION

Boone, D.R.

, Weisz, H., Parsley, M., Bolding, I., Prough, D., DeWitt,

D., Hellmich, H.

University of Texas Medical Branch, Department of Anesthesiology,

Galveston, USA

We and others have shown that TBI alters expression of several mi-

croRNAs (miRNAs) – small, non-coding RNAs that negatively reg-

ulate the expression of target genes involved in cell death/survival.

Here we test the hypothesis that miR-15b, a TBI-induced miRNA

suppresses the expression of prosurvival genes in dying neurons.

Adult male Sprague-Dawley rats (350–400 g) were prepared for se-

vere lateral fluid percussion brain injury, their brains removed 24

hours later, and total RNA containing microRNA isolated. We per-

formed quantitative real-time PCR analysis (using TaqMan probes) of

individual miRNAs in total RNA samples isolated from whole hip-

pocampus and from laser capture microdissected samples of Fluoro-

Jade-positive (dying) and Fluoro-Jade-negative (surviving) cells. To

determine if dying neurons had higher levels of miR-15b, we per-

formed

in situ

hybridization experiments using an Alexa 594 antibody

to the digoxigenin-labeled locked nucleic acid (LNA) probe to miR-

15b, and then stained the sections with Fluoro-Jade C. To confirm the

negative regulation of BDNF, a predicted gene target of miR-15b, we

cloned the sequence of the miR-15b binding site in the 3

¢

UTR of

BDNF into the pmirGlo Dual Luciferase reporter vector, and per-

formed the assay using a miR-15b mimic and antagomir. We have

confirmed the up- and down regulation of several miRNAs in both

whole rat hippocampi and individual dying and surviving neurons.

miR-758, miR-379 and miR-181c were confirmed to be down regu-

lated after TBI, and miR-18a and 19a were up-regulated in total RNA

samples from whole hippocampi. We confirmed that dying FJ

+

neu-

rons expressed higher levels of miR-15b than adjacent FJ- surviving

neurons. miR-15b mimics reduced the expression of BDNF (reduced

luciferase activity) and addition of LNA antagomir for miR-15b re-

stored normal levels of luciferase activity, confirming that BDNF

expression is negatively regulated by miR-15b. These studies are

expected to aid in developing miRNA-based therapeutics that can be

used to treat TBI.

Key word

miRNA

C3-03

ADENOSINE KINASE GENE ASSOCIATED WITH POST-

TRAUMATIC EPILEPSY DEVELOPMENT

Diamond, M.L.

1

, Ritter, A.C.

1

, Conley, Y.P.

2

, Boison, D.

7

, Kochanek,

P.M.

3,5

, Jackson, E.K.

4

, Wagner, A.K.

1,5,6

1

Department of PM&R, Pittsburgh,

2

Department of Health Promotion and Development, Pittsburgh,

3

Department of Critical Care Medicine, Pittsburgh,

4

Department of Pharmacology and Chemical Biology, Pittsburgh,

A-89