Reference values for peptidergic and non-peptidergic intra-epidermal nerve fiber density in the human hand and distal leg.

Neuropathic pain yearly develops in around 1% of the Dutch general population
and has a worldwide prevalence estimated between 6.9% and 10%. As neuropathic
pain responds poorly to standard pain therapies, patients have a lower
health-related quality of life. Treatment specifically for neuropathy and
neuropathic pain, like anticonvulsants and anti-depressants, will only be
initiated after diagnostic tests have been performed. Skin biopsy for
diagnosing peripheral neuropathy in the distal leg by objectively quantifying
nerve fibers in the epidermis is a reliable and safe technique and has become a
valuable addition to existing neurophysiologic tests. Tests like
electromyography and nerve conduction studies focus mainly on large nerve
fibers, whereas QST and skin biopsy focus on small nerve fibers. An advantage
of the skin biopsy is that it can detect changes in the quantity of nerve
fibers while conventional neurophysiologic tests cannot. Furthermore, the skip
biopsy provides a quantitative value, making this technique suitable for
follow-up of progression or recovery of neuropathy.5 The advantage of skin
biopsy over QST is that skin biopsy is completely objective whereas QST is
dependent on participation of the patient.
In the skin biopsy the small nerve fibers are counted by using an
immunohistochemical staining with the pan-axonal marker anti-protein gene
product 9.5(PGP9.5). Unfortunately, the PGP9.5 staining cannot discriminate
between subgroups of small nerve fibers. These fibers detect painful stimuli in
the skin and can be divided in peptidergic A-delta and C fibers and in
non-peptidergic C fibers. A-delta fibers are thinly myelinated, whereas C
fibers are unmyelinated. Peptidergic and non-peptidergic fibers can be further
divided by expression of neuropeptides and receptors: Calcitonin gene-related
peptide(CGRP) and Substance P(SubP) are predominantly expressed on peptidergic
C-fibers but can also be found on A-delta fibers. Neurofilament 200(NF-200) is
expressed on peptidergic A-delta fibers but also on larger myelinated A fibers
and can therefore be used as a marker for myelination. Non-peptidergic C-fibers
express receptor P2X3. Recent studies in rats have shown that this distinction
may be of clinical importance, as regeneration after nerve injury is mostly
done by peptidergic fibers. Along with the increase in peptidergic fibers,
hypersensitivity develops. Eventually the increase of peptidergic fibers leads
to recovery of the total amount of small nerve fibers, but hypersensitivity is
still present and the ratio of peptidergic and non-peptidergic fibers is
changed as compared to the physiological situation. Furthermore, Schüttenhelm
et al. studied the intra-epidermal nerve fiber density in two itch models and
one inflammation model of the skin. They found significant changes in
peptidergic fibers in all three models and significant changes in
non-peptidergic fibers in one model. This shows that peptidergic and
non-peptidergic fibers play roles in different pathologies, like the
development of neuropathic pain and skin disorders. Therefore, it is important
to discriminate between peptidergic and non-peptidergic fibers. As this
distinction cannot be made with the PGP9.5 staining, there are no reference
values of peptidergic and non-peptidergic fibers in the human skin. Clinically,
this could be problematic for patients with complaints of neuropathic pain but
skin biopsy with PGP9.5 staining showing a normal nerve fiber density, while
the ratio of peptidergic and non-peptidergic fibers is changed. Because they
cannot be diagnosed adequately, they might not receive optimal treatment for
their neuropathic pain complaints. Previous studies of intra-epidermal nerve
fibers density have shown different nerve fiber densities between different
biopsy sites all over the human body, thus it is not possible to use the
reference values of the distal leg for other parts of the body. No reference
values adjusted for age and sex have been provided for the hand, so diagnosing
neuropathies in the hand affecting only the small nerve fibers is problematic.
As there might be a difference in intra-epidermal nerve fiber density between
the innervation territories of the median, ulnar and radial nerve, we will take
to biopsies from the palm of the hand and one of the dorsum of the hand
respectively. We will compare the intra-epidermal nerve fiber density of the 3
biopsy sites of the hand and if they differ from each other, we will give
reference values of all 3 sites. If they do not differ from each other, we will
give reference values for only 1 site of the hand.

New information provided by this study will be reference values of the
intra-epidermal nerve fiber density for the hand with the distinction between
peptidergic and non-peptidergic nerve fibers, which will also be provided for
the distal leg. In order to provide these reference values we need healthy
volunteers without any signs of neuropathy or risk factors for neuropathy.
Since intra-epidermal nerve fiber density is age dependent, we will need to
include volunteers of all ages(above 17 years old).

The aim of this study is to provide reference values for peptidergic and non
peptidergic intra-epidermal nerve fiber density of the hand and distal leg.
Furthermore, we will compare the intra-epidermal nerve fiber density of three
biopsy sites of the hand to analyse whether there is a difference.

The study design is observational with invasive measurements. We will approach
volunteers of 18 years or older to participate. All volunteers will have to
fill out a questionnaire about their medical history regarding possible
neuropathic conditions and will undergo neurological exam. When they do not
meet the exclusion criteria, volunteers will be included in the study and get
an appointment for taking 4 skin biopsies from the hand and distal leg. One
biopsy will be taken from the distal leg, 10cm above the lateral malleolus. The
other 3 biopsies will be taken from the hand from areas innervated by the
median, ulnar and radial nerve. When we have data on nerve fiber density of the
different biopsy sites of 20 volunteers, we will compare them to find out
whether there is a difference. If that is the case, we will only take biopsies
from the sites that are different from each other. If that is not the case, we
will compare the three sites again after 41 volunteers. If we find no
difference then, we will only take one biopsy from the hand and the distal leg.
After obtaining the skin we will use immunohistochemical stainings to determine
the nerve fiber density, which will be expressed in fibers per millimeter. We
will use the protocol for the staining and counting as described by Lauria et
al.
Reference values will be stratified by age and gender.

After volunteers have signed the informed consent form, they will be asked to
fill out a questionnaire about their medical history regarding possible
neuropathic conditions. If they do not meet any of the exclusion criteria they
will undergo neurological exam. Then, if neurological exam is normal, the
volunteer will be included and four skin biopsies will be taken; 3 from the
hand and 1 from the distal leg. Skin will be locally anesthetized before taking
3mm circular biopsies. The injection will give physical discomfort and might be
painful, especially in the hand. The biopsies will leave 4 small wounds.
Volunteers will be advised not to lift heavy things with the biopsied hand
until 7 days after the biopsies are taken. The wounds do not need suturing and
heal within 7-10 days. Nevertheless, if volunteers wish to get the wounds
sutured or if the wounds keep bleeding, we will uses sutures.
Obtaining a skin biopsy from the distal leg has been shown to be a safe
procedure for determining intra-epidermal nerve fiber density, with an
incidence of adverse events of 1.9 out of 1000 biopsies and no serious adverse
events. There are no numbers of adverse events in the hand, but we estimate
these will be comparable to adverse events in the distal leg. Possible adverse
events are mild infection and excessive bleeding.
Participating in this study will take volunteers approximately 1 hour and the
risk for adverse events is very low. With the reference values for peptidergic
and non-peptidergic nerve fibers for the leg and hand this study provides new
information that can extend the use of the skin biopsy as diagnostic test for
neuropathy, therefore we believe that the benefits will outweigh the
disadvantages.