Dietary Administration of Diquat for 13 Weeks Does Not Result in a Loss of Dopaminergic Neurons in the Substantia Nigra Pars Compacta (SNpc) of C57BL/6J Mice
Dietary Administration of Diquat for 13 Weeks Does Not Result in a Loss of Dopaminergic Neurons in the Substantia Nigra Pars Compacta (SNpc) of C57BL/6J Mice
Therapeutic Club on Tauopathy. Its pathophysiology and therapeutic targets. Interactive session held at All India Institute of Medical Sciences, New Delhi - 110029 on 28th October 2017.
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1. STUDY DESIGN
Five treatment groups:
1. Control
2. Low DQ 12.5 ppm DQ∙Br2 in diet (~1.5 mg DQ ion / mg BW / day)
3. High DQ 62.5 ppm DQ∙Br2 in diet (~7.5 mg DQ ion / mg BW / day)
4. Low MPTP 4 x 10 mg/kg/dose i.p. at 2-hr intervals during week 12
5. High MPTP 4 x 16 mg/kg/dose i.p. at 2-hr intervals during week 12
Three Assessment Subsets / Group:
Subset I: Pathology (after 4, 8 and 13 weeks of DQ exposure)
Subset II: Stereology (after 13 weeks of DQ exposure)
Subset III: Neurochemistry (after 13 weeks of DQ exposure)
STUDY OBJECTIVE
This study assessed the potential effects of diquat (DQ) on the nigrostriatal
dopaminergic system of male and female C57BL/6J mice continuously
exposed to DQ in the diet for 13 weeks. Neuropathology, stereology, and
neurochemistry endpoints were evaluated for effects of DQ by comparison to
control and MPTP.
CONCLUSIONS
Mice exposed to DQ in their diets for up to 13 weeks showed no
evidence of effects on the nigrostriatal dopaminergic system based on
stereological, neuropathological and neurochemical assessments.
MPTP, the positive control, produced changes in all three assessment
endpoints (stereology, neuropathology and neurochemistry).
The achieved DQ dose level in the study (~7.5 mg DQ ion / mg BW / day
at 62.5 ppm) was approximately 15 times the chronic DQ reference dose
of 0.5 mg DQ ion / kg BW / day.
Dietary Administration of Diquat for 13 Weeks Does Not Result in a Loss of Dopaminergic Neurons in the Substantia Nigra pars compacta (SNpc) of C57BL/6J Mice
Daniel J. Minnema 1, Nicholas C. Sturgess 2, Kim Z. Travis 2, Charles B. Breckenridge1, Mark T. Butt 3, Jeffrey C. Wolf 4, Dan Zadory 4, Andrew Cook 2, and Philip A. Botham 2
1 Syngenta Crop Protection LLC, Greensboro, NC, US; 2 Syngenta Limited, Bracknell, UK; 3 Tox Path Specialists, Frederick, MD, US; 4 EPL, Sterling, VA, US Abstract #2745
NEUROPATHOLOGY – SNpc and Striatum
Brains were block-randomly assigned to matrices/slides (25 sections/slide).
A qualitative grading system was used by the pathologist who was blinded to treatment
group during the evaluation.
Neuropathology Results
DQ: Exposure to DQ was not associated with any morphological (neuronal,
astrocytic or microglial) changes in the SNpc or the striatum.
MPTP: The SNpc &/or striata of the MPTP-treated mice displayed:
• reduced TH immunostaining intensity (decreased dopamine neurons)
• increased AmCuAg staining (neuronal degeneration)
• increased IBA-1 immunoreactivity (microglial activation)
• increased GFAP immunoreactivity (astrocytic activation)
Mean severity scores
STRIATUM SNpc
STEREOLOGY – SNpc
Following in situ perfusion fixation, sections of midbrain (SNpc) were
prepared and stained at EPL (Sterling VA).
• Section thickness: 40 μm
• Section frequency: every third section through SNpc region
• Staining: TH immunostain - avidin-biotin complex (ABC) and
diaminobenzidine (DAB) chromogen
• Stereology: optical fractionator [Stereo Investigator (Micro-Brightfield)]
• Disector Height: entire depth of section
• Stereologist: blinded to treatment group
• Statistical Analysis: one-sided two-sample t-test
Stereology Results
DQ: There were no statistically-significant decreases in the
mean number of TH+ (dopamine) neurons in the SNpc after 13
weeks of dietary exposure to DQ∙Br2 at either 12.5 or 62.5 ppm.
MPTP: The mean number of TH+ (dopamine) neurons in the
SNpc was significantly decreased in MPTP-treated mice.
Mean number of TH+ neurons at each depth of SNpc sections
Effect of treatment on mean number of TH+ neurons in SNpc of male mice
** p ≤ 0.01
ACKNOWLEDGEMENTS
Sielken and Associates Consulting Inc. (Bryan, TX) performed the statistical analyses of the
stereological data. Scott Watson and Jim Mathews (RTI International, Research Triangle
Park, NC) provided technical assistance for the striatal neurochemical analysis. Melissa
Beck, Pragati Coder and Mark Herberth (WIL Laboratories, Ashland, OH) provided direction for
the in-life and necropsy phases of the study.
Stain: Diagnosis Grade: Normal (0) Slight (1) Minimal (2) Mild (3) Moderate (4) Severe (5)
AmCuAg: Necrosis No necrotic neurons/ terminals < 1% 1 – 5% 5 – 15% 15 – 40% >40%
TH+: Decreased TH+ neurons
/ synaptic terminals
Densely stained neurons or
synaptic terminals
1 – 20%
↓ staining
20 – 40%
↓ staining
40 – 60%
↓ staining
60 – 80%
↓ staining
80 – 100%
↓ staining
GFAP: Reactive Astrocytosis No reactive astrocytes 1 – 20% 20 – 40% 40 – 60% 60 – 80% 80 – 100%
IBA-1: Reactive Microgliosis No reactive gliosis 1 – 20% 20 – 40% 40 – 60% 60 – 80% 80 – 100%
+ + + + + + + + + + + + + +
0 1 2 3 4 5 6 7 8 9 10 11 12 13 weeks
Pathology
5 mice/sex/group
48 hrs after MPTP
Group 1: Control (basal diet)
Group 2: Low DQ 12.5 ppm DQ·Br2∙H2O
Group 3: High DQ 62.5 ppm DQ·Br2∙H2O
Group 4: Low MPTP
Group 5: High MPTP MPTP ip
4x16mg/kg
MPTP ip
4x10mg/kg
Pathology
5 mice/sex in
Groups 1 – 3
Pathology
5 mice/sex in
Groups 1 – 3
Stereology
20 mice/sex/group
1 week after MPTP
Striatal
Neurochemistry
6 mice/sex/group
1 week after MPTP
NEUROPATHOLOGY – SNpc and Striatum
Following in situ perfusion fixation, sections of striatum and/or SNpc were
stained (or immuno-stained, as appropriate) at Neuroscience Associates, Inc.
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
NEUROCHEMISTRY – STRIATUM
Flash-frozen striatal tissues were analyzed at RTI (RTP, NC). Striatal
concentrations of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC),
and homovanillic acid (HVA) were determined by HPLC coupled with
electrochemical detection. DA turnover was calculated: [DOPAC+HVA] / [DA].
Neurochemistry Results
DQ: There were no DQ-related effects on striatal dopamine concentration,
dopamine metabolite (DOPAC, HVA) concentrations or dopamine turnover.
MPTP: Statistically-significant reductions in mean striatal concentrations
of dopamine and its metabolites (DOPAC, HVA) and a statistically-significant
increase in mean dopamine turnover (relative to controls) was noted for the
mice dosed with MPTP.
Representative photomicrographs of sections of the SNpc
AmCuAg GFAP IBA-1 TH
MPTPControl
MeanSeverityScoreMeanSeverityScore
MeanSeverityScoreMeanSeverityScore
DQ·Br2
AmCuAg+ Terminals Decreased TH AmCuAg+ Neurons Decreased TH
Increased GFAP Increased IBA-1 Increased GFAP Increased IBA-1
WEEK 4 WEEK 8 WEEK 13 WEEK 4 WEEK 8 WEEK 13 WEEK 4 WEEK 8 WEEK 13 WEEK 4 WEEK 8 WEEK 13
WEEK 4 WEEK 8 WEEK 13 WEEK 4 WEEK 8 WEEK 13 WEEK 4 WEEK 8 WEEK 13 WEEK 4 WEEK 8 WEEK 13
** p ≤ 0.01