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SHORT COMMUNICATION
Heterogeneity of Skin Re-innervation After Burns and Factors Involved in its Regulation: A Pilot Study
Virginie BUHÉ 1 , Alexandra TRIMAILLE 2 , Martine SCHOLLHAMMER 3 , Florence MORVAN 4 , Weiguo HU 2 , Christophe EGLES 5 ,
Alexis DESMOULIÈRE 6 and Laurent MISERY 1,3 *
Laboratory of Neurosciences of Brest (EA4685), Faculty of Medicine, University of Brest, FR-29200 Brest, Departments of 2 Plastic Surgery and
Dermatology and 4 INSERM CIC 1412, University Hospital of Brest, Brest, 5 CNRS UMR 7338, Biomechanics and Bioengineering, Technological
University of Compiègne, Compiègne, and 6 Myelin maintenance and peripheral neuropathies (EA6309), University of Limoges, Limoges,
France. *E-mail: [email protected]
1
3
Accepted Oct 19, 2017; Epub ahead of print Oct 23, 2017
Deep burn injuries alter the integrity of skin innervation
and have a significant impact on patient’s quality of life
by compromising perceptions of touch, temperature and
pain. Moreover, patients can develop long-term disabili-
ties, ranging from loss of cutaneous sensibility to chronic
pain, itch and other paraesthesia and dysaesthesia (1, 2).
The mechanisms involved in skin reinnervation fol-
lowing burns remain unclear. Depending on the depth
of the burn, nerve sprouting can occur from the wound
bed or surrounding healthy tissue, but this process fails
to provide correct re-innervation of the wound during
scarring (1). Although nerve fibres may regenerate after
spontaneous wound healing and/or skin grafting, abnor-
mal nerve fibre density is often encountered, resulting in
altered skin sensation (3). More precisely, a significant
reduction in the axonal regrowth of small fibres within
the wound occurs (4).
This pilot study compared the expression of genes
involved in the regulation of innervation in healthy skin
and in post-burn scars from the same patients. In addi-
tion, the intra-epidermal density of nerve endings was
measured in these samples.
PATIENTS AND METHODS
The study was approved by an ethics committee (CPP Ouest II
n°2014-37) and registered on clinicaltrials.gov (NCT02356354).
Patients evaluated pain and/or pruritus with the help of a visual
analogue scale (VAS) then answered the Douleur Neuropathique-4
(DN4) questionnaire (5) and the Hospital Anxiety and Depression
Scale (HADS) (6) to measure the levels of anxiety and depres-
sion. The quality of the scar tissue was assessed with the help of
the Patient and Observer Scar Assessment Scale (POSAS) (7).
Patients underwent 4-mm diameter skin biopsies in the post-burn
scar and in the contralateral healthy skin. Each skin sample was
identified by a code number to allow blinded analyses and cut into
2 half-biopsies; one was fixed overnight in a 4% paraformaldehyde
bath and then preserved in a phosphate-buffered saline (PBS) –
10% sucrose bath for an additional 24 h prior to being frozen and
stored at –80°C. The biopsies were cut into 7-μm or 30-μm-thick
sections, spaced at least at 98 μm apart.
Determination of nerve fibre density was performed as described
previously (8, 9). The other half-biopsy was preserved at –80°C in
RNAlater (Thermo Fisher, Carlsbad, CA). RNA was later extracted
with a Purelink RNA Micro kit (Invitrogen, Carlsbad, CA) after
lysis with a rotor stator in lysis buffer. RT-PCR was performed
using the High-capacity cDNA reverse transcription Kit (AB App-
lied, Foster City, CA). The expression of genes that can regulate
neuronal regeneration was studied with TaqMan technology and
doi: 10.2340/00015555-2826
Acta Derm Venereol 2018; 98: 280–281
the kit TaqMan Fast Universal PCR Master Mix (AB Applied)
associated with a couple of specific gene assays. Actin B was
used as a housekeeping gene standard. For each half-biopsy, the
expression of each gene was first normalized to the expression of
actin B, which was stable (ΔCt). The levels of expression were
normalized in scars and then compared with those in healthy skin
(ΔΔCt) for all patients. The final results were expressed according
to the multiplication factor of each gene in scars by comparison
with contralateral healthy skin (calculation of “×-fold change”
according to 2-ΔΔCt).
Although this is a pilot study, statistical analysis was performed.
RESULTS
Six patients were included in the study (3 men and 3
women, ranging from 22–66 years old). The burn was
chemical in one case and thermal in 5 cases. The total
body surface area of burns (TBSA) was higher than
40% in one patient, but lower than 15% in the 5 others.
The age of the burns ranged from 1 to 4 years. Using
POSAS (thickness ≥ 6/10), 3 patients had hypertrophic
scars (patients 3, 5 and 6). The mean intensity of pain
was 5.2/10 (from 2 to 9) and that of itch was 5/10 (from
4 to 8). All patients could be considered to present with
neuropathic pain, with a mean DN4 score of 6.2 (from 4
to 8; score > 4 for the diagnosis). Patients did not show
pathological scores on the HADS depression subscale
(scoreHADS < 7), but 3 patients had a pathological score
of anxiety (score > 7).
The profiles of expression of the studied genes (arte-
min, BDNF, FGF-2, GDNF, HSP27, IL6, NGF, neurturin,
NT-3, NT-4, Netrin 1, semaphorin 3A, persephin and
VEGF) were variable in each patient. Overexpression
was assessed by a level of expression that was increased
2-fold or more. Nine genes were overexpressed accor-
ding to this rule: artemin (ARTn), BDNF, IL6, neurturin
(NRTN), NT3, NT4, semaphorin 3A (SEMA3A) and
persephin (PSPN). Among them, IL6 and SEMA3A were
highly overexpressed in the scars of 2 patients. There
was no significant difference between patients with
hypertrophic and non-hypertrophic scars.
The results were also heterogeneous. Two patients
had notably higher densities of intra-epidermal fibres
in scars