Institute of Anatomy, Technische Universität Dresden
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Pan-Montojo, F. J., Funk, R. H. Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System. J. Vis. Exp. (44), e2123, doi:10.3791/2123 (2010).
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 disease4,5. 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 exposure6-8. 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.
1) Intragastric Administration of Rotenone
2) Immunostaining Procedures
3) Alpha-synuclein Inclusion Image Analysis
It is recommended that an external person unaware of images' origin performs the analysis. Therefore, the data should be codified. Image analysis was performed using FIJI software.
4) Representative Results
When gavage administration protocol is performed correctly the inconveniences for the animal are minimal. Treatment using this concentration of rotenone enables a local effect of rotenone on the ENS without rotenone levels in blood and no inhibition of muscle or brain mitochondrial Complex I even after 1.5 months of treatment (see Figure 1). If image analysis is performed correctly a rigorous analysis of alpha-synuclein pattern should be possible. It gives reliable data on the total amount of alpha-synuclein (total surface), alpha-synuclein pattern in the cell (number of inclusions) and the presence of alpha-synuclein inclusions (inclusion size) (Figure 2).
Figure 1. Motor dysfunction in rotenone treated mice without detection of rotenone in blood or CNS. A, standard (50 ng/mL) and chromatogram from brain samples of 20, 10 and 5 mg/kg treated mice. B and C, quantification of rotenone levels in blood (B) and CNS (C). B, blood levels 1, 2 and 3 hours after treatment. Mice were divided in three groups (n=3) and treated with 2.5, 5, 10 and 20 mg/kg rotenone (n=3), 300 μL of blood was extracted 1, 2 and 3 hours after rotenone administration and pooled together for HPLC analysis. C, mice were treated for one week once a day with 5 (n=3), 10 (n=3) and 20 (n=1) mg/kg rotenone, brain and brainstem were extracted 1 and 2 hours after last administration and prepared for HPLC analysis. D, mitochondrial Complex I activity in muscle and brain samples of 1.5 month treated mice.
Figure 2. Locally administered rotenone induces alpha-synuclein phosphorylation, accumulation and aggregation with gliosis in ENS ganglia. (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 in C, 3 months treatment induced formation of larger alpha-synuclein inclusions (ø>6 μm). D, immunofluorescence staining using anti-alpha-synuclein, Thioflavine S and DAPI. Arrow in D, only 3 month treated mice showed aggregation of these larger alpha-synuclein accumulations. E, E', E'', image analysis quantification steps. E, example of image used in quantification showing an ENS ganglion immunostained against alpha synuclein showing different inclusions. E', example of image obtained after image analysis protocol. E'', overlap between some of the original inclusions (green) and the representation obtained after performing the protocol (yellow lines). F-G, quantification of the experiment shown in A-C was made using automatic segmentation and entropy-based thresholding methods. Single-asterisk, P<0.05, and double-asterisk, P<0.01. F, each column represents total alpha-synuclein surface/ganglion surface. G, each column represents total number of alpha-synuclein inclusions/ganglion surface. All graphs show mean ± s.e.m.
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 volume of administered solution is also significantly decreased. Thus, reducing complications in the administration.
We recommend the use of straight gavages. The esophagus of the mouse is straight, ending directly on one side of the stomach. Therefore, straight gavages are more adequate for this task.
Image analysis of confocal images was done using FIJI software. The plug-ins for this software can be downloaded online. The immunofluorescent staining for image analysis was performed as previously described 9. Images were acquired using a LSM 510 Zeiss Confocal Microscope. It is important that images are acquired using the same signal/noise ratio. This can be achieved by adjusting gain and offset before acquisition.
Francisco Pan-Montojo has a patent application pending for this animal model (Application number PCT/EP 2009/005688).
The Pedro Barrie de la Maza Foundation supported this work.
|Chloroform||Carl Roth GmbH||3313.1||Germany|
|Gavage 1,2 mm x 60 mm||Unimed||Switzerland|
|LSM 510||Carl Zeiss, Inc.|