Study type: Medical/biological study (experimental study)

A novel in vitro assay for electrophysiological research on human skin fibroblasts: degenerate electrical waves downregulate collagen I expression in keloid fibroblasts med./bio.

By: Sebastian A, Syed F, McGrouther DA, Colthurst J, Paus R, Bayat A
Published in: Exp Dermatol 2011; 20 (1): 64-68
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Aim of study (acc. to author)

A new in vitro model system was developed for testing the effects of precisely defined, different types of electrical stimulation on the collagen expression of normal and keloid human skin fibroblasts (keloid fibroblasts show excessive collagen production).

Background/further details

Electrical stimulation is used for the treatment of wounds and has been shown to alter gene expression and protein synthesis in skin fibroblasts in vitro.

Endpoint

Exposure

Exposure Parameters
Exposure 1: 10–60 Hz
Exposure duration: continuous for 1, 4, 8 or 12 h
Exposure 2:
Exposure duration: continuous for 1, 4, 8 or 12 h
Exposure 3: 10–60 Hz
Exposure duration: continuous for 1, 4, 8 or 12 h

Exposure 1

Main characteristics
Frequency 10–60 Hz
Type
Waveform
Exposure duration continuous for 1, 4, 8 or 12 h
Additional info AC stimulation
Additional info 10 Hz or 60 Hz applied
Exposure setup
Exposure source
Chamber 44 mm x 13 mm x 11 mm exposure chamber made of a rectangular glass Petri dish covered on the inside with silicone resin; two 10 mm diameter holes for the agar bridges drilled into the glass cover lid of the Petri dish; current conducted into the chamber through Ag-AgCl electrodes inserted in synthetic rubber, agar bridges filled with Steinberg's saline gelled with 1% agar
Setup six 11 mm x 25 mm slides made of microscopic cover slips placed in this chamber
Parameters
Measurand Value Type Method Mass Remarks
electric field strength 200 mV/cm - measured - -
electric field strength 750 mV/cm - measured - -
electric field strength 1,500 mV/cm - measured - -

Exposure 2

Main characteristics
Frequency
Type
Exposure duration continuous for 1, 4, 8 or 12 h
Additional info DC stimulation
Exposure setup
Exposure source
Setup inverted U-shaped agar bridges conducted the current from silver electrode-dipped Steinberg's saline solution into the exposure chamber
Parameters
Measurand Value Type Method Mass Remarks
electric field strength 200 mV/cm - measured - -
electric field strength 750 mV/cm - measured - -
electric field strength 1,500 mV/cm - measured - -

Exposure 3

Main characteristics
Frequency 10–60 Hz
Type
Exposure duration continuous for 1, 4, 8 or 12 h
Additional info degenerate electrical wave stimulation
Additional info 10 Hz or 60 Hz applied
Exposure setup
Exposure source
Parameters
Measurand Value Type Method Mass Remarks
electric field strength 200 mV/cm - measured - -
electric field strength 750 mV/cm - measured - -
electric field strength 1,500 mV/cm - measured - -

Exposed system:

Methods Endpoint/measurement parameters/methodology

Investigated system:
Time of investigation:
  • before exposure
  • after exposure

Main outcome of study (acc. to author)

Apoptosis was increased by DC stimulation (field 2) at higher and lower electric fields when compared to all other wave forms. At 1500 mV/cm and 60 Hz, keloid fibroblasts exposed to degenerate electrical waves stimulation (field 3) underwent higher level of apoptosis than by AC stimulation (field 1). Degenerate electrical waves were the least cytotoxic to normal fibroblasts when compared to all other stimulations.
At lower electric field frequency (10 Hz), all types of electrical stimulation upregulated collagen I gene expression in both cell types compared to controls. However, at higher electric field strength (1500 mV/cm) and frequency (60Hz), degenerate electrical waves maximally downregulated collagen I in keloid fibroblasts.
Additionally, data of ELISA showed a decreased protein expression of collagen I in keloid fibroblasts at 750 mV/cm and 60 Hz.
The data of immunocytochemistry showed for both normal skin and keloid fibroblasts a significant decrease in collagen I expression after 12 h of degenerate electrical waves and AC stimulation compared to unstimulated cells (controls). In contrast, increasing amplitude of DC stimulation upregulated collagen I and PAI-1 gene expression in normal and keloid fibroblasts, along with increased cytotoxicity.
Thus, this new preclinical assay system showed highly differential effects of specific types of electrical stimulation on human fibroblast collagen expression and cytotoxicity and identified degenerate electrical waves as a promising, novel therapeutic strategy for suppressing excessive collagen I formation in keloid disease.

Study character:

Study funded by

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