This protocol can be used to build 3D models of metabolite and parasite distribution. This can help in understanding the relationship between local tissue metabolic perturbation, parasite distribution, and clinical disease symptoms. The main advantage of this method is that it enables live scale cross-organ mapping of metabolite distribution versus pathogen distribution and its statistical analysis.
Theoretically, chemical cartography can explore the development and impact of the disease on the host by directly examining the metabolites responsible for the pathogenesis and the effects of treatment. Begin by weighing and labeling the tubes. Section the tissues systematically with one section per tube.
Weigh the tubes containing tissue samples to determine sample weight and record the weight. To perform water-based homogenization of the tissue samples, add one five millimeter stainless steel bead to the two milliliter microcentrifuge tubes containing tissue samples. Make one blank tube containing LCMS grade water.
Make up the volume to 500 microliters per 50 milligrams of the sample by adding chilled LCMS grade water, then homogenize samples at 25 hertz for three minutes. Samples should be thoroughly homogenized. Collect 1/10th of the homogenization volume for DNA extraction and other analyses.
Add ice cold LCMS grade methanol spiked with four micromolar of sulfachloropyridazine to the homogenate. Homogenize samples in a tissue homogenizer at 25 hertz for three minutes, followed by centrifugation for 10 minutes. Collect an equal volume of supernatant into a 96 well plate and keep solid residue on ice while collecting the supernatants.
Dry the aqueous extraction supernatant completely using maximum speed and no heating. Perform organic extraction by adding 1, 000 microliters of pre-chilled dichloromethane methanol spiked with two micromolar sulfachloropyridazine per 50 milligram of solid residue sample. Homogenize samples at 25 hertz for five minutes, followed by centrifugation for 10 minutes.
Collect an equal volume of supernatant into a 96 well plate. Take a picture of the organ of interest. Click on File, then Import in the SketchUp software to import a picture of the organs of interest.
Click on the lines tool and select the freehand option. Use the pencil tool to trace and draw the outlines of the organs of interest. Select the push/pull tool and pull up on the shaded area to convert the drawing from 2D to 3D.
Export the file in dae format by clicking on File Export and then 3D model. To improve the model's realism, import the model into MeshLab software by selecting File, then clicking on Import Mesh. Select Wireframe on the top menu, then select Filters, click on Remeshing Simplification and Reconstruction, and then on Subdivision Surfaces Midpoint.
Leave all values as default and select Apply twice. Export the model in sdl format by clicking on File, then on Export Mesh As, and then selecting SDL File Format in the files of type dropdown menu. Save this and select OK in the next popup menu.
Open the Meshmixer software and import the sdl file generated at the previous step. Use the sculpt, brushes, dragon sculpt, brushes, inflate tools to pull out the model surfaces that need to be rounded out. Save it in sdl format once the model has the desired appearance by clicking on File and Export.
Name the file as desired, select the SDL Binary Format and save. Open the 3D model in the MeshLab software. Click on OK in the post open processing popup window.
Obtain X, Y, and Z coordinates for each sampling spot by selecting the pick points tool and then right-click at regularly spaced intervals across the 3D model surface. Once all the desired coordinates have been selected, click on the topmost Save button in the form popup window. In spreadsheet software, open the pp file generated in the previous step.
Adjust data display by clicking on Data Text to Columns, then Delimited. Click on Next and then select Space and click on Finish. Reformat so that only numerical values remain in the spreadsheet cells by selecting Home Find and Select Replace.
In the find what box, enter Y equal coat and leave the replace box empty. Click on Replace All and then on OK.Repeat for X equal coat and for Z equal coat and for coat slash greater than. In the spreadsheet software table, rows correspond to each position and columns to data.
Paste the appropriate metadata and metabolite feature abundance in the subsequent spreadsheet columns. In the radius column, enter the desired size of the sampling spots to be visualized on the model. Determine the values for the radius empirically and save the file in CSV format.
Navigate to ELI Plot software, select Surface, then drag and drop the created 3D model into the browser window. Drag and drop the created feature table into the same browser window. Use the legend at the bottom right corner to project the desired data column on the 3D model.
Consecutively select each data column to assess the distribution of this metabolite feature on the 3D model. This analysis led to a table of 5, 502 features and their visualization in 3D. This approach enables the visualization of metabolite features in individual animals that are high at the site of high parasite load, metabolites with differential distribution across tissue regions and metabolite features that are found at comparable levels across small and large intestines.
It is important to remember to use only LCMS solvents and to collect all the adjacent tissue and extract the metabolites for all collected samples to avoid gaps in spatial maps. This technique can be used to explore nutrient requirements for tissue colonization by Trypanosomatid parasites to understand disease pathogenesis through comparing 3D metabolite distribution maps upon infection.
