Progression of Parkinson's Disease Pathology Is Reproduced by Intragastric Administration of Rotenone in Mice methods
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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...
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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.
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