Division of Neurotoxicology
Merle G. Paule, PhD, ATS Director
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
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% S t r e t c h
De
ad
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lls
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% 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
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% L
DH
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% 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
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Left Right
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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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7 days after exposure
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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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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