Division of Neurotoxicology Merle G. Paule, PhD, ATS Director - PowerPoint PPT Presentation

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

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 2

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

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 5

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 6

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. 7

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 8

Primary Cultured Rat BMECs NCTR ABBOTT, et. al. Division of Neurotoxicology Journal of Cell Science 103

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. 10

High Speed Stretcher (UAF) 0% stretch PDMS “chip” X% stretch PDMS “chip” Mechanical 11 damage

1 0 0 0 ** 8 0 0 D e a d C e l l s ( % o f c o n t r o l ) 6 0 0 * 4 0 0 2 0 0 0 0 5 1 0 1 5 % S t r e t c h % Stretch 0 5 10 15 Mean 100.0 112.8 440.9 650.3 Std. Deviation 108.3 121.5 36.14 288.8 12 Std. Error of Mean 54.14 60.73 18.07 144.4

**** 1 2 0 % L D H r e l e a s e 1 0 0 8 0 6 0 4 0 2 0 0 0 5 1 0 1 5 % S t r e t c h % 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 13

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) – T 2 mapping • Follow-up neuropathology (Neuroscience Associates) – 80 slices/brain – Silver cupric (AgCu) stain 14

List of Compounds Tested Compound route dose frequency dosing Imaging time Experiment duration point(s) duration 3 rd day Kainic acid (KA) IP 12 mg/kg Once 1 day 3 days Domoic acid (DA) IP 2 mg/kg Once 1 day 4 th day 4 days 6 th day Hexachlorophene (HC) Oral 30 mg/kg Daily 5 days 6 days Trimethyltin (TM) IP 12 mg/kg Once 1 day 1,2,3 weeks 3 weeks 8 th day Cytarabine (AC) IP 400 mg/kg Daily 5 days 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 4 th 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

T 2 relaxation as a measure of neurotoxicity – Kainic Acid case Kainic acid induces measurable T 2 changes in the brain 16

Kainic Acid– 2 hrs T 2 before KA T 2 2 hrs after KA CA3 histology 2 hrs after KA Obvious changes in T 2 after KA and challenged architecture in CA3 region 17

Kainic Acid– 2 days Averaged Baseline Actual MRI Statistical Difference 18

Longitudinal T 2 MRI monitoring of hexachlorophene toxicity 30 mg/kg, po, 5 x daily treatment Baseline 3 days 6 days 13 days 20 days T 2 values peaked at 6 days (1 day post-treatment) and faded out to baseline level in 2 weeks 19

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 Dorsal Dorsal Right Left Right Left Ventral Ventral

1 2 CT 1 2 microPET/CT microPET

18 Dynamic Uptake of [ F]-FEPPA (Frontal Cortex ) Dynamic Uptake of FEPPA (Frontal Lobe) A Control 1.8 Sevoflurane 1 day after exposure Sevoflurane+ALC Control+ALC 1.6 1 day after exposure C 1.4 * * * * ^ ^ ^ 1.8 # ^ * * 21 days after exposure * # # SUV # 1.2 * ^ * ^ 1.6 1.0 1.4 0.8 SUV 1.2 0.6 1.0 1.8 B 7 days after exposure 7 days after exposure 0.8 1.6 * * * * * * * * * # ^ # ^ # ^ * ^ ^ # ^ 0.6 1.4 * ^ # ^ # ^ # ^ # * ^ # # 0 20 40 60 80 100 120 140 ^ # # SUV Time (min) 1.2 1.0 0.8 0.6