ORIGINAL RESEARCH PAPER

Comparison of electrically induced flare response patternsin human and pig skin

M. Dusch Æ M. Schley Æ O. Obreja Æ E. Forsch ÆM. Schmelz Æ Roman Rukwied

Received: 22 January 2009 / Revised: 4 March 2009 / Accepted: 4 March 2009 / Published online: 25 March 2009

� Birkhauser Verlag, Basel/Switzerland 2009

Abstract

Objective We compared the characteristics of neurogenic

flare responses in human and pig skin to establish a

translational research animal model.

Material and subjects Eight domestic pigs and six male

subjects were investigated.

Treatment Electrical pulses were delivered transcutane-

ously with increasing current intensities, pulse frequencies

and pulse widths.

Methods Inflammatory skin responses were recorded by

laser Doppler imaging and analyzed by ANOVA and

Fisher’s (LSD) post hoc test.

Results Transcutaneous stimuli of 5 mA onward induced

a significant flare development in humans. In the pig, sig-

nificantly lower currents of 2.5 mA already induced a flare

response. Smaller flare sizes of about 3.5 cm2 were ana-

lyzed. The flare continuously declined despite ongoing

stimulation.

Conclusions Lower excitation thresholds and smaller

receptive fields of nociceptors can be suggested in pigs.

Impaired neuropeptide release, altered vesicle replenish-

ment, different neuropeptide sensitivity, or insufficient

peripheral decoding of action potentials may contribute to

steadily decreasing flare responses. These attributes may be

objectives of pre-clinical anti-hyperalgesic studies and

their accurate analysis in pigs reveals a particularly sensi-

tive translational animal model for nociceptor researches.

Keywords Axon reflex flare �Mechanically insensitive nociceptors � Heat nociceptors �Electrical thresholds

Introduction

Pig skin is closely related to human skin considering

structure, vascularization epidermal enzyme patterns [1],

peptide nerve fiber immunoreactivity (e.g., CGRP, VIP,

substance P) [2], and thus is highly suitable for inflam-

matory research. In addition, pig skin heat sensitive

nociceptors, of which some are also mechanically insen-

sitive, are involved in the progress of the flare development

[3]. It was shown previously in humans that the activation

of heat sensitive nociceptors [4] and mechano-insensitive

C-fibers [5] contribute considerably to the flare reaction.

One characteristic of mechano-insensitive nerve fiber

classes is that they can increase their responsiveness to

external stimuli and thereupon contribute to the develop-

ment and maintenance of hyperalgesia [6]. Thus, focused

investigation and characterization of these nociceptors

helps to understand the mechanisms behind hyperalgesia.

Here, we compare the flare development upon noci-

ceptor activation at matching anatomical sites of human

and pig skin in response to transcutaneously delivered

electrical stimuli. Supra-threshold excitation of skin noci-

ceptors induces the generation of action potentials, which

are conducted retrograde at branching points of the nerve

toward the terminal endings to evoke a depletion of

M. Dusch and M. Schley contributed equally to the manuscript.

Responsible Editor: G. Geisslinger.

M. Dusch � M. Schley � O. Obreja � E. Forsch � M. Schmelz �R. Rukwied (&)

Department of Anaesthesiology and Intensive Care Medicine,

University Medicine Mannheim, Ruprecht-Karls-University

Heidelberg, Theodor-Kutzer-Ufer 1-3,

68167 Mannheim, Germany

e-mail: Roman.Rukwied@medma.uni-heidelberg.de

Inflamm. Res. (2009) 58:639–648

DOI 10.1007/s00011-009-0029-3 Inflammation Research

neuropeptides, particularly calcitonin gene-related peptide

(CGRP), causing widespread vasodilatation (axon reflex

flare) [7, 8]. Spatial extension of this flare response has

been explored with accuracy as objective output measures

of nociceptor activation [9, 10]. In the present study, the

parameters of the administered current intensity, pulse

width and pulse frequency were kept identical in human

and pigs. Electrical thresholds for flare development,

maximum flare sizes, and flare progress over time were

identified to characterize and compare in both species the

excitation of skin nociceptors, i.e., mechano-insensitive

and heat sensitive C-fibers. Their comparison between the

species serves the establishment of the pig as translational

experimental animal model for anti-hyperalgesic research,

even though elevated resources may be needed for their

keeping compared to commonly used laboratory animals.

Materials and methods

The animal protection authorities of the regional council of

Baden-Wurttemberg, Germany, and the central animal

research unit at the University of Heidelberg approved the

study protocol and experimental procedure performed on

pig skin according the European Communities Council

Directive and the Guide for the Care and Use of Laboratory

Animals. The local Ethic Committee of the Medical Fac-

ulty Mannheim, University of Heidelberg, approved the

experimental procedure on human volunteers according to

the Code of Ethics of the World Medical Association,

Declaration of Helsinki and its amendment in Tokyo and

Venice.

Animal preparation

Investigations were performed on eight domestic pigs (Sus

scrofa) supplied by a local breeder (Sommerhof, Dielheim-

Unterhof, Germany). The age of the animals was on

average 12 ± 4 weeks and body weight 20–27 kg.

According to a previous investigation, pig skin at this age

is most similar to human skin [11]. All pigs were used

twice with a time interval of at least 7 days between the

experiments. Prior to the experiments animals were

allowed to familiarize with the new environmental condi-

tion for at least 7 days and fed daily with Muskator EM DE

BW 200060 (MuskatoWerke, Mannheim, Germany) and

water, the latter ad libidum. Six hours prior to the experi-

ment the food was withdrawn.

As described previously [12], pigs were pre-medicated

by injections of 2 mg/kg Stresnil� (Azaperon, Janssen

Pharmaceutica, Beerse, Belgium), 0.05 mg/kg Atropin

(Atropinsulfat-1-Hydrat, Eifelfango, Bad Neuenahr,

Germany) and 0.3 mg/kg Dormicum� (Midazolam, Roche,

Basle, Switzerland) into the trapezius muscle. Following a

sedation period of approximately 20 min animals were

anesthetized with an intravenous infusion of 1.5 mg/kg

Propofol� (Fresenius, Bad Homburg, Germany) into an ear

vein. Subsequently, pigs were intubated by means of

a 4.0 mm ID microlaryngeal tubus (Rusch, Gauting,

Germany) and artificially ventilated at a tidal volume of

2.5–3 l/min with O2 (25–50%), which was adjusted indi-

vidually according to the continuously measured O2

consumption and exhaled CO2 concentration, the latter

kept at 40 mmHg (Narkomat�, Heyer Anesthesia, Bad

Ems, Germany). Following intubation, i.e., approximately

5 min after Propofol� infusion, anesthesia was achieved by

an intravenous infusion of 20 mg/kg Narcoren� (Pento-

barbital-Natrium, Rhone Merieux, Laupheim, Germany)

administered as bolus using a 20G 11/400 infusion cannula

(Vasofix�, B. Braun, Melsungen, Germany) placed into the

saphenous vein of the lower leg. The cannula was con-

nected via a three-way valve to a drip and continuously but

slowly perfused with saline (NaCl 0.9%, Delta Select,

Pfullingen, Germany) using an administration set R 87

RLS Luer-Lock IG-P (Becton Dickinson, Heidelberg,

Germany).

Electrocardiogram, body core temperature, pulse rate

and O2 blood concentration were monitored throughout the

experiment (Lohmeier M211-371, Munchen, Germany).

Body core temperature was maintained constant at

37.2 ± 1.1�C throughout the experiments. This tempera-

ture is lower as compared to unrestrained pig temperature

(38–39�C) but has no effect on peripheral blood flow [13].

Sufficient depth of anesthesia was estimated by heart fre-

quency, physiological reflexes evoked by touching the eye

lid, or spontaneous muscular movements. Additional doses

of pentobarbital were administered on demand through a

three-way valve system.

Volunteers

Six healthy male volunteers (mean age 39 ± 8 years)

participated in the study, after having been informed about

the experimental procedure and given their written

informed consent. Subjects receiving any medication

influencing vascular responses (e.g., anti-histamines, cor-

ticosteroids, etc.) were excluded from the study. All

experiments were performed in a temperature-controlled

environment (23�C).

Electrical stimulation

For transcutaneous electrical stimulation, a pair of self-

adhesive rectangular (3 9 10 mm) surface electrodes

(Pierenkemper, Wetzlar, Germany) was attached to the

dorso-lateral back skin 5 cm lateral to the spine of the

640 M. Dusch et al.

volunteer and the animal, respectively. The dorsum area

was investigated in both pig and human to allow direct

comparisons of flare sizes between the species. A distance

of 3 mm between each electrode was kept constant.

Only in pig skin intradermal electrical currents were

delivered and compared to transcutaneous nerve stimula-

tion in order to characterize the extension of the axon reflex

flare in relation to the depth of the neuronal activation.

Intradermal stimulation was performed by means of a

pair of 27G injection cannulae (outer diameter 0.41 mm,

Neoject�, Dispomed, Gelnhausen, Germany) inserted

intradermally at a distance of 3 mm and a length of 0.5–

1.5 mm into the dorso-lateral back skin of the animal.

Insertion depth of the intradermal electrodes was approxi-

mately 1 mm.

Both, self-adhesive electrodes and injection cannulae

were connected to a constant current stimulator (DS7,

Digitimer, Hertfordshire, UK) delivering rectangular elec-

trical stimuli triggered by an external pulse generator

(Rimkus Pulsgenerator PG1, Parsdorf, Germany). The

current stimulator was equipped with an overload indicator

light to show if the load exceeded its capacity.

Study design

Current intensity, pulse width, and frequency of the

administered current pulses were systematically studied in

randomized order in both species. For each investigated

parameter the other remaining variables of the current were

kept constant throughout the experiments (Table 1). If cur-

rent intensity was explored, the electrical pulse duration and

frequency was kept at 0.5 ms and 1 Hz. If pulse width was

studied, the current intensity and the pulse frequency were

kept at 5 mA and 1 Hz. If pulse frequency was investigated,

the current intensity and pulse width was kept at 5 mA and

1 ms. The influence of the frequency order with which the

current pulses were delivered, i.e., increasing or decreas-

ing frequency, was assessed by administering pulses at

0.1–100 Hz (increasing) or 100–0.1 Hz (decreasing).

In the pig, continuous stimuli were applied for 30 min at

current pulses delivered at 0.5 and 50 Hz, respectively. The

concept of an increasing number of stimuli was investi-

gated also by delivering 75, 150, and 225 pulses at

frequencies of 0.3–100 Hz. Finally, the development of

flare responses was studied upon transcutaneously and

intradermally delivered stimuli at varying current intensi-

ties and pulse widths, respectively (Table 1).

Duration of stimulus administration was 4 min and

increased successively for each parameter with increasing

current intensity, pulse width, or discharge frequency. This

stimulation paradigm was applied to both human and pig,

thus any effect of the previously delivered stimulus on the

analyzed response would affect both species equally.

Current intensity

Intensities of constant current pulses were increased in 4-

min intervals in steps of 2.5 mA and up to a maximum of

15 mA. Current was delivered transcutaneously to both

species, human and pig, at a pulse frequency of 1 Hz and a

pulse duration of 0.5 ms. In pig skin, intradermal current

pulses were delivered in steps of 2.5 mA to a maximum of

15 mA and flare development was compared to those

evoked after transcutaneous current stimuli. For the com-

parisons of intradermal versus transcutaneous stimuli,

pulse widths were set at 0.2 and 0.5 ms, respectively, at a

pulse frequency of 1 Hz.

Pulse width

Duration of electrical pulses was increased at 4-min

intervals from 0.05 to 0.1 to 0.2 to 0.5 to 1 to 2 ms at a

constant current intensity of 5 mA and a pulse frequency of

1 Hz. In pig skin, transcutaneous and intradermal current

delivery was investigated.

Pulse frequency

Transcutaneous electrical stimuli were administered at

pulse frequencies of 0.1–0.2–0.3–0.5–1–5–10–50–100 Hz

at 5 mA current intensity and 1 ms pulse width. Duration

of stimulation was constant at 4 min for each frequency.

Pulses up to 1 Hz were delivered biphasic, whereas

electrical pulses at higher frequencies (5–100 Hz) were

delivered via monophasic bursts. Each burst consisted of

five single current stimuli. The time interval between each

stimulus within the burst determined the pulse frequency

(5–100 Hz). Bursts of electrical pulses were administered

in 2-s intervals and both from low to high frequencies

(0.1–100 Hz) and in reversed order (100–0.1 Hz). The

area under the curve (AUC) of the flare response was

calculated as sum total of all recorded flare areas for each

individual and depicted as mean AUC for the investigated

species.

Continuous stimulation and number of current pulses

assessed in pigs

In three animals continuous constant current pulses were

delivered transcutaneously for 30 min at frequencies of 0.5

and 50 Hz, respectively. The current intensity was set at

5 mA and pulse duration at 1 ms. In addition, the influence

of the number of stimuli on the flare development was

assessed further in three pigs by delivering 75, 150 and 225

pulses at frequencies of 0.3–100 Hz. Similarly, in these

experiments the current intensity was 5 mA at a pulse

width of 1 ms.

Electrically induced flare response pattern 641

Flare assessment

Superficial skin blood flow was recorded online by a series

of color-coded laser Doppler images (MoorLDI2-IR Laser

Doppler Imager, Axminster, UK) covering an area of

32 cm2. Scan resolution was set at 132 9 155 pixels;

image capture required 115 s and recording interval was

set at 120 s. The distance between laser scanner and skin

surface was kept constant at 40 cm. For off-line analysis of

vasodilatation, mean flux values and standard deviation

were determined in the baseline image recorded prior to the

onset of the electrical stimulation. A rectangular area of

approximately 12 cm2 was defined in the vicinity of the

stimulation site and mean flux values were calculated

within this area using the manufacturers’ software (Moor-

LDI 3.08, Axminster, UK). The threshold for a significant

increase of blood flow was defined as mean flux

value ? 2 times standard deviation. Flux values exceeding

the threshold were identified as significant flare reaction

and included into the calculation. Total flare area was

assessed in each image of the laser Doppler sequence by

means of the calculated number of pixels and their size,

respectively.

Statistical analysis

Data were analyzed using Statistica� 6.0 software package

(Statsoft, Tulsa, US). Values were calculated by ANOVA

multi-variant analysis and repeated measures. Signifi-

cant differences were identified between the factors

‘‘species’’ - ‘‘current intensity’’ - ‘‘pulse width’’ - ‘‘fre-

quency’’ by Fisher’s least-significant difference (LSD) post

hoc test. P values \0.05 were considered significant. Data

are depicted as mean ± standard error of the mean (SEM)

throughout the manuscript.

Results

Transcutaneous nerve stimulation

Current intensity

In human skin, transcutaneously delivered currents from

2.5 to 15 mA, which had been administered at a constant

pulse width of 0.5 ms and a frequency of 1 Hz, evoked a

dose-dependent and almost linear increase of flare area

without reaching a plateau. Statistically significant flare

sizes (P \ 0.0001, ANOVA) were identified from 5 mA

onwards. Flare size was analyzed on average at

2.5 ± 0.7 cm2 following 5 mA stimuli with maximum

flare sizes of about 13.4 ± 1.4 cm2 following 15 mA

stimulation. Half-maximum flare size developed during the

7.5 mA administration period. Vasodilatation declined

when current supply had been terminated, but was still

significantly elevated (P \ 0.0001, ANOVA) 6 min post-

stimulation when compared to baseline value. Similar to

human skin, transcutaneous electrical nerve stimuli evoked

in pig skin a linear increase of flare development from

0.99 ± 0.27 cm2 at 2.5 mA to a maximum flare size of

about 3.23 ± 0.42 cm2 after 7.5 mA. Further increase of

the current intensity to 10, 12.5, and 15 mA, however, did

not additionally enhance the flare size. Thus, maximum

flare development was significantly smaller as compared to

humans (P \ 0.0001, ANOVA). Half-maximum flare

development was analyzed after the 2.5 mA stimulation

period, which induced already significantly elevated

vasodilatation in comparison to baseline value (P \ 0.001,

Fisher’s LSD post hoc). Thus, when compared to human

skin, flare area was significantly larger in pig skin

(P \ 0.02, Fisher’s LSD post hoc) in response to 2.5 and

5 mA stimulation (Fig. 1a). No significant difference

Table 1 Summary of the study design performed in humans (n = 6) and pigs (n = 8) to explore the dependency of flare development in

response to the administered current parameters (left column) and the corresponding current variables that had been kept constant

Assessed parameter Parameters kept constant

Human Pig

Current intensity 2.5–15 mA Pulse width 0.5 ms, frequency 1 Hz

Pulse width 0.05–2 ms Current intensity 5 mA, frequency 1 Hz

Frequency increase 0.1–100 Hz, and decrease 100–0.1 Hz Current intensity 5 mA, pulse width 1 ms

Continuous stimulation 30 min at 0.5 and 50 Hz n/a Current intensity 5 mA, pulse width 1 ms

Number of stimuli (75 or 150 or 225 pulses at frequencies of 0.3–100 Hz) n/a Current intensity 5 mA, pulse width 1 ms

Intracutaneous versus transcutaneous stimulation current intensity 2.5–

15 mA

n/a Pulse width 0.2 mA, 1 Hz and pulse width 0.5 ms, 1 Hz

Intracutaneous versus transcutaneous stimulation pulse width 0.05–2 ms n/a Current intensity 5 mA, frequency 1 Hz

Note that the parameters ‘‘continuous stimulation’’ and ‘‘number of stimuli’’ was performed in pigs only (n = 3). The development of flare

responses upon transcutaneously versus intradermally delivered stimuli was investigated in pigs (n = 6) at varying current intensities (2.5–

15 mA) and pulse widths (0.05–2 ms), respectively

642 M. Dusch et al.

was obtained between male and female pigs (P [ 0.5,

ANOVA), which may be attributed to the pre-menstrual

cycle of the female animals given at an average age of

12 weeks.

Pulse width

Increase of the pulse duration (0.2–2 ms) significantly

enhanced flare development in a dose dependent manner in

both human and pig skin (P \ 0.0001, ANOVA). In

humans, flare size increased linearly in response to elec-

trical stimuli delivered at a constant current of 5 mA and a

frequency of 1 Hz from 0.6 ± 0.2 cm2 (0.2 ms pulse

width) to a maximum level of 9.5 ± 0.7 cm2 (2 ms pulse

width). Compared to baseline value, a significantly larger

flare area (P \ 0.0001, ANOVA) was recorded at the end

of the experimental protocol. Half-maximum flare size of

4.9 ± 0.5 cm2 was obtained following a pulse width of

1 ms, even though the flare response does not reach a

plateau in this experimental setting. In pig skin a linear

increase of flare size was analyzed in response to the pulse

duration. Flare development, however, levelled following

0.5 ms pulse width with a peak maximum flare of about

3.5 ± 0.5 cm2, which did not further increase even though

pulse duration was extended up to 2 ms. After termination

of the electrical stimulation flare development returned to

baseline values within 6 min. In comparison to the baseline

value, half-maximum and significantly elevated flare size

of about 1.2 ± 0.3 cm2 (P \ 0.001, Fisher’s LSD post

hoc) was analyzed after the 0.2 ms stimulation period.

When compared to human skin, flare sizes analyzed in pigs

were significantly smaller (p \ 0.02, Fisher’s LSD post

hoc) at pulses exceeding 1-ms duration and after current

was switched off (Fig. 1b).

Pulse frequency

Flare development was dependent on the pulse frequency

of the administered current stimuli (P \ 0.0001, ANOVA),

which were delivered at a constant intensity of 5 mA and a

pulse width of 1 ms. In humans, when compared to base-

line value, a significant increase of flare development was

induced from 0.3 Hz onward (P \ 0.035, Fisher’s LSD

post hoc). Maximum flare size was determined at

9.2 ± 0.08 cm2 in response to 50 and 100 Hz stimulation

frequency, with half-maximum flare sizes developing

during 1 Hz stimuli. Current offset caused a slight but

continuous reduction of the flare size to 7.6 ± 0.6 cm2 at

Fig. 1 Flare responses in

human (n = 6) and pig skin

(n = 8) upon transcutaneous

electrical nerve stimulation.

a The parameter ‘‘current

intensity’’ (2.5–15 mA) was

investigated at a pulse width of

0.5 ms and 1 Hz frequency.

b ‘‘Pulse widths’’ (0.05–2 ms)

were studied at a current

intensity of 5 mA and 1 Hz

frequency. c The dependency of

‘‘pulse frequency’’

(0.1–100 Hz) was assessed at a

current of 5 mA and 1 ms pulse

width at increasing

(0.1–100 Hz, left) or decreasing

(100–0.1 Hz, right) frequencies.

d Area under the curve (AUC)

of the frequency dependent flare

recording in human (left) and

pig (right). Significant

differences were identified

between the species in response

to the analyzed parameters

(P \ 0.05, ANOVA, marked by

asterisks) and in comparison to

the baseline recordings

(P \ 0.001, Fisher’s LSD post

hoc, marked by crosses)

Electrically induced flare response pattern 643

the end of the observation period. In pig skin, sustained

stimulation at 0.1 and 0.2 Hz induced already a significant

flare development in comparison to baseline values

(P \ 0.05, Fisher’s LSD post hoc) and human skin

(P \ 0.01, Fisher’s LSD post hoc), respectively. Half-

maximum flare developed at a pulse frequency of 0.3 Hz

and further increase of current frequency to 5 Hz evoked

already a maximum flare response of 3.2 ? 0.07 cm2 on

average. High frequent stimuli of 10–100 Hz caused a

continuous and in comparison to human skin significant

(P \ 0.001, Fisher’s LSD post hoc) diminution of vaso-

dilatation that reached baseline levels after current

termination (Fig. 1c, left).

Administration of electrical stimuli starting at 100 Hz

evoked an immediate and significant steep incline of flare

development, causing already half-maximum flare sizes in

both human and pig skin (P \ 0.05, Fisher’s LSD post

hoc). In humans, a maximum flare of 10.7 ± 1.1 cm2 was

recorded when 1 Hz stimuli were delivered, and less fre-

quent stimulation caused a linear decrease of the flare of

finally 4.1 ± 0.9 cm2 after current offset. In pig skin,

maximum flare sizes of 4.5 ± 1.3 cm2 were obtained

during the 50 Hz stimulation period, which was followed

by a linear reduction of vasodilatation in response to fur-

ther decreased pulse frequencies, reaching baseline levels

following 0.5–0.1 Hz stimuli. In comparison to human

skin, frequency-dependent flare development was sig-

nificantly smaller in pig skin after 50 Hz stimulation

(P \ 0.05, Fisher’s LSD post hoc) and until the end of the

observation period (P \ 0.001, Fisher’s LSD post hoc)

(Fig. 1c, right).

Comparing the order of the administered pulses, i.e.,

increasing versus decreasing frequencies, analyses of the

AUC of the recorded flare revealed no significant differ-

ence in human (P = 0.08, ANOVA) and pig skin

(P = 0.32, ANOVA) (Fig. 1d).

Transcutaneous versus intradermal nerve stimulation

in pig skin

Current intensity

A comparison of the flare development upon transcutane-

ous and intradermal electrical nerve stimulation was

investigated in pig skin only. Stimuli were administered

with increasing current intensities from 2.5 to 15 mA at a

frequency of 1 Hz and a pulse width of 0.2 and 0.5 ms,

respectively. In general, a significantly larger flare was

determined at a pulse width of 0.5 ms as compared to

pulses of 0.2 ms duration (P \ 0.001, ANOVA). In addi-

tion, transcutaneous current application induced

significantly larger flare in comparison to intradermally

delivered stimuli at 0.2 ms (P \ 0.002, ANOVA) and

0.5 ms (P \ 0.05, ANOVA). Significant differences

between transcutaneous and intradermal stimulation were

determined at 0.2 ms pulse width at current intensities of

7.5 mA onward, and in response to 0.5 ms pulse duration

at currents exceeding 2.5 mA (P \ 0.05, Fisher’s LSD post

hoc). Half-maximum flare size developed in both transcu-

taneous and intradermal stimulation at current intensities of

5 mA (0.2 ms pulse width) and 2.5 mA (0.5 ms pulse

width), respectively (Fig. 2a).

Pulse width

No significant differences were obtained between transcu-

taneous and intradermal stimulation (P = 0.27, ANOVA)

when investigating the pulse widths of the administered

current (5 mA, 1 Hz). Compared to baseline condition, a

significant increase of flare development was analyzed

following transcutaneous (P \ 0.001, Fisher’s LSD post

hoc) and intradermal (P \ 0.05, Fisher’s LSD post hoc)

stimuli delivered at 0.2 ms pulse width and reaching almost

half-maximum flare size. Ongoing increase of pulse dura-

tion to 1 ms evoked maximum flare responses of

3.5 ± 0.5 cm2 and 2.4 ± 0.5 cm2 to transcutaneous and

intradermal stimuli. Pulses of 2 ms duration, however, did

not further enhance flare sizes and current offset caused an

immediate and linear drop of vasodilatation to baseline

conditions (Fig. 2b).

Continuous transcutaneous stimulation in pig skin

Transcutaneous electrical stimuli of 5 mA and a pulse

width of 1 ms were administered continuously over a time

period of 30 min at a frequency of 0.5 and 50 Hz.

Regardless the frequency and compared to baseline level a

significant increase of flare development was determined

immediately after current onset and throughout 14 min of

continuous electrical stimulation (P \ 0.05, Fisher’s LSD

post hoc). Maximum flare sizes of 3.1 ± 0.97 cm2 (0.5 Hz)

and 3.1 ± 0.88 cm2 (50 Hz) were found about 5 min fol-

lowing stimulation onset. Thereafter, an almost linear

reduction of the flare was observed even though electrical

stimuli continued, and after approximately 20 min of

continuous stimulation flare sizes had returned to baseline

levels of 0.08 ± 0.05 cm2 (0.5 Hz) and 0.22 ± 0.19 cm2

(50 Hz) (Fig. 2c). The AUC of the recorded flare was

analyzed on average at 18.9 ± 1.16 cm2 for 0.5 Hz and

17.2 ? 1.02 cm2 for 50 Hz (NS; P = 0.21, ANOVA)

(Fig. 2c, inlet).

Number of electrical pulses

Increasing the number of stimuli from 75 to 150 and further

to 225 pulses caused a significant increase of the flare area

644 M. Dusch et al.

(P \ 0.05, ANOVA). No significant difference of the AUC

was identified between 150 and 225 pulses (P = 0.37,

ANOVA). Regardless the administered frequency of cur-

rent pulses (0.3–100 Hz) the AUC of flare development

was analyzed on average at 8.9 ± 0.5 cm2 (75 pulses),

21.0 ± 1.12 cm2 (150 pulses) and 29.9 ± 1.43 cm2 (225

pulses) (data not shown), respectively.

Discussion

In the present study we compared the flare response pat-

terns in pig and human skin upon transcutaneous electrical

stimulation. In the pig, significantly lower excitation

thresholds, smaller flare sizes and a ‘‘fatigue’’ of the flare

response was observed. Thus, ongoing electrical stimula-

tion tends to result in a gradually declining flare area

irrespective the frequency, pulse width or current intensity.

This characteristic attribute in pig skin was most prominent

when electrical stimuli were delivered continuously for

half an hour at a constant pulse duration and current

intensity. By contrast, in human skin, we determined an

almost linear increase of the flare area with enhanced

current intensity, pulse duration, or current frequency.

Noteworthy, the flare response does not reach a plateau

under the given experimental settings. Similarly, previous

Fig. 2 Comparison between

transcutaneous and intradermal

electrical nerve stimulation in

pig skin. a The impact of the

current intensity on the flare

development was assessed at a

pulse frequency of 1 Hz and

pulse durations of 0.2 ms (left)or 0.5 ms (right), respectively.

Flare development upon

transcutaneous stimulation was

significantly larger as compared

to intradermal stimulation

(P \ 0.05, ANOVA, marked by

asterisks) and dependent on the

administered current intensity

(P \ 0.001, Fisher’s LSD post

hoc, marked by asterisks).

b Analysis of the pulse duration

on the flare development

assessed at 5 mA and 1 Hz. No

significant difference was

identified between

transcutaneous and intradermal

stimuli (P = 0.27, ANOVA).

c Ongoing transcutaneousstimuli (30 min) administered at

0.5 and 50 Hz at 5 mA and

1 ms pulse width evoked a

maximum flare response within

5 min, which declined to

baseline level within 20 min of

stimulation [inset in c, area

under the curve (AUC)].

Significant differences

compared to baseline recordings

are marked by crosses

Electrically induced flare response pattern 645

human studies also demonstrated a linear increase of flare

development, which does not saturate at current intensities

of 30 mA [14, 15]. If current intensities are further

increased, the flare area gains a maximum after 50 min of

continuous stimulation at about 50–80 mA [16]. Only

when the stimulation period exceeded 100 min at this

current intensity the flare area significantly decreases [16],

which suggests that an evacuation of the neuropeptide

stores in both human and pigs may have caused the

observed declining flare responses.

Similar to humans [17, 18], the neuropeptide CGRP is

one of the major neurotransmitter in the pig and contributes

to the vasodilatation evoked by, e.g., histamine or brady-

kinin [19]. CGRP-containing nerves were identified in

close association around blood vessels [20] and electrically

induced vasodilatation could be inhibited in pigs by

reducing the neurotransmitter release from capsaicin-sen-

sitive nerves after opioid receptor stimulation [21]. As

shown previously, the proportion of CGRP-positive nerve

fibers has been demonstrated similar in pig and human [2].

Further, in comparison to sparsely haired domestic mam-

mals, only the domestic pig revealed morphological and

functional skin characteristics comparable to human skin in

terms of, e.g., structure, vascularisation, or epidermal

enzyme patterns [1]. The innervation density of nerve

fibers between the species, unfortunately, was not analyzed

in these studies [1, 2]. The recorded differences between

the species presented here, particularly plateau of flare and

declined vasodilatation, therefore may be due to varying

dynamics of neuropeptide release.

Apart from emptied vesicle stores, the decrease of the

flare area observed in pigs might be attributed to alterations

of synaptic vesicle docking, or to changes in receptors and

their coupling to blood vessels, or to altered levels of

extracellular enzymes involved in mediator degradation. In

addition, impaired action potential propagation at the ter-

minal endings of the stimulated nerve fibers may be

enhanced in pig skin and contribute to the apparent ‘‘fati-

gue’’ of flare development in the animal. As described

previously in man in response to high-frequent trains of

electrical pulses [22], impaired stimulus decoding also

might contribute in pigs to an affected action potential

generation and thus reduced neuropeptide release, leading

to a plateau of the flare response.

In addition, decreased flare response upon ongoing

stimulation might be explained by counteracting influences

of the autonomic nervous system. Electrical stimuli

delivered at high frequencies caused a short-lasting vaso-

constriction in humans, probably due to the activation of

sympathetic neurons [23, 24]. In the pig, cutaneous glands

responded to injections of adrenaline and noradrenaline

[25], and in guinea pig ear skin sympathetically induced

vasoconstriction revealed maximum responses at electrical

stimulation frequencies of 10–20 Hz [26]. The frequency-

dependent vasoconstriction is in accordance to the weak-

ened flare responses observed herein in pig skin upon

electrical stimulation exceeding a frequency of 10 Hz.

Comparing the flare development in human and pig skin

in dependence on the administered current intensity, we

obtained in pigs a significant increase of vasodilatation

already when electrical stimuli were administered at low

current intensities. Apparently, electrical thresholds to

induce a flare reaction are lower in pigs than in humans. In

addition, maximum flare responses were analyzed to be

significantly smaller in pig skin. These observations might

have been expected considering the smaller size of the

animals. Receptive field sizes of heat-sensitive and

mechanically insensitive units of the saphenous nerve in

pigs were relatively small indeed, on average 0.25 cm2

[27]. In contrast, mechano-insensitive units from the

peroneal nerve innervating the lower leg and the dorsum of

the foot of humans showed considerably larger receptive

fields of on average 5.34 cm2 [28]. Thus, the variations of

the receptive field sizes between the investigated species

might be responsible for the different flare sizes analyzed

in pig and human skin.

Previously, Lynn and colleagues [3] described the flare

development in pigs upon antidromic nerve stimulation.

The authors demonstrated that polymodal nociceptors

apparently are not involved in the mediation of vasodila-

tation, whereas all units responsive to heat did evoke

vasodilatation upon their stimulation. Of these, the majority

were unresponsive to mechanical stimuli [3]. In agreement

with this study, persistent vasodilatation in human skin was

dependent on the excitation of mechano-insensitive [5] and

heat-sensitive [4] nociceptors. Therefore, we assume that in

both human and pig skin mechano-insensitive and heat-

sensitive nociceptors mediate widespread flare responses,

but additional electrophysiological studies still are required

to fully characterize the properties of axon reflex mediating

nerve fibers in pig skin.

An impact of the sedation of the pig on these observa-

tions appears unlikely, as recent reports demonstrated that

sedation had no adverse effect on the neurogenic-mediated

flare development [29, 30]. In anesthetized pigs the

peripheral administration of a local anesthetic completely

attenuates the flare development [12]. This observation

indicates that in the present experimental setting the flare

development depends on the conduction of action poten-

tials at axonal branches in the periphery.

In experimental human studies, current stimuli of higher

intensity were needed for mapping the receptive fields of

mechano-insensitive units [28] and for evoking a wide-

spread flare reaction [5]. Apparently, high electrical

thresholds are functional attributes of those nerve fibers

that contribute to the flare reaction. By contrast, current

646 M. Dusch et al.

intensities required to induce a flare reaction in pigs were

significantly lower [3]. Confirming the study of Lynn and

colleagues [3], we demonstrate in the present investigation

an instant flare development upon low currents, indicating

its independence from the activation of electrical high-

threshold nociceptors. We further obtained that intradermal

electrical stimuli elicited in pig skin a significantly smaller

flare reaction as compared to transcutaneous nerve stimu-

lation. It may be suggested that the administration of

electrical currents in different skin layers evoke varying

flare responses. Given that the type of stimulation probes,

i.e., intradermal needles versus transcutaneous surface

electrodes, however also may influence the dynamic of

flare development, histologic studies of pig skin would be

needed to evaluate whether structural profiles of vascular-

ization or nerve fiber distribution contribute to the smaller

flare size.

In conclusion, we compared in human and pig skin

specific nociceptors sharing the similarity to induce a

neurogenic-mediated flare response. Particular nerve fiber

attributes were identified between the species. In the ani-

mal, excitation thresholds of nociceptors to evoke a flare

response were significantly lower, the flare area was

smaller, and a continuously declining vasodilatation

(‘‘fatigue’’) was identified upon ongoing electrical stimu-

lation. These parameters may be specific targets for pre-

clinical anti-hyperalgesic studies. Thus, a particularly

sensitive assessment is required for its exploration. Having

demonstrated that these nociceptor attributes can be

examined in pigs with high sensitivity, this laboratory

species may be a promising translational animal model for

anti-hyperalgesic researches and modulation processes of

specific skin nociceptor sup-classes.

Acknowledgments This work was supported by grants from the

Deutsche Forschungsgemeinschaft, KFG 107, and the Kompetenz-

zentrum Schmerz, State Baden-Wurttemberg.

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