High frequency low intensity current effects: bio-stimulation and cellular regeneration

Transcription

1 UNIVERSITA DEGLI STUDI DI PADOVA Dipartimento di Anatomia e Fisiologia Umana Department of Human Anatomy and Physiology High frequency low intensity current effects: bio-stimulation and cellular regeneration Department of Human Anatomy and Physiology, University of Padua Telea Electronic Engineering Srl Introduction Electrical currents, electrical fields and electromagnetic fields are themselves kind of energy which can produce important biological effects on cells or on the extra cellular matrix. Apart from generating thermal effects, the flow of current through the biological tissues is able to modify the distribution of charges on the surface of the membrane, inducing modifications of proteins of the membranes. Such effects can force some voltage dependent ionic channels to open or close. At a proper intensity, currents can provoke the appearance of pores on the surface of the membranes (electroporetion), allowing the transfer of relatively large molecules through the membrane. These direct effects can cause subsequently important biological response: think about stimulation with low frequency currents, used to control pain or used in order to improve the trophism and the muscular performance. The biostimulation apparatus developed by Telea Electronic Engineering S.r.l., generates high frequency and low intensity currents so that the thermal effect on the tissues is very modest. This is a new approach, much different from those usually used and because of this of relevant interest. The main intention of the investigations discussed in this paper is a first characterization on biological effects of currents. Objective Our purpose was to investigate the effects on tissues of the high frequency and low intensity currents generated from the apparatus mentioned above. The effects of the currents have been studied on three different levels: on live animals (in vivo), on cellular culture (in vitro) and on a high density proteins suspension. In particular, our attention was focused on verifying if current flow may 1) cause cellular damages able to trigger a cellular regeneration process and 2) cause modification in the structure of wide molecules such as proteins. In vivo experimentation

2 2 In vivo experimentation was made on rats (with the authorization of departmental ethical committee) applying 1 minute stimulation with intensity of 2, based on an arbitrary scale from 1 to 9, with an approximate output power of 6W. Animals, after being shaved and anaesthetized, were stimulated with the electrode applied on the lower part of the abdomen. Another group of animals was not stimulated but kept as a control group. Tissue samples were taken from their abdomen 1, 3 and 7 days after the treatment. Each sample included epidermis, dermis and muscle beneath the treated region. From the observation and the comparison between the acquired images of tissues samples (from the control group and treated group), no significant structural differences imputable to the treatment are visible, both at the skin and at the abdomen muscle. However, relevant differences could be found on the cutaneous muscle, where on the treated animals it is possible to detect scattered fibres and a modest amount parvicellular infiltration. These images are indicative of a regenerative process that could be an evolution of a small damage induced from the current. Figure 1: On the circle parvicellular infiltration is visible on the cutaneous muscle. Coloured by ematoxylin-eosin. A different approach to monitor the muscular degenerative-regenerative processes triggered by currents is represented by the appearance of the isoforms of myosin that are typical in immature cells (neonatal and embryonic myosin). These proteins can be considered as molecular markers of regenerative processes and their presence on adult muscle pointed out a cellular damage occurred some days before. In order to detect these proteins, the stimulation was made with an intensity of 2 (about 6W of power) for 1 minute of treatment on the skin above the anterior tibial muscle of the posterior paw. The counter-lateral paw s muscle, not stimulated, was used for comparison. Tissue samples were taken 1, 3 and 7 days after the treatment. Electrophoretic test of proteins did not point out bands that could be referred to the neonatal and embryonic myosin or a smear due to a wide proteolysis. Via F. Marzolo, Padova Telefono Fax

3 3 Figure 2: Electrophoresis on Polyacrylamid showing isoforms of myosin (molecular weight 200 kd) Figure 3: Electrophoresis on polyacrylamid gel showing all the principal proteins with molecular weight over 10kD. In vitro experimentation on cellular cultures The possible effect on apoptosis and cellular deaths has been verified on myoblastic and fibroblastic culture. Main cultures were made from the posterior paw muscle of newborn rats. The stimulation with high frequency current was carried out 5 days after the cells have been put on culture, and before the culture came to confluence. The stimulation was conducted at different parameters of duration and intensity. Culture cells were fixed at 2 hours, 1 day and 2 days after the stimulation. After the fixation, cultures were coloured Via F. Marzolo, Padova Telefono Fax

4 4 with Hoechst colour. The morphology of the nuclei of the cells has been observed with a fluorescence microscope (wave length of 350nm). In these conditions the apoptotic nuclei can be identified from the condensed chromatin collected on the periphery of the nuclear membrane or from the totally irregular morphology of the nuclear structures while nuclei emitting an intense and homogeneous light are considered normal. Under no circumstances cases of apoptosis were observed. This suggests that high frequency currents have no harmful effect on cell cultures (myoblast and fibroblast). Figure 4: Group of coloured nuclei with Hoechst. A regular aspect can be observed. Experimentation on proteins in solution High frequency current effect has been studied on a solution of albumin at a concentration of 0.5%. Current was applied at different intensity, starting from 1 and up to 8 on a conventional scale of 1-9 (practically from powers of 0W up to about 15W) and duration from 1 to 7 minutes. Notice that these values are higher than those used with in vivo and in vitro cell cultures and tissues. The composition of the proteinic solution has been examined with electrophoretic gel and no degradation of proteins has been observed. Conclusion From the experimental investigations, considered together, follows : 1) We can exclude that high frequency currents (for the intensities and the durations considered) may cause damages to the proteinic structure (see experiment on solution of albumin), and may trigger apoptotic phenomena (see experiments on cellular cultures), and may cause relevant phenomena of degeneration-regeneration at the tissues level (see experiment on isoforms of myosin of anterior tibial). 2) Histological analysis points out that a modest amount of parvicellular infiltration may be observed on the cutaneous muscle of the abdomen suggesting the possibility of a biological response to degenerative localized processes. Such phenomena were not observed nor on the abdomen s muscle nor on the tibial muscle. Via F. Marzolo, Padova Telefono Fax

5 The presence of degenerative-regenerative phenomena on the superficial muscles deserves a comment. These degeneration-regeneration phenomena, which overlapped to the normal cellular regeneration processes of the muscle, but which however do not unsettle its structure (because of their modest intensity) could be an important mechanism of tissues regeneration triggered by high frequency, low intensity currents stimulation. More studies should be addressed to clarify: 1) if similar phenomena occurs also on connective tissues 2) which is the determinant mechanism of the cellular degeneration-regeneration process 5 Professor Carlo Reggiani Responsabile Scientifico della Ricerca Padova 4 aprile 2005 Via F. Marzolo, Padova Telefono Fax