Pulsed radiofrequency for chronic pain: In vitro evidence of an electroporation mediated calcium uptake
Introduction
Pulsed radiofrequency (PRF) treatments consist in the delivery of a train of sinusoidal electrical bursts (5–20 ms length) in the radiofrequency range (500 kHz) at a repetition rate of a few hertz (2–5 Hz). This treatment modality has been applied at various locations along the afferent pain pathways such as directly to the affected nerve [1], adjacent to the dorsal root ganglion [2] or in intra-articular fashion [3] and it proved to be effective in managing pain in patients suffering chronic pain in all cases. Nowadays the most common pathologies where PRF is indicated are radicular pain, trigeminal neuralgia, occipital neuralgia and shoulder and knee pain [4].
PRF originated from conventional thermal radiofrequency as clinical researchers were seeking for a less destructive radiofrequency based treatment to be applied to the afferent nervous pathways [5]. Two decades after its conception and despite multiple evidence of its effectiveness [4], the exact mechanism of action of PRF has not been revealed yet. There is evidence that the analgesic effect of PRF is neither related to thermal effects nor to a permanent physical neural damage [6], [7], [8], [9]. Currently most studies suggest that PRF induced pain relief takes place through a neuromodulatory-type process that alters the synaptic transmission or the excitability of C fibers [10], [11], [12]. These fibers carry pain and temperature sensations and are involved in most neuropathic pain syndromes [13].
Animal studies have exhibited several biological effects of PRF. Some studies have shown morphological changes in the inner structures of axons [9], [14], [15], [16]. Other studies have reported molecular effects such as: alterations of cellular activity [17] and gene expression [18], [19], [20], [21], an increase in the expression of inflammatory proteins [16] and the inhibition of extracellular signal-regulated kinasses [22]. Also, evidence of a long-term depression effect was found in a recent study [23]. These findings have lead to several possible explanations on how PRF inhibits the transmission of pain signals from a biological perspective. However, there has been little progress in elucidating the underlying biophysical mechanisms by which the electric bursts produce these effects.
Interestingly, most of the effects reported after PRF treatments can be triggered by an increase in the cytosolic free calcium (Ca2+) concentration, a second messenger involved in many short and long term cellular processes. This may link PRF effects with a direct effect of the electric fields. We hypothesize that PRF causes a permeabilization of the neural membrane through a mild electroporation process leading to a Ca2+ influx. Given that this hypothetical Ca2+ increase could occur in the same manner in different parts of the neuron, it would explain the fact that PRF can be effectively applied at different regions of the afferent nervous pathways.
Electroporation is a biophysical phenomenon in which cell membrane permeability to ions and molecules is increased when the cell is briefly exposed to high electric fields [24]. This increase in permeability has been attributed to the formation of nanometric pores in the cell membrane, hence the term electro-poration. Electroporation is typically performed by delivering a series of short (100 µs) DC pulses but it also appears when radiofrequency bursts are delivered [25], [26], [27].
In the present study, we have performed a series of in vitro experiments and we have used numerical models in order to investigate the possibility that PRF electric fields cause a Ca2+ influx into neuronal cells and elucidate the pathways of this influx.
Section snippets
Cell culture
Human embryonic kindey cells, HEK-293, were grown in Dubelcco’s modified Eagle’s medium (Gibco, Dublin, Ireland) supplemented with 10% fetal bovine serum (Biosera, Ringmer, East Sussex, UK) and 1% streptomycin/penicillin (Panreac, Barcelona, Spain). Cells were incubated at 37 °C in a humidified environment containing 5% CO2. The day before performing the experiments, the cells were seeded into 4 well NuncTM Lab-TekTM chambered coverslips (Thermofisher, Waltham, MA, USA) and incubated overnight.
Electric burst delivery
Ca2+ uptake occurs at lower electric fields than Yo-pro-1 uptake
First of all we exposed the cells to PRF bursts (500 kHz, 20 ms length) and compared the effects of a single burst with the effects of 120 bursts (delivered at a frequency of 2 Hz) in terms of the free cytosolic Ca2+ concentration and the uptake of Yo-pro-1. Fig. 3a-c show representative examples of the time evolution of the fluorescence in single cells. For convenience, when comparing the different sets of experiments, we will define the E50 as the electric field that has a probability of 0.5
Discussion
Our results show that the PRF bursts can cause an increase in the intracellular Ca2+ at electric fields significantly lower than those required to internalize Yo-Pro-1. The fact that no increase was detected in the absence of extracellular Ca2+ suggests that the observed increase in Ca2+ concentration is due to an uptake from the extracellular medium. One of the characteristic features of PRF treatments in the clinical practice is the large amount of bursts that are delivered and, according to
Conclusion
We showed that PRF bursts can cause a Ca2+ influx. Our results are consistent with the hypothesis that PRF causes a Ca2+ uptake mediated by a “mild” electroporation of the cell membrane. The results of this study may link the biological effects of PRF reported by other authors with a direct action of the electric fields in neurons.
Declaration of Competing Interest
None.
Acknowledgments
This work was supported by the Ministry of Economy and Competitiveness of Spain through the grants TEC2014-52383-C3-2-R and SAF2014-52228-R. Antoni Ivorra gratefully acknowledges the financial support by ICREA under the ICREA Academia programme.
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