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Protocol Steps
View Abstract
In patients with Parkinson's disease (PD), the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS), the dorsal motor nucleus of the vagus (DMV), the intermediolateral nucleus of the spinal cord and the substantia nigra, providing the basis for the neuropathological staging of the disease. Here we report that intragastrically administered rotenone, a commonly used pesticide that inhibits Complex I of the mitochondrial respiratory chain, is able to reproduce PD pathological staging as found in patients. Our results show that low doses of chronically and intragastrically administered rotenone induce alpha-synuclein accumulation in all the above-mentioned nervous system structures of wild-type mice. Moreover, we also observed inflammation and alpha-synuclein phosphorylation in the ENS and DMV. HPLC analysis showed no rotenone levels in the systemic blood or the central nervous system (detection limit [rotenone]<20 nM) and mitochondrial Complex I measurements showed no systemic Complex I inhibition after 1.5 months of treatment. These alterations are sequential, appearing only in synaptically connected nervous structures, treatment time-dependent and accompanied by inflammatory signs and motor dysfunctions. These results strongly suggest that the local effect of pesticides on the ENS might be sufficient to induce PD-like progression and to reproduce the neuroanatomical and neurochemical features of PD staging. It provides new insight into how environmental factors could trigger PD and suggests a transsynaptic mechanism by which PD might spread throughout the central nervous system.
1
Animal housing and acclimation
One-year old C57BL/6J mice were housed at room temperature under a 12-h light/dark cycle with ad libitum food and water access
Not specifiedRoom temperature
Note: Mice were divided into four groups (n=5)
View evidence from paper
“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)”
2
Treatment administration
Rotenone solution or vehicle control administered via stomach tube at 0.01 ml/g animal weight. Treatment groups received rotenone at 5 mg/kg dose; control groups received vehicle only
5 days per week for 1.5 and 3 monthsNot specified
Note: Rotenone solution composition: 0.625 mg/ml rotenone, 4% carboxymethylcellulose, 1.25% chloroform. Control vehicle: 4% carboxymethylcellulose and 1.25% chloroform
View evidence from paper
“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”
3
Rotarod test initiation
Mice were placed on a rotating rod starting at an initial speed of 4 rpm
Not specifiedNot specified
Note: Test performed after 1.5 and 3 month treatment periods
View evidence from paper
“The rotarod test was performed after 1.5 and 3 month treatment as already described by others [37]. Briefly, mice were place on a rotating rod with a initial speed of 4 rpm”
4
Speed increase protocol
The speed of rotation was gradually increased at a rate of 0.3 rpm/sec throughout the test
Not specifiedNot specified
Note: Speed increase continues until mouse falls from rod
View evidence from paper
“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”
5
Measurement of latency to fall
Time from rod start until the first fall of the mouse was recorded as the primary outcome measure
Not specifiedNot specified
Note: This measurement reflects motor coordination and balance ability
View evidence from paper
“the rodent's ability to remain on the rotating rod (time to the first fall) was recorded”
6
Test repetition schedule
The rotarod test was repeated three times per day on each animal over three consecutive days
Three times daily for three consecutive daysNot specified
Note: Multiple trials allow for assessment of learning and consistency of motor performance
View evidence from paper
“The test was repeated three times a day on each animal over three consecutive days”
7
Blood extraction
Blood was extracted from the retina plexus using a glass capillary under general anesthesia with ketamine for HPLC analysis
Not specifiedNot specified
Note: Performed under general anesthesia (0.01 ml/g ketamine i.p.)
View evidence from paper
“Blood extraction for High Performance Liquid Chromatography (HPLC) analysis was obtained from the retia plexus using a glass capillary under general anaesthesia (0.01 ml/g of Ketamin i.p.)”
8
CNS tissue collection
Central nervous system tissue was obtained after cervical dislocation
Not specifiedNot specified
Note: Performed after blood extraction
View evidence from paper
“CNS-tissue was obtained after cervical dislocation”
Subjects / Specimens
Species
mouse
Strain
C57BL/6J
Age
One-year old
Sex
unknown
Count
20
Weight
Not specified
Mice were divided into four groups (n=5). Housed at room temperature under a 12-h light/dark cycle with ad libitum food and water