NNS 2015 Program & Abstract Book

antioxidant glutathione (GSH). Probenecid can maintain intracellu-

lar GSH stores and potentially increase brain exposure to NAC via

inhibition of drug transporters. We sought to determine whether the

combination resulted in detectable CSF drug concentrations and in

any adverse events.

IRB approved, randomized, double-blind, placebo controlled

Phase I study in children (2–18 y) after severe TBI (GCS

8). In-

clusion criteria: externalized ventricular drain and indwelling vas-

cular catheters to obtain CSF and blood, respectively. After informed

consent patients were randomized to receive probenecid (25 mg/kg

load then 10 mg/kg/dose q6h

11) and NAC (140 mg/kg load then

70mg/kg/dose q4h

17), or placebo of equal volume and timing.

Serum and CSF samples were drawn pre-bolus and at 1, 24, 48, 72,

and 96h after randomization and drug concentrations were measured

(HPLC-MS/MS).

Fourteen patients were enrolled (7/group). Age, initial GCS, gen-

der, and race weren’t different between groups. In the drug treated

group serum NAC concentrations ranged from 19.8

–

13.7 to

16.8

–

8.7

g/ml and CSF NAC concentrations ranged from

214.6

–

239.0 to 467.9

–

695.0 ng/ml (1 to 96h post-bolus; mean

–

SEM). There were no adverse events attributable to drug treatment.

Temperature, blood pressure, intracranial pressure (ICP), and the use

of ICP-directed therapies were not different between groups.

Treatment with NAC and probenecid resulted in detectable con-

centrations of NAC in CSF and was not associated with undesirable

effects after TBI in children. Our data coupled with other clinical

studies support larger trials evaluating pharmacokinetics and out-

come with combination therapy, in addition to evaluating NAC

alone.

Support:

R01NS069247

Keywords: Phase I Clinical Trial, N-acetylcysteine, Probenecid,

Drug Transporters

A4-02

FATTY ACID OXIDATION IS INCREASED SELECTIVELY IN

ASTROCYTES OF INJURED HIPPOCAMPUS AFTER

TRAUMATIC BRAIN INJURY

Jennifer Jernberg

, Caitlyn Bowman

, Michael Wolfgang

, Susanna

Scafidi

Johns Hopkins, Anesthesiology and Critical Care Medicine, Balti-

more, USA

Johns Hopkins, Biological Chemistry, Baltimore, USA

Traumatic brain injury (TBI) is the leading cause of permanent life-

long disability in children and is characterized by deficits in cog-

nition, attention and sensory-motor integration. Impaired oxidative

glucose metabolism following TBI further contributes to cell death.

The role of fatty acid (FA) oxidation after TBI, however, is un-

known. The developing brain utilizes fatty acids for energy and

metabolism, while the adult brain only uses FA under pathologic

conditions. Mitochondrial

-oxidation of fatty acids supports energy

production and metabolic homeostasis, especially during the periods

of fasting and stress. To be oxidized in mitochondria, fatty acids

must be converted to acyl-carnitine esters by transfer of acyl groups

to l-carnitine. This reaction is catalyzed by Carnitine Palmitoyl

Transferases (CPT1a and CPT2), which are located on the outer and

inner mitochondrial membranes respectively. Only then are acyl-

carnitines transported into mitochondria to support oxidative me-

tabolism. This study is the first to examine fatty acid oxidation after

TBI.

Postnatal day 21–22 male rats were anesthetized with isoflurane

and TBI was administered using a controlled cortical impact to the

left parietal cortex. Fatty acid oxidation in the cortex and hippo-

campus was measured using [1-14C] oleic acid, and oxidation was

increased after TBI in the ipsilateral hippocampus but not in the

cortex. The concentration of carnitines in both the cortex and hip-

pocampus was unchanged after TBI. Using immunofluorescence, we

determined that astrocytes are the only cells expressing CPT1a and

CPT2, thus the only cells able to use fatty acids for energy and

metabolism. There were no significant differences in the amount of

CPT1a and CPT2, quantified via western blotting, at 6 and 24 hrs

after TBI. This study provides strong evidence that astrocytes se-

lectively utilize fatty acids for energy and metabolism and this early

astrocytic support leads to improved recovery after TBI in the de-

veloping brain.

Keywords: Metabolism

A4-03

20-HETE INHIBITION IMPROVES OUTCOME IN A PEDI-

ATRIC RAT MODEL OF TRAUMATIC BRAIN INJURY

Courtney Robertson

, Shiyu Shu, Manda Saraswati, Dawn Spicer,

Zhi Zhang, Xiaoguang Liu, Sujatha Kannan, Raymond C. Koehler

Johns Hopkins School of Medicine, Dept. of Anesthesiology & Critical

Care Medicine, Baltimore, USA

Previous work has shown that inhibition of 20-hydro-

xyeicosatetraenoic acid (20-HETE) formation by cytochrome P450

(CYP) omega-hydoxylation of arachidonic acid can protect imma-

ture and mature brain from ischemia. We tested the hypothesis that

post-treatment with the 20-HETE synthesis inhibitor N-hydroxy-N-

4-butyl-2-methylphenylformamidine (HET0016) can protect the

immature brain from traumatic brain injury (TBI). Male Sprague

Dawley rats (postnatal day 9–10) were subjected to controlled cor-

tical impact (CCI; 3 mm impactor; velocity 5.5 m/s; depth 1.5 mm),

and studied in 3 groups: 1) sham-operated, 2) vehicle-treated TBI,

and 3) HET0016-treated TBI (1 mg/kg, ip, at 5 min and 3 h post-

injury). Lesion volume and microglia morphology (Neurolucida)

were measured. Expression of inflammatory factors (Real-time PCR

of TNFalpha, IL-1beta, IL-4, IL-10), microglia activation (CD68/

Iba-1), neuron loss (NeuN/DAPI), and astrocyte activation (GFAP)

were evaluated. Neurologic testing (foot fault, novel object recog-

nition) was performed at 30 and 90 days. Lesion volumes in the

vehicle-treated TBI group were 12.9

–

1.9, 15.4

–

4.9 and

18.2

–

0.8% of hemisphere at 3, 7 and 30 days. HET0016-treated TBI

groups had significantly reduced lesion volumes (6.2

–

1.9, 5.5

–

1.3

and 8.8

–

0.9%). HET0016 treatment significantly increased number

and length of processes of microglia (3d, 30d), and significantly

decreased microglia cell body area (7d, 30). Peri-lesion gene ex-

pression of pro-inflammatory cytokines (TNFalpha, IL-1beta) was

lower at 1d, and reparative cytokines (IL-4, IL-10) expression was

higher at 3d with HET0016-treatment. HET0016 decreased the

number of CD68-positive microglia in peri-lesion cortex, neuronal

loss in ipsilateral thalamus, and GFAP intensity of astrocyte staining

around the contralateral hippocampus and cortex. Contralateral hind

limb foot faults were reduced, and discrimination index for explor-

ing a novel object was improved at 30 days post-injury with

HET0016. In conclusion, HET0016 reduced lesion volume and im-

proved neurologic outcome after TBI in immature rats. The potential

protective mechanism may be related to 20-HETE-induced pro-

inflammatory state of microglia, as evident by HET0016 attenuation of

pro-inflammatory cytokines, later augmentation of reparative cytokines,

and an accelerated recovery of microglia to a ramified state.

Keywords: 20-hydroxyeicosatetraenoic acid, arachidonic acid,

microglia

A-27