Advanced Glycation End-Products Sensitize Human Sensory-Like Neuron Cells to Capsaicin-Induced Calcium Influx

Increased collagen derived advanced glycation end products or AGEs are consistently linked to painful diseases. Here we investigated whether the glycation process sensitizes sensory neurons to capsaicin excitation.

Our results show that collagen glycation increases causing flux compared to cells treated with a normal collagen suggesting that sensory-like neurons express functionality TRPV1 channels and that glycation increases capsaicin excitation.

This research demonstrate that mimicking a prognos susceptive environment with glycated collagen leads to upregulation of capsaicin induced calcium influx. Confirming the functionality of TRPV1 channel and suggesting that AGE may sensitize this channel.

Since glycation of collagen occurs with aging and pathological conditions, this approach may serve as a viable tool for investigating new molecular targets for treating various degenerative diseases.

[Narrator] To begin, prepare one milliliter of FLU-08 dye loading solution by adding two microliters of FLU-08 stock solution to 998 microliters of ASE buffer. Pipette 250 microliters of this solution into each plated dish containing sensory-like neuron cells that are previously differentiated and treated with glycated or non-glycated extracellular collagen matrix. Incubate for 30 minutes in a humidified atmosphere of 5% carbon dioxide at 37 degrees Celsius. After 30 minutes, remove the dish from the incubator and keep it at room temperature in the dark for another 30 minutes. For calcium influx imaging, prepare a syringe with 250 microliters of two micromolar capsaicin solution. Connect the syringe to a sterile 23 gauge butterfly scalp vein set, With extreme caution, place the scalp needle into the Petri dish positioned on the confocal microscope stage without touching the bottom. Identify a field containing more than 20 cells and adjust the focus using bright field light. Start live mode with a 488 nanometer laser and adjust the focus and illumination parameters. Start the acquisition recording. After the baseline period of T equals zero to T equals 60 seconds, gently push the syringe plunger to release 250 microliters of capsaicin into the Petri dish. Open the Microscopy software and select Quantification Mode to perform the cell analysis. Analyze fluorescent spike cells before and after the capsaicin stimulus. Create a region of interest, or ROI in each responsive cell, ensuring that the entire cell area is included. Export the data as a CSV file for further analysis. To perform the analysis with Fiji software, open the image files by selecting File, followed by Import, Bio Formats, and then choose the relevant file. Use the magnifying glass tool to zoom in and the freehand selection tool to draw an ROI around the responsive cells. Press the T key to add it to the ROI manager. Then go to the main menu and choose Analyze, select Mean Gray Value, and click Okay. Go to the ROI Manager window and select all ROIs. Select More, followed by Multi Measure. A new window displaying fluorescence intensity measurements across multiple frames will appear. Navigate to File, Save As, and export the results as a CSV file. Now, import the CSV file into the Google sheet. Normalize the intensity value for each time point. Calculate average intensity as F zero for all images captures between zero to 60 seconds. Calculate FT, The average of the maximum fluorescence intensity as observed after the stimulus. Calculate the delta F over F zero for each ROI using the given equation. Now paste the data in the GraphPad Prism software and select Scatterplot to visualize the data. Beta three tubulin expression in undifferentiated and differentiated cells is presented in this figure. From the results, it is evident that differentiated cells exhibited neuron-like morphology with elongated neurites, while undifferentiated cells retained a rounded shape. Differentiated cells also showed an increased expression of TRPV1 compared to undifferentiated cells. Cells treated with glycated collagen exhibited a higher capsaicin induced calcium influx compared to normal collagen treated cells.