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Method Article
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Increased collagen-derived advanced glycation end products (AGEs) are consistently linked to painful diseases. Here, we investigated whether glycation sensitizes sensory neurons to capsaicin excitation.
Increased collagen-derived advanced glycation end products (AGEs) are consistently linked to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. Human sensory-like neurons differentiated from the SH-SY5Y cell line gain pro-nociceptive functions when exposed to AGEs by releasing substance P and upregulating the transient receptor potential vanilloid 1 (TRPV1) expression. Here, we investigated whether this receptor was functionally active and whether the glycation process sensitizes sensory neurons to capsaicin excitation. Sensory-like neuron cells were obtained from the differentiation of SH-SY5Y cells with all-trans-retinoic acid and brain-derived neurotrophic factor. Incubation with glycated collagen extracellular matrix (ECM-GC) simulated a pro-nociceptive stimulus. Control cells were incubated with a non-glycated extracellular collagen matrix (ECM-NC). Fluo-8 Calcium Flux Assay Kit was used to assess calcium influx, which was stimulated by capsaicin. The results show that glycation increases calcium influx compared with cells treated with normal collagen, suggesting that sensory-like neurons express functional TRPV1 channels and that glycation increases capsaicin excitation. These data indicate AGEs hypersensitive sensory-like neuron cells, triggering pro-nociceptive signaling. Together, our results suggest that we established a functional model responsive to capsaicin that can be useful for screening candidates for managing painful conditions.
Glycation is a non-enzymatic, irreversible, and spontaneous process in which proteins, such as collagen, bind to reducing sugar molecules, resulting in advanced glycation end products (AGEs). AGEs may activate cellular membrane receptors, triggering intracellular pathways activation, such as extracellular signal-regulated protein kinase (ERK) 1/2, p38 mitogen-activated protein kinase (MAPK), and c-jun n-terminal kinases (JNKs), rho-GTPases, phosphoinositol-3-kinase (PI3K), Janus kinase/signal transducer and activator of transcription (JAK/STAT), and protein kinase C (PKC), increasing proinflammatory molecules release and oxidative stress1. Glycated collagen also impairs the structure and properties of the extracellular matrix, and increased collagen-derived AGEs are consistently linked to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders2,3.
Our group previously demonstrated that the SH-SY5Y cell line can be differentiated into sensory-like neuron cells since these cells express channels involved in nociception, such as sodium channels (Nav 1.7, Nav 1.8, and Nav 1.9) and transient receptor potential vanilloid type 1 (TRPV1), markers typically found in peripheral sensory neurons4. TRPV1 is a nonselective cation channel permeable to calcium ions and sensitive to capsaicin stimulus. Importantly, when the sensory-like neuron cells are exposed to glycated collagen matrix (ECM-GC), they gain pro-nociceptive functions by increasing c-Fos expression, a transcription factor involved in neuronal activation, and substance P release, a neuropeptide widely involved in neuroinflammation and pain. These cells respond to analgesics, such as morphine, the prototype opiate, decreasing ECM-GC-induced substance P release. Together, these data indicate that this model is responsive to a pro and anti-nociceptive molecule4,5.
Monitoring intracellular Ca2+ concentration changes is essential for studying numerous cellular processes. In neurons, it can be a useful tool to predict neuronal damage and neuroprotective properties of drugs. Capsaicin, the pungent active ingredient of hot chili peppers, is the most studied agonist of the TRPV1 receptor5 and a valuable tool for studying the pain mechanisms and screening potential new analgesics. Previous studies demonstrated that primary sensory neurons from the dorsal root ganglia of rodents incubated with high glucose exhibit a significant increase in capsaicin-induced calcium influx6. However, whether the TRPV1 channel was functionally active in our cell model and whether the glycated collagen sensitizes sensory-like neuron cells to capsaicin excitation, which may activate nociceptive signaling pathways, remain unknown. Therefore, we aimed to develop a cost-effective protocol utilizing simple tools for real-time calcium monitoring in sensory-like cells while ensuring reliable analysis. Here, we provide a comprehensive protocol for helping researchers go through the steps to differentiate SH-SY5Y cells in sensory-like neuron cells and how to sensitize them to pro-nociceptive stimuli. This method can contribute to the discovery of new analgesic or neuroprotective compounds.
1. SH-SY5Y culture and differentiation into sensory-like neuron cells
NOTE 1: All steps present in this section need to be done under a laminar flow hood, and all solutions and supplies need to be sterile.
2. Glycated collagen and glycation process
NOTE: All steps in this section must be done under a laminar flow hood, and all solutions and supplies must be sterile.
3. Calcium influx assay
4. Capsaicin induction
NOTE: Capsaicin, a TRPV1 agonist, was used to induce calcium influx in the cells.
5. Calcium influx imaging and confocal microscopy analysis
NOTE: Imaging was performed in a confocal microscope equipped with a 20x/0.75NA objective and a 488 nm excitation laser (0.5% intensity). Emission was detected at 520 nm. Cells were scanned in xy axes (512 x 512 pixels) over time (t) at a speed of 600 Hz with an acquisition interval of 433 ms and a total acquisition time of 5 min. Imaging was performed at 37 °C to maintain the physiological condition of the cells using the microscopy software.
6. Post-processing/data analysis
Figure 1: Example of ROI in the selected cells for calcium influx analysis in LAS X software. (A) LAS X interface in quantify mode. The pink rectangle shows the quantification tab. The yellow rectangle shows the draw polyline tool, and the cyan rectangle shows the zoom-out and zoom-in tools. (B) Field of view (FOV) captured. (C) Zoom in on FOV to facilitate the drawing of ROI in the entire cell. Please click here to view a larger version of this figure.
Figure 2: Example of ROI in the selected cells for calcium influx analysis in FIJI software. (A) FIJI interface showing the main menu and tool. The red square shows the magnifying glass tool, and the green square shows the Freehand selection. (B) Field of view (FOV) captured with zoom-in and a drawn ROI. (C) The ROI manager window. Please click here to view a larger version of this figure.
7. Troubleshooting
SH-SY5Y cells differentiation into sensory-like neuron
High-content screening images demonstrate that the protocol of neuronal differentiation changes SH-SY5Y cell morphology. The sensory-like neuron cells (differentiated cells) display a rounded cell body that projects an extensive network of neurofilaments. They form branches of more elongated neurite projections connecting surrounding neurons, which is consistent with mature neuron features (Figure 3)
Nociceptors are specialized subsets of sensory neurons that mediate pain. These cells express voltage-gated and ligand ion channels, such as TRPV1, whose activation leads to calcium influx and the release of neuropeptides and neurotransmitters that regulate nociceptive transmission. Here, we describe a protocol for differentiating SH-SY5Y into sensory-like neuron cells to evaluate capsaicin-induced calcium influx8,9. Importantly, we showed that mimicking a pro-no...
MCB, AMCT, and VOZ own a patent on the process of identifying molecular entities involved in osteoarthritis pain (BR102018008561-1).
This work was supported by Fundação Amparo à Pesquisa do Estado de São Paulo FAPESP Grant number 2015/50040-4 and 2020/13139-0, São Paulo Research Foundation and GlaxoSmithKline, FAPESP 2022/08417-7 and 2024/04023-0.
Name | Company | Catalog Number | Comments |
All-trans retinoic acid | Tocris | 695 | |
BDNF | Tocris | TOCR-2837 | |
BDNF | Sigma-Aldrich | B3795 | |
Butterfly type 23GA sterile | Beckton Dickinson Asepto | 38833814 | Scalp vein set |
Capsaicin | Sigma-Aldrich | M2028 | |
D-glucose | Sigma-Aldrich | G5767 | |
DMEM/F12 | Gibco | 12500062 | Basal medium |
D-ribose | Sigma-Aldrich | R7500 | |
D-threose | Sigma-Aldrich | T7392 | |
Fluo-8 Calcium Flux Assay Kit | Abcam | ab112129 | No wash |
Heat-inactivated fetal bovine serum | Gibco | A5670801 | |
High Content Screening | Molecular Devices | ||
LASX software | Leica Microsystems | Microscopy software | |
Leica TCS SP8 | Leica Microsystems | Leica TCS SP8 | Confocal microscope |
Penicillin-streptomycin | Gibco | 15140130 | |
Petri dish (35/10 mm) | Greiner bio-one | 627965 | |
Rat tail type I collagen | Corning | 354236 | |
SH-SY5Y | Merck | 94030304-1VL | Neuroblastoma cell line |
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