Progression of Parkinson's Disease Pathology Is Reproduced by Intragastric Administration of Rotenone in Mice methods
Aim. Evidence-backed execution summary for Progression of Parkinson's Disease Pathology Is Reproduced by Intragastric Administration of Rotenone in Mice methods from Progression of Parkinson's Disease Pathology Is Reproduced by Intragastric Administration of Rotenone in Mice.
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mouse
Subject model for the experiment.
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Materials and Methods
reagent used in the protocol.
- Use
- One-year old C57BL/6J mice (Janvier, France) were housed at room temperature under a 12-h light/dark cycle. Food and water was provided ad libidum. Mice were divided into four groups (n = 5) and treated 5 days a week for 1.5 and 3 months. A stomach tube was used to administer 0.01 ml/g animal weight of...
Measurement of Rotenone Levels in Plasma and CNS from Mice
reagent used in the protocol.
- Use
- Blood from 8 week old control mice and 8 week old mice treated with 2.5, 5, 10 or 20 mg/kg rotenone for one week (n = 3) was extracted 1, 2 and 3 hours after rotenone administration and pooled in the same tube. Serum samples were obtained by centrifugation at 3000 rpm (Micro 12-24, Hettich Zentrifu...
Effects of Rotenone on the ENS
(scale bars 20 µm). A, B, C, anti βIII-tubulin, alpha-synuclein and DAPI staining in duodenum ( B ) and ileum ( A, C ) sections. Arrow in B, 1.5 months treatment induced an increased alpha-synuclein punctate pattern inside enteric nervous system ganglia when compared to 3 months controls ( A ). Arrow...
- Use
- (scale bars 20 µm). A, B, C, anti βIII-tubulin, alpha-synuclein and DAPI staining in duodenum ( B ) and ileum ( A, C ) sections. Arrow in B, 1.5 months treatment induced an increased alpha-synuclein punctate pattern inside enteric nervous system ganglia when compared to 3 months controls ( A ). Arrow...
Stereological Procedures
Every sixth section was stained against TH or ChAT and alpha-synuclein and used for stereological analysis. The number of TH + /ChAT + neurons in the SN and the DMV was estimated using the Optical Fractionator principle, with StereoInvestigator software (MicroBrightField Inc., Williston, USA) on a Zeiss Axioplan mi...
- Use
- Every sixth section was stained against TH or ChAT and alpha-synuclein and used for stereological analysis. The number of TH + /ChAT + neurons in the SN and the DMV was estimated using the Optical Fractionator principle, with StereoInvestigator software (MicroBrightField Inc., Williston, USA) on a Zeiss Axioplan mi...
Image Analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Immunofluorescence image analysis was made using ImageJ and performed by a third independent researcher, unaware of images origin. Z-stacks from gut sections were used for quantifying the alpha-synuclein droplet size. For each stack, the ganglion region was selected and isolated. Automatic segmentation of alpha-synu...
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Discussion
Our results show that chronic treatment with intragastrically administered rotenone reproduces some of the reported PD pathological features - accumulation and aggregation of alpha-synuclein within ENS, DMV, IML and SN. These alterations occur without detectable levels of the pesticide in the blood or the brain and no inhibition of Complex I activity in muscle or brain even after 1.5 months of treatment. The HPLC method we employed was able to detect as little as 20 nM rotenone, but we were unable to detect rotenone in the blood with 5 mg/kg treatment, or in the CNS with 10 mg/kg treatment, see ), which argues against systemic effect of rotenone being a causative factor for observed changes. Also, the results from other studies using animal models with higher systemic concentrations of rotenone, and the absence of systemic Complex I inhibition speak against this possibility. Ri...
Materials and Methods
One-year old C57BL/6J mice (Janvier, France) were housed at room temperature under a 12-h light/dark cycle. Food and water was provided ad libidum. Mice were divided into four groups (n = 5) and treated 5 days a week for 1.5 and 3 months. A stomach tube was used to administer 0.01 ml/g animal weight of rotenone solution (0.625 mg/ml rotenone (Sigma-Aldrich, Germany), 4% carboxymethylcellulose (Sigma-Aldrich, Germany) and 1.25% chloroform (Carl Roth, Germany)) corresponding to a 5 mg/kg dose. Controls were treated only with the vehicle (4% carboxymethylcellulose and 1.25% chloroform). Blood extraction for High Performance Liquid Chromatography (HPLC) analysis was obtained from the retina plexus using a glass capillary under general anaesthesia (0.01 ml/g of Ketamin i.p.). CNS-tissue was obtained after cervical dislocation. The rotarod test was performed after 1.5 and 3 mon...
Measurement of Rotenone Levels in Plasma and CNS from Mice
Blood from 8 week old control mice and 8 week old mice treated with 2.5, 5, 10 or 20 mg/kg rotenone for one week (n = 3) was extracted 1, 2 and 3 hours after rotenone administration and pooled in the same tube. Serum samples were obtained by centrifugation at 3000 rpm (Micro 12-24, Hettich Zentrifugen, Germany). Serum was analysed using a modified HPLC protocol as previously described. Brain and brainstem from control, 5 (n = 3), 10 (n = 3) and 20 mg/kg (n = 1) rotenone 5 day-treated one-year old animals were collected 1 and 3 hours after the last rotenone administration and processed as already described by others. The detection threshold was determined by adding different concentrations of rotenone to control plasma before extraction. The methods were linear over a concentration range from 10 to 500 ng/ml, the inter-assay preci...
Measurement outputs
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One-year old C57BL/6J mice (Janvier, France) were housed at room temperature under a 12-h light/dark cycle. Food and water was provided ad libidum. Mice were divided into four...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Immunofluorescence image analysis was made using ImageJ and performed by a third independent researcher, unaware of images origin. Z-stacks from gut sections were used for quant...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Every sixth section was stained against TH or ChAT and alpha-synuclein and used for stereological analysis. The number of TH + /ChAT + neurons in the SN and the DMV was estimate...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
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Acquisition
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inferred from protocolPreprocessing / cleaning
(scale bars 20 µm).
from paperScoring or quantification
Quantify the primary readouts for this experiment: One-year old C57BL/6J mice (Janvier, France) were housed at room temperature under a 12-h light/dark cycle. Food and water was provided ad libidum. Mice were divided into four...; Immunofluorescence image analysis was made using ImageJ and performed by a third independent researcher, unaware of images origin. Z-stacks from gut sections were used for quant...; Every sixth section was stained against TH or ChAT and alpha-synuclein and used for stereological analysis. The number of TH + /ChAT + neurons in the SN and the DMV was estimate....
from paperStatistical comparison
(scale bars 20 µm). A, B, C, anti βIII-tubulin, alpha-synuclein and DAPI staining in duodenum ( B ) and ileum ( A, C ) sections. Arrow in B, 1.5 months treatment...; Immunofluorescence image analysis was made using ImageJ and performed by a third independent researcher, unaware of images origin. Z-stacks from gut sections were used for quant...
from paperReporting output
Report representative outputs alongside summary comparisons for One-year old C57BL/6J mice (Janvier, France) were housed at room temperature under a 12-h light/dark cycle. Food and water was provided ad libidum. Mice were divided into four..., Immunofluorescence image analysis was made using ImageJ and performed by a third independent researcher, unaware of images origin. Z-stacks from gut sections were used for quant..., Every sixth section was stained against TH or ChAT and alpha-synuclein and used for stereological analysis. The number of TH + /ChAT + neurons in the SN and the DMV was estimate....
inferred from protocolStructured statistical methods
(scale bars 20 µm). A, B, C, anti βIII-tubulin, alpha-synuclein and DAPI staining in duodenum ( B ) and ileum ( A, C ) sections. Arrow in B, 1.5 months treatment...; Immunofluorescence image analysis was made using ImageJ and performed by a third independent researcher, unaware of images origin. Z-stacks from gut sections were used for quant...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (3)
Our results show that chronic treatment with intragastrically administered rotenone reproduces some of the reported PD pathological features - accumulation and aggregation of alpha-synuclein within ENS, DMV, IML and SN. These alterations occur without detectable levels of the pesticide in the blood or the brain and no inhibition of Complex I activity in muscle or brain even after 1.5 months of treatment. The HPLC method we employed was able to detect as little as 20 nM rotenone, but we were unable to detect rotenone in the blood with 5 mg/kg treatment, or in the CNS with 10 mg/kg treatment, see ), which argues against systemic effect of rotenone being a causative factor for observed changes. Also, the results from other studies using animal models with higher systemic concentrations of rotenone, and the absence of systemic Complex I inhibition speak against this possibility. Richter and colleagues have shown that subcutaneous administration of 2.5-4 mg/kg of rotenone for 30-40 days does not induce striatal degeneration or neuronal loss in the CNS of C57BL/6J mice. On the other hand, Betarbet and colleagues observed striatum degeneration before the SN was affec...
One-year old C57BL/6J mice (Janvier, France) were housed at room temperature under a 12-h light/dark cycle. Food and water was provided ad libidum. Mice were divided into four groups (n = 5) and treated 5 days a week for 1.5 and 3 months. A stomach tube was used to administer 0.01 ml/g animal weight of rotenone solution (0.625 mg/ml rotenone (Sigma-Aldrich, Germany), 4% carboxymethylcellulose (Sigma-Aldrich, Germany) and 1.25% chloroform (Carl Roth, Germany)) corresponding to a 5 mg/kg dose. Controls were treated only with the vehicle (4% carboxymethylcellulose and 1.25% chloroform). Blood extraction for High Performance Liquid Chromatography (HPLC) analysis was obtained from the retina plexus using a glass capillary under general anaesthesia (0.01 ml/g of Ketamin i.p.). CNS-tissue was obtained after cervical dislocation. The rotarod test was performed after 1.5 and 3 month treatment as already described by others. Briefly, mice were place on a rotating rod with a initial speed of 4 rpm. The speed of rotation was gradually increased (0,3 rpm/sec) and the rodent's ability to remain on the rotating rod (time to the first fall) was recorded. The test was repeated thre...
Blood from 8 week old control mice and 8 week old mice treated with 2.5, 5, 10 or 20 mg/kg rotenone for one week (n = 3) was extracted 1, 2 and 3 hours after rotenone administration and pooled in the same tube. Serum samples were obtained by centrifugation at 3000 rpm (Micro 12-24, Hettich Zentrifugen, Germany). Serum was analysed using a modified HPLC protocol as previously described. Brain and brainstem from control, 5 (n = 3), 10 (n = 3) and 20 mg/kg (n = 1) rotenone 5 day-treated one-year old animals were collected 1 and 3 hours after the last rotenone administration and processed as already described by others. The detection threshold was determined by adding different concentrations of rotenone to control plasma before extraction. The methods were linear over a concentration range from 10 to 500 ng/ml, the inter-assay precision expressed as coefficient of variation was <10%. The limit of detection was 10 ng/ml.
Machine-readable layer
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