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

The symptoms of Parkinson's disease, including motor and speech dysfunction, result from reduced activity of dopamine secreting cells in the brain. The enteric nervous system and the olfactory bulb are the most exposed nervous structures and the first ones to be affected. Parkinson's disease has been associated with pesticide exposure.

In order to analyze the effects of pesticides on the enteric nervous system, the pesticide roone is administered to mice by oral gavage. The mice are then intracardiac perfused with 4%paraform, aldehyde brain and enteric nervous system. Tissue is extracted, prepared and immunostain for the PD associated proteins.

Beta three tubulin and alpha synuclein images acquired by confocal microscopy demonstrate the accumulation of alpha synuclein inside enteric nervous system neurons in pesticide exposed animals. The main advantage of this technique over existing methods like systemic administration of rotenone, is that using this technique, it is ensured that the effect of rotenone is confined to the enteric nerve system. This method can help answer key questions in Parkinson's disease, such as its etiology and mechanisms underlying its progression.

The implications of this technique extends tower therapy and diagnosis of Parkinson's disease because they will allow the development of new therapeutic targets and diagnostic methods. Though this method can provide insight into Parkinson's disease pathophysiology, it can also be applied to other systems such as the study of the effect of other environmental toxins on the enteric, sympathetic and parasympathetic nervous systems as well as the intestinal wall. Generally, individuals new to this methods will struggle because it takes a while to master the gavage.

I first had the idea of these methods when I read about the linkage between pesticides and Parkinson's disease. It was also helpful to see high CORAs conference at the World Parkinson Congress in Berlin in 2005. Peaceful demonstration of this method is necessary.

The method to administrate ol with the gavage is difficult as mice have got a different anatomy from humans on the other side, image analysis was done with customized steps for these images. Begin the protocol by weighing the animal. Calculate the appropriate volume of rotenone.

0.01 milliliters is needed per gram of the animal weight, which corresponds to a five milligram per kilogram roone dose. Charge a one milliliter syringe with the roone solution, then charge a second syringe with vehicle solution. 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. Once the head is fixed between the two fingers, lean it against the palm of the hand and turn it upside down. Fix the tail.

Using the fourth and fifth fingers, keeping the head towards the back, press the palate to open the mouse's mouth. Gently introduce the gavage into the mouth. Slowly move the gavage in the direction of the stomach until its full length is inserted.

In order to avoid material getting in the lungs, administer the roone solution or vehicle by gently pressing the embolus. When completed, slowly extract the gavage, place the animal back in another cage until all the animals from one cage are done. Following the treatment mice are intracardiac profused with 4%paraform aldehyde.

In PBS, the guts are then extracted by dissection and placed in 15%sucrose overnight. The next day, the tissue is transferred to 30%sucrose and incubated again overnight at four degrees Celsius. The tissue is then placed in a minus 80 degree Celsius freezer and stored until use Using a cryostat 20 micron intestine.

Cross sections are transferred to rost slides, blocking and immunostaining procedures. Using anti beta three tubulin and Antifa synuclein antibodies are then performed. It is recommended that an external person perform a blind analysis of the data.

To begin the analysis, open Fiji software. Open the confocal image file. Then split the channels so that as possible to work on each channel independently.

Close those channels that are not going to be used and select, analyze, set, scale, and set the pixel size to one. Set the known distance to 0.13 and click on global. The size of the image will now be shown in microns.

Select alpha synuclein channel and select ganglion. Making sure that the selection encloses the ganglion in all slices. Duplicate the image with the ganglion selected only an image the size of the ganglion will be duplicated.

Press edit clear outside the part of the image outside the selected region will become black. Duplicate the image again to determine how well the procedure went. Images that have a low signal to background noise ratio should not be used for image analysis.

Select image adjust threshold. Then select max entropy. Then move over the slices in the cropped image and the original one to ensure that the procedure went well.

The tropic threshold selects an intensity signal level based on the image histogram, and only those pixels with intensities higher than this threshold will be displayed in a white over black image select process. Then click on smooth. This step transforms the pixelated edges of the images into smooth edges select process.

Then click on binary and choose Make binary. The image will be displayed as a black over white image duplicate image to perform the next step on the duplicated image, click on analyze, analyze particles. Then select show outlines and click on summarize.

The rest of the setting should be left in default. This function recognizes the total number of pixels with a signal and those that are grouped together the total surface of pixels with signal. Plus the number of groups of pixels and the mean size are reported.

Compare and select the slice with the largest total area that adjusts well. The slice will be marked. Copy the data table with the total alpha synuclein surface the number of inclusions and the mean particle size.

Paste the data into an Excel spreadsheet or annotated elsewhere. Return to Fiji. Find the previous selected slice in the beta tubulin channel and select it.

Select ganglion area and click on analyze. Then select measure. Another table will appear showing the total surface of the ganglion.

Extract the data from the table to an Excel spreadsheet near the alpha synuclein measurement or copy it down elsewhere using Excel. Divide the different parameters obtained in the alpha synuclein measurement by the ganglion surface to obtain total alpha-synuclein surface and inclusion number normalized by ganglion size in order to analyze the effects of roone acting locally on the enteric nervous system, five milligram per kilogram of the pesticide rot known was administered by oral gavage to 1-year-old C 57 black six mice. The chromatogram shown here shows the presence of a rotan known peak one hour after administration controls mice treated with 20 milligrams per kilogram of rotenone 10 and five milligrams per kilogram of rotenone.

Rotenone levels were determined for blood and brain and brainstem extractions by HPLC as shown here. Administration of five milligrams per kilogram of rotten by oral gavage does not result in measurable levels in the blood one, two, or three hours following treatment as determined by H plc. This graphic shows rot levels in brain and brainstem of treated mice.

One hour after extraction administration of 10 milligrams per kilogram or less of rotenone shows, no roton levels in brain or brainstem samples. One hour after administration mitochondrial complex, one activity was assessed in muscle and brain samples of 1.5 month treated mice. There is no significant difference between groups showing no inhibition of mitochondrial complex one, thus confirming that there was no rone in these regions.

Analysis of the Parkinson's disease, Lewy body component alpha synuclein following administration of rone allows for reliable determination of the total surface amount of alpha synuclein, the presence of alpha synuclein inclusions, and the alpha synuclein pattern in the cell. This figure shows anti beta three tubulin, alpha synuclein, and DAPI staining in duodenum and ileum sections as can be seen here. 1.5 month treatment with roach known induced an increased alpha synuclein punctate pattern inside enteric nervous system ganglia, even one compared to three months controls.

This pattern can be seen in both ileum and duodenum. Following three months treatment formation of larger alpha synuclein inclusions can be seen immunofluorescent staining using anti alphas synuclein, theof, flavin, and DPI demonstrates aggregation of larger alpha synuclein accumulations only at three months. This experiment was analyzed using automatic segmentation and entropy based thresholding methods.

In the graph on the left, each column represents total alpha synuclein surface to ganglion surface. In the graph on the right, each column represents the total number of alpha synuclein inclusions per ganglion surface taken. Together, these data suggest that local administration of rotenone induces alpha synuclein phosphorylation, accumulation, and aggregation with gliosis in enteric nervous system ganglia Once mastered, this technique can be done in one minute per animal if performed correctly.

While attempting this procedure, it's important to introduce the gavage gently, and if any obstacles are found, it is better to get it out and introduce it again following these procedures. Other methods like HPLC, immuno stain and image analysis can be performed. This will enable to analyze other regions, the effects of the substance, or even the presence of the substance in the blood after its development.

This technique paved the way for neuroscientists to investigate the effects of environmental neurotoxins on the enteric nervous system. After watching this video, you should be able to apply the gage correctly and analyze the images that come from the immunochemistry. Don't forget that working with rotten can be extremely hazardous.

Therefore, we recommend the use of gloves and masks while performing this procedure.The.