Division of Neurotoxicology

Merle G. Paule, PhD, ATS Director

Merle.Paule@fda.hhs.gov

The views expressed here are those of the author and not necessarily of the U.S. Food and Drug Administration

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Division Staff • Government Positions ― 39 full-time employees (FTE)

– Research Scientists, Staff Fellows & Visiting Scientists : 20 FTE – Support Scientists : 16 FTE – Administrative : 2 FTE – FDA Commissioner’s Fellows: 1 FTE

• ORISE Post Docs, Graduate Students, etc.: 7 staff members • Total = 46 FTE

Division Mission and Research Themes … to develop and validate quantitative biomarkers and identify biological pathways associated with the expression of neurotoxicity….…employing fundamental research efforts in several focal areas designed to broadly examine the involvement of:

• N-methyl-D-aspartic acid (NMDA) and gamma amino butyric acid (GABA) receptor complexes as a mediators of adult and developmental neurotoxicity (general anesthetics; excitotoxins).

• Monoamine neurotransmitter systems as targets for neurotoxicity (drugs of abuse; affective and movement disorders).

• Mitochondrial dysfunction and oxidative stress as mechanisms of neurotoxicity (final common pathways).

• Aß and α-synuclein aggregation in the expression of neurotoxicity (Alzheimer’s Disease and Parkinson’s Disease models).

Approaches/Systems • Cell culture: primary, organotypic and neural stem cells

– Rodent and human (nonhuman primate underdevelopment) – Health and differentiation – Mechanistic studies – Organ-on-a-chip: modeling blood-brain barrier (BBB)-on-a-chip (Traumatic Brain Injury studies)

• Whole animals: zebrafish, rodents, nonhuman primates – Morphology: light and confocal microscopy – Neuropathology: light, fluorescent and confocal microscopy, PET/CT, MRI – Functional

• Nerve conduction • Observational/non-operant behavior • Trained/operant; behavior: cognition, executive functions

• Humans – Translational studies: NCTR Operant Test Battery (OTB) performance as functional biomarker

• Validation studies • Population characteristics: ADHD, Anxiety, Depression • Drug effects • Clinical Collaborations/outreach: Mayo Clinic pediatric anesthetics; Mt. Sinai/Mexico: Pb

exposures

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Outreach Examples • Collaborations

– NCTR Divisions • Systems Biology: MALDI-MS Brain Imaging • Bioinformatics and Biostatistics: Consortium study on biomarkers of

neurotoxicity • Biochemical Toxicology: Arsenic

– FDA Regulatory Centers • CDER: Pediatric anesthetics; magnetic resonance imaging (MRI) and

biomarkers of neurotoxicity; gadolinium retention MRI; Neurotoxicity assessment subcommittee.

• CTP: Behavioral pharmacology of tobacco products.

MALDI-MS = Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

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Outreach Examples • Collaborations (continued)

– Government agencies • ILSI/HESI multi-institute/agency consortium: fluidic biomarkers of

neurotoxicity • DEA/University of Arkansas for Medical Sciences: street drugs of abuse • University of AR at Fayetteville: microphysiological systems development • Coalition Against Major Diseases (CAMD): Alzheimer’s and Parkinson’s

• Global Leadership/Outreach – OECD Adverse Outcome Pathway (AOP) Identification: Developmental

neurotoxicity – ILSI/HESI Developmental and Reproductive Toxicology (DART): Neonatal

Pediatrics

ILSI = International Life Science Institute HESI = Health and Environmental Sciences Institute DEA = Drug Enforcement Agency OECD = Organization for Economic Cooperation and Development

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Three Recent Accomplishments

1. Development of BBB-on-a-chip: TBI modeling. 2. Progress on qualification of MRI T2 images as

biomarkers of neurotoxicity and beyond. 3. Sevoflurane general anesthesia-induced cognitive

deficits in the nonhuman primate model.

BBB = Blood-brain Barrier TBI = Traumatic Brain Injury MRI = Magnetic Resonance Imaging is a dynamic and flexible technology that allows one to tailor the imaging

study to the anatomic part of interest and to the disease process being studied. Strong magnetic pulses perturb the orientation of protons (typically hydrogen atoms) and the instrument records the time it takes for the perturbed protons to return or relax to their pre-perturbed state. Longitudinal relaxation time is referred to as T1 and transverse relaxation time as T2.

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Development of BBB-on-a-chip: TBI modeling Collaboration with the University of Arkansas at Fayetteville

• Isolation of primary Brain Microendothelial Cells (BMECs) • Collect gray matter from fresh rat, cow, or nonhuman primate

cerebral cortices • Mechanical and enzymatic digestion • Several centrifugal separations • Seeded on collagen/fibronectin-coated tissue culture plates

Primary Cultured Rat BMECs

ABBOTT, et. al. Journal of Cell Science 103

NCTR Division of Neurotoxicology

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Hypothesis

• High speed and biaxial stretch mimics the damage induced by TBI in primary cultures and commercially available brain endothelial cells and can be used to study TBI in vitro.

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High Speed Stretcher (UAF) 0% stretch

PDMS “chip”

PDMS “chip”

X% stretch

Mechanical damage

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0 5 1 0 1 5

0

2 0 0

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1 0 0 0

% S t r e t c h

De

ad

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lls

(%

of

co

ntr

ol)

*

**

% Stretch 0 5 10 15 Mean 100.0 112.8 440.9 650.3 Std. Deviation 108.3 121.5 36.14 288.8 Std. Error of Mean 54.14 60.73 18.07 144.4

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0 5 1 0 1 5

02 04 06 0

8 0

1 0 0

1 2 0

% S t r e t c h

% L

DH

re

lea

se

****

% Stretch 0 5 10 15 Mean 100.0 102.5 108.0 119.6 Std. Deviation 6.969 9.817 6.123 5.074 Std. Error of Mean 2.464 3.471 2.165 1.794

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Progress on qualification of MRI T2 images as biomarkers of neurotoxicity and beyond: an

NCTR/CDER Project

• Mature Sprague-Dawley rats • 10 known neurotoxic compounds • In vivo MRI @ 7 tesla (the strength of the MRI magnet)

– T2 mapping • Follow-up neuropathology (Neuroscience Associates)

– 80 slices/brain – Silver cupric (AgCu) stain

List of Compounds Tested Compound route dose frequency dosing

duration Imaging time

point(s) Experiment

duration

Kainic acid (KA) IP 12 mg/kg Once 1 day 3rd day 3 days

Domoic acid (DA) IP 2 mg/kg Once 1 day 4th day 4 days

Hexachlorophene (HC) Oral 30 mg/kg Daily 5 days 6th day 6 days

Trimethyltin (TM) IP 12 mg/kg Once 1 day 1,2,3 weeks 3 weeks

Cytarabine (AC) IP 400 mg/kg Daily 5 days 8th day 8 days

3-Acetylpyridine (AP) IP 30 mg/kg Once 1 day 1,2,3 weeks 3 weeks

Pyrithiamine (PT) IP 0.25 mg/kg Daily 2 weeks 5,6,7,8 weeks 8 weeks

3-Nitropropionic acid (NP) SQ 20 mg/kg Daily 3 days 4th day 4 days

Methamphetamine (MA) IP 5 mg/kg x4 every 2 hrs 6 hrs 2 days 2 days

MK-801 (MK) SQ 1 mg/kg once 4 hrs 1 day 1 day

IP = intraperitoneally SQ = subcutaneously

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T2 relaxation as a measure of neurotoxicity – Kainic Acid case

Kainic acid induces measurable T2 changes in the brain

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Kainic Acid– 2 hrs T2 before KA T2 2 hrs after KA CA3 histology 2 hrs after KA

Obvious changes in T2 after KA and challenged architecture in CA3 region

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Kainic Acid– 2 days

Averaged Baseline Actual MRI Statistical Difference

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Longitudinal T2 MRI monitoring of hexachlorophene toxicity

Baseline 3 days 6 days 13 days 20 days

treatment 30 mg/kg, po, 5 x daily

T2 values peaked at 6 days (1 day post-treatment) and faded out to baseline level in 2 weeks

Hexachlorophene causes transient changes in white matter in rats

Diffusion Tensor Imaging (DTI) Probes the anisotropy of water diffusion pattern in each voxel Probabilistic reconstruction of restricted diffusion pathways (fibers)

Sevoflurane general anesthesia-induced cognitive deficits in the nonhuman primate model

Sevoflurane-induced general anes thesia during development: PET/CT imaging of neural effects and NCTR Operant Test Battery demonstration of long-term cognitive deficits in nonhuman primates (CDER collaboration). PET: Positron Emission Tomography CT: Computerized tomography, a type of x-ray. PET/CT: Combined CT with PET provides the power to better locate the PET signals.

Sevoflurane-Induced General Anesthesia

• PND 5-6 nonhuman primates • 2.5% sevoflurane • 8 hour exposures • Sevoflurane caused significant neuronal

damage: neuronal cell death; glial cell activation

Demonstration of Neuroinflammation

PET marker of glial activation: FEPPA PET/CT imaging post anesthesia to describe time-course.

A B

Left Right

Dorsal

Ventral

Left Right

Dorsal

Ventral

CT

microPET/CT

microPET

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Dynamic 18Uptake of [ F]-FEPPA (Frontal Cortex)

1 day after exposure

7 days after exposure

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Time (min)

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1.8 21 days after exposure

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1.8 ControlSevofluraneSevoflurane+ALCControl+ALC

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7 days after exposure

1 day after exposure

A

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Dynamic Uptake of FEPPA (Frontal Lobe)

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National Center for Toxicological Research Operant Test Battery (OTB) Assessments

• Learning • Motivation • Color and Position Discrimination • Short-term Memory

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 330

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70 Control (n = 6)Ketamine (n = 6)

Blocks of 5 Sessions1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33

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70 Control (n = 8)Sevoflurane (n = 5)

Ketamine IRA Percent Task Completed

Sevoflurane IRA Percent Task Completed

PTC

Sevoflurane-induced general anesthesia during development: long-term cognitive deficits in

nonhuman primates

• A single episode of 8 sevoflurane-induced general anesthesia during a sensitive period of brain growth can cause subsequent cognitive deficits in nonhuman primates.

• These effects appear to be permanent and worsen with age.

• Effects are seen in behaviors thought to reflect aspects of brain function related to IQ (learning; concept formation) and motivation.

• Preliminary data suggest protection by acetyl-l-carnitine.

Help ensure the safety of anesthetics used in infants

and young children, donate today

www.SmartTots.org

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Future Directions • Use of Fluoro-Jade in fresh (unfixed) tissue to

elucidate cell death. • Use of Fluoro-Jade in live tissue culture to

elucidate dying cells. • MALDI-MS imaging of brain slices. • Add larger bore MRI.

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Future Directions: Rare Earth Metal (REM) Chelates

• Euro-Glo: [europium (III), 1, 10-phenanthroline-5-

amine] • Fluorescent: high intensity emission; resistance to

fading; compatible with multiple labeling protocols

• Myelin and amyloid plaques • Paramagnetic …tracer for in vivo MRI?

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Examples illustrating the staining of amyloid plaques with Euro-Glo as well as other specific histochemical markers. Examples of Euro-G staining of plaques is seen at survey magnification in the perirhinal cortex (A) and high magnification in the hippocampus (B) revealing roughly circular flocculent staining as well as the presence of some vesical-like structures (arrows). Comparing amyloid plaque staining with the myelin specific tracer, Black Gold II (D) it is apparent that the nearly black myelinated fibers reach the most distal borders of the plaques, but are not actually incorporated into the plaques. Plaques were stained with Amylo-Glo (C), which generally co-localizes with the anti-A-beta immunohistochemistry. FJ-C (E) staining amyloid plaques (arrows). Periodic Acid Schiff reagent histochemistry (F) labels both myelin (olfactory tract, top of panel) and both cores and peripheries of amyloid plaques.

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Feedback Requested

• How important would you consider the addition of electrophysiological endpoints (EP, NCV, EEG, MEA) to our toolbox?

• Are there emerging sciences/technologies that

you would suggest we pursue?

Division of Neurotoxicology

Syed F. Ali, PhD Melissa Beckmann Zbigniew Binienda, DVM., PhD John F. Bowyer, PhD John J. Chelonis, PhD Elvis Cuevas-Martinez, PhD Sherry A. Ferguson, PhD Timothy Flanigan, PhD C. Matthew Fogle Amy Goodwin, PhD Bobby Gough Qiang Gu, PhD Zhen He, PhD Hector Rosas-Hernandez, PhD Takato Hiranita, PhD Syed Imam, PhD Jyotshnabala Kanungo, PhD Susan Lantz C. Delbert Law Mi Li, PhD Serguei Liachenko, MD, PhD Fang Liu, PhD Shuliang Liu, PhD Waqar Majeed Katelin Matazel Jennifer Naylor, PhD Chinna Orish, PhD Merle G. Paule, PhD Tracy Pearsall James (Bryan) Raymick Bonnie Robinson Natalya Sadovova, PhD Sumit Sarkar, PhD Lawrence C. Schmued, PhD Melody Smith Andrea Sutton, MAP John Talpos, PhD Crystal Thomas David Thorn, PhD Karen Tranter Jennifer Walters, PhD Cheng Wang, MD, PhD Pam Wilson Qi Yin, PhD Xuan Zhang, MD, PhD

3D-Imaging/University of Arkansas for Medical Sciences Marc Berridge, PhD Scott Apana