Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

Summary

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

Abstract

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) ^1,2, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)³, the intermediolateral nucleus of the spinal cord ⁴ and the substantia nigra, providing the basis for the neuropathological staging of the disease^4,5. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure^6-8. Here we show in detail two methods used in our previous study ⁹. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I ¹⁰. It is highly lipophylic and poorly absorbed in the gastrointestinal tract ¹¹. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.

Protocol

1) Intragastric Administration of Rotenone

1. Weigh the animal.
2. Calculate the total volume of rotenone/vehicle solution needed. In this case 0.01 ml per gram of the animal weight.
3. Charge a 1 mL syringe with the previously calculated volume of rotenone solution (0.625 mg/mL rotenone, 4% carboxymethylcellulose and 1.25% chloroform) or vehicle solution (4% carboxymethylcellulose and 1.25% chloroform). In this case it corresponds to a 5 mg/kg rotenone dose.
4. Pick up the mouse and, holding the mouse's tail with one hand, stretch the skin of the neck by pulling it with the first two fingers of the other hand.
5. Once the head is....

Discussion

Intragastric administration has been previously performed ¹². However, according to Inden's manuscript, rotenone was administered dissolved in 0.5% carboxymethylcellulose sodium salt (CMSS) alone. Rotenone is a high liphophylic substance. Therefore, rotenone cannot be dissolved in 0.5% CMSS alone and will precipitate if done so. The use of chloroform creates an evenly distributed rotenone suspension avoiding precipitation. Moreover, due to the higher concentrations of the pesticide in the CMSS, the final volu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Rotenone	Sigma-Aldrich	R8875	Germany
Chloroform	Carl Roth GmbH	3313.1	Germany
Carboxymethylcellulose	Sigma-Aldrich	C5678	Germany
Gavage 1,2 mm x 60 mm	Unimed		Switzerland
LSM 510	Carl Zeiss, Inc.
FIJI	http://pacific.mpi-cbg.de