Pulsed Radiofrequency Decreases pERK and Affects Intracellular Ca2+ Influx, Cytosolic ATP Level, and Mitochondrial Membrane Potential in the Sensitized Dorsal Root Ganglion Neuron Induced by N-Methyl D-Aspartate

Abstract

Background:The molecular mechanism of pulsed radiofrequency (PRF) in chronic pain management is not fully understood. Chronic pain involves the activation of specific N-Methyl D-Aspartate receptors (NMDAR) to induce central sensitization. This study aims to determine the effect of PRF on central sensitization biomarker phosphorylated extracellular signal-regulated kinase (pERK), Ca 2+ influx, cytosolic ATP level, and mitochondrial membrane potential (Δψm) of the sensitized dorsal root ganglion (DRG) neuron following NMDAR activation. Methods: This study is a true experimental in-vitro study on a sensitized DRG neuron induced with 80 µM NMDA. There are six treatment groups including control, NMDA 80 µM, Ketamine 100 µM, PRF 2Hz, NMDA 80 µM + PRF 2 Hz, and NMDA 80 µM + PRF 2 Hz + ketamine 100 µM. We use PRF 2 Hz 20 ms for 360 seconds. Statistical analysis was performed using the One-Way ANOVA and the Pearson correlation test with α=5%. Results: In the sensitized DRG neuron, there is a significant elevation of pERK. There is a strong correlation between Ca 2+ , cytosolic ATP level, and Δψm with pERK intensity (p<0.05). PRF treatment decreases pERK intensity from 108. 48 ± 16.95 AU to 38.57 ± 5.20 AU (p<0.05). PRF exposure to sensitized neurons also exhibits a Ca 2+ influx, but still lower than in the unexposed neuron. PRF exposure in sensitized neurons has a higher cytosolic ATP level (0.0458 ± 0.0010 mM) than in the unexposed sensitized neuron (0.0198 ± 0.0004 mM) (p<0.05). PRF also decreases Δψm in the sensitized neuron from 109.24 ± 6.43 AU to 33.21 ± 1.769 AU (p<0.05). Conclusion: PRF mechanisms related to DRG neuron sensitization are by decreasing pERK, altering Ca 2+ influx, increasing cytosolic ATP level, and decreasing Δψm which is associated with neuron sensitization following NMDAR activation.

Pulsed Radiofrequency 2 Hz Preserves the Dorsal Root Ganglion Neuron Physiological Ca2+ Influx, Cytosolic ATP Level, Δψm, and pERK Compared to 4 Hz: An Insight on the Safety of Pulsed Radiofrequency in Pain Management [Response to Letter]

“…Our study found that 2Hz 20-millisecond pulsed radiofrequency for 600 seconds significantly decreased the pERK level compared to the control, indicating decreased sensitization. 2 This result shows the scientific evidence that pulsed radiofrequency could directly reduce one of the biological processes of pain generation. However, in clinical practice, the overall outcome of PRF is achieved at two weeks, one month, three months, or six months after the procedure.…”

Pulsed Radiofrequency 2 Hz Preserves the Dorsal Root Ganglion Neuron Physiological Ca2+ Influx, Cytosolic ATP Level, Δψm, and pERK Compared to 4 Hz: An Insight on the Safety of Pulsed Radiofrequency in Pain Management

“…This study uses DRG neurons that are differentiated from F11 cell line (08062601; Sigma, USA). Cell culture method according to Hashemian et al 21 with modifications described in a previous study by Laksono et al 22 We use DRG neurons that differentiated from F11 cell line on day 5. In this study, there were three treatment groups with nine replicates in each group ( Figure 1 ), including the control, 2 Hz PRF, and 4 Hz groups.…”

“…PRF treatment was carried out by transmitting pulsed frequencies of 2 Hz and 20 ms for 360 s (2 Hz group) and 4 Hz and 10 ms for 360 s (4 Hz group). PRF treatment is based on Laksono et al 22 study. Both PRF frequencies delivered the same energy, following the formula derived from Ohm’s law:…”

“…Calcium influx measurement was done according to the Laksono et al 22 study. Calcium influx was marked using Fluo-3/AM (F4897-1MG, Sigma-Aldrich, USA) and observed using a confocal laser scanning microscope (CLSM) in 340 nm and 380 nm.…”