OBJECTIVE These studies assessed the potential effects of paraquat (PQ) on the number of dopaminergic neurons in the substantia nigra pars compacta (SNpc) of C57BL/6 mice when treated twice weekly with PQ·Cl2 i.p. for 3 weeks, as assessed using stereology and neuropathology.

CONCLUSIONS

In these studies, mice exposed to PQ showed no effect on the nigrostriatal dopaminergic system based primarily on stereological assessments conducted under a variety of conditions. The results are consistent with those reported by Breckenridge et al. (2013), but in contrast to other investigators (e.g., McCormack et al., 2002) who reported reductions in the number of TH+ neurons in the SNpc. Jiao et al. (2012), who conducted a study with PQ in the same laboratory (St. Jude) using essentially identical methods to those employed here, also reported reductions in the number of TH+ neurons in the SNpc; the basis for the differences obtained between studies conducted in the same laboratory remains unclear.

Twice-Weekly Administration of Paraquat for 3 Weeks Does Not Result in a Loss of Dopaminergic Neurons in the Substantia Nigra of C57BL/6 Mice Richard J. Smeyne 1, Jeffrey C. Wolf 2, Dan Zadory 2, Nicholas C. Sturgess 3, Kim Z. Travis 3, Charles B. Breckenridge 4, Daniel J. Minnema 4, Andrew Cook 3, and Philip A. Botham 3

1 St. Jude Children’s Research Hospital, Memphis, TN, US; 2 EPL, Sterling, VA, US; 3 Syngenta Limited, Bracknell, UK; and 4 Syngenta Crop Protection LLC, Greensboro, NC, US Abstract #2744

Acknowledgements: Neuropathological assessments were performed by Dr. Mark Butt (Tox Path Specialists). Sielken and Associates Consulting Inc. (Bryan, TX) performed the statistical analyses of the stereological data. Melissa Beck, Pragati Coder & Mark Herberth at WIL (Ashland, OH) provided direction for the in-life and necropsy phases of the study.

STUDY VARIABLES

• Source of Male Mice • C57BL/6J (Jackson Labs) • C57BL/6NHsd (Harlan)

• Age at Initiation of Dosing • 9 weeks (~14 weeks at necropsy) • 16 weeks (~20 weeks at necropsy)

• Source of Paraquat Dichloride • Syngenta (anhydrous PQ·Cl2) • Sigma (PQ·Cl2 hydrate)

• Animal Housing Conditions / Facilities • 1 per cage (WIL Laboratories) • 5 per cage (St. Jude CRH)

• Stereological Methods for TH+ Neurons (performed “blinded” to treatment) • 2D stereology: SNpc sections prepared and assessed at St. Jude CRH

[thickness: 10 μm; frequency: every 5th section; staining: TH immunostain and counterstained with Nissl stain, Neutral Red; stereology: standard microscopy (Baquet et al., 2009); disector height: entire depth].

• 3D stereology: SNpc sections prepared and assessed at EPL [thickness: 40 μm; frequency: every 3rd section; staining: TH immunostain (ABC and DAB chromogen); stereology: optical fractionator (Stereo Investigator: Micro-Brightfield); disector height: entire depth].

• Neuropathology (performed “blinded” to treatment) Sections of striatum and/or SNpc were prepared at Neuroscience Associates, Inc. and assessed by the study pathologist (Dr. Mark Butt) using a qualitative grading system (0=normal to 5=severe).

Stain Pathologic Assessment Parameter • amino cupric silver necrosis of neurons / synaptic terminals • GFAP astrocytic responses • IBA-1 microglial responses • TH (tyrosine hydroxylase) dopamine neuronal processes • Caspase 3 apoptotic cell death • TUNEL DNA fragmentation • Thionine nuclear changes

BASIC DOSING REGIMEN

PQ·Cl2 Dose = 10 mg/kgBW/dose i.p. twice weekly for 3 weeks MPTP·HCl Dose = ~16 mg MPTP/kgBW/dose i.p. x 4 doses at 2-hr intervals

+ + + + + + + + + + + + + + + + + + + + + + + + + 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 DAY

PQ

PQ

PQ

PQ

PQ

PQ

In situ Perfusion

Fixation for Stereology

STUDY I. VARIABLES

• Source of Male Mice: C57BL/6J (Jackson Labs) • Age: 9 weeks at initiation of dosing • Source of Paraquat Dichloride: Syngenta (anhydrous PQ·Cl2 ) • Animal Housing: Single housing at WIL Laboratories • Stereology: both 2D (St. Jude CRH) and 3D (EPL) • Neuropathology

STUDY II. VARIBLES

• Source of Male Mice: C57BL/6J (Jackson Labs) • Age: 16 weeks at initiation of dosing • Source of Paraquat Dichloride: Sigma • Animal Housing: 5 mice/cage, St. Jude Children’s Research Hospital • Stereology: 2D (St. Jude Children’s Research Hospital)

STUDY III. VARIBLES

• Source of Male Mice: C57BL/6NHsd (Harlan) • Age: 16 weeks at initiation of dosing • Source of Paraquat Dichloride: Sigma • Animal Housing: 5 mice/cage, St. Jude Children’s Research Hospital • Stereology: 2D (St. Jude Children’s Research Hospital)

MP

TP

In situ Perfusion Fixation for Neuropathology

STUDY DESIGN • Three Treatment Groups: • Control • PQ • MPTP • Three Assessment Subsets / Group:

Subset I: 3D Stereology (at 168 hours after final dose) Subset II: 2D Stereology (at 168 hours after final dose) Subset III: Neuropathology (at 8, 16, 24, 48, 96 & 168 hours after final PQ dose) (at 48 hours after final MPTP dose)

RESULTS - STEREOLOGY • PQ did not produce a decrease in the number of TH+ neurons in the SNpc. • MPTP did result in a decrease in the number of TH+ neurons in the SNpc. • Similar results were obtained with the two different stereological techniques.

RESULTS - NEUROPATHOLOGY

• PQ did not result in any neuropathological findings • MPTP produced neuropathological changes in the SNpc and striatum: • reduced TH immunostaining intensity (decreased dopamine neurons) • increased AmCuAg staining (neuronal degeneration) • increased IBA-1 immunoreactivity (microglial activation) • increased GFAP immunoreactivity (astrocytic activation)

RESULTS - STEREOLOGY • PQ did not produce a decrease in the number of TH+ neurons in the SNpc. • MPTP did result in a decrease in the number of TH+ neurons in the SNpc.

(% change from control)

** p ≤ 0.01

RESULTS - STEREOLOGY • PQ did not produce a decrease in the number of TH+ neurons in the SNpc. • MPTP did result in a decrease in the number of TH+ neurons in the SNpc.

Mean

Sev

erity

Sco

re

Neuropathology Findings (Mean Severity Scores) in the SNpc

Neuropathology findings in the striatum were similar to those in the SNpc (data not shown)

Baquet ZC, et al. (2009) A comparison of model-based (2D) and design-based (3D) stereological methods for estimating cell number in the substantia nigra pars compacta (SNpc) of the C57BL/6J mouse. Neuroscience. 2161:1082-90. Breckenridge CB, et al. (2013) Pharmacokinetic, neurochemical, stereological and neuropathological studies on the potential effects of paraquat in the substantia nigra pars compacta and striatum of male C57BL/6J mice. Neurotoxicol. 37, 1-14. Jiao Y, et al. (2012) Genetic dissection of strain dependent paraquat-induced neurodegeneration in the substantia nigra pars compacta. PLoS ONE, 7, e29447, 1-6. McCormack A, et al. (2002) Environmental risk factors and Parkinson’s disease: selective degeneration of nigral dopaminergic neurons caused by the herbicide PQ. Neurobiol. Dis., 10, 119-127.

(% change from control)

** p ≤ 0.01

(% change from control)

** p ≤ 0.01

(% change from control)

** p ≤ 0.01