To the Editor
Chronic pruritus affects millions of patients worldwide and has a significant impact
on quality of life similar to that of chronic pain (Kini et al., 2011; Stander et
al., 2007). Significant advances have been made in the last five years to elucidate
the molecular pathways of acute itch (Liu et al., 2009; Sun and Chen, 2007). However,
experimental approaches investigating the pathogenesis of pruritus and the ability
to test novel therapeutic agents are largely limited to rodent models. Although these
models offer some advantages, their translational potential to human disease remains
to be established (Seok et al., 2013; Jeffry et al., 2011). Furthermore, most animal
models focus on acute itch, which displays significant pathophysiological differences
in comparison with chronic itch (Yosipovitch et al., 2007). Therefore, there is an
unmet need for better animal models for chronic itch research.
Recently, gastrin-releasing peptide (GRP) and its receptor (GRPR) were discovered
to play a key role in itch transmission, but not in nociception (Sun and Chen, 2007).
GRP was found in a subset of unmyelinated dorsal root ganglion (DRG) neurons, while
GRPR was expressed in lamina I of the dorsal horn of the spinal cord in mice. In the
skin, GRP is present in primary afferent nerve fibers and found to be increased in
mice with chronic dermatitis (Tominaga et al., 2009). The GRP/GRPR signaling pathway
is considered to be the first molecular pathway specific to itch transmission, but
there is no evidence that this pathway has an analogous role in primates or humans.
We identified a subgroup of adult female Cynomolgus macaques (Macaca fascicularis)
suffering from idiopathic chronic itch and observed their scratching behaviors twice
every week for 10 minutes for four years using a focal observation technique (Altmann,
1974). The population pattern of GRP and GRPR expression in the skin and spinal cord
were assessed in a blinded manner, respectively. The expression of GRP and GRPR was
quantified by immunohistochemistry and analyzed with a 1-way ANOVA and Bonferroni
post hoc test, while an Unpaired t-test was use to compare the amount of PGP9.5+ never
fibers between groups. The scratching behavior of each animal was then analyzed for
potential correlations with GRP and GRPR expression levels using a 2-tailed Spearman
correlation test and linear regression.
The frequency (number of scratching episodes per hour) and duration (percent time
of scratching during focal observation) of scratching were recorded over four years,
totaling 68 hours of observation per animal. The animals were euthanized and histological
sections from six randomly selected animals with different itch severities were collected.
For each primate, the total count of nerve fibers expressing GRP in lichenified (chronically
damaged skin lesion) and non-lichenified skin from thoracic dermatomes were correlated
with itch severity. Skin cryosections (20 μm thickness) were double stained with antibodies
for GRP (Immunostar, Hudson, WI) and the neuronal marker Product Gene Protein 9.5
(PGP9.5; Neuromics, Edina, MN) to examine the amount of co-localization at the dermal-epidermal
junction. Although fibers were found in both the epidermis and dermis, they were mainly
present in the dermal-epidermal junction so all quantification was focused on this
area. The population of GRPR expressing cells in the dorsal horn of the thoracic spinal
cord of the same animals was also analyzed for a potential correlation to itch intensity
by staining the spinal cord cryosections with the GRPR antibody (MBL International,
Woburn, MA). For a detailed methods section, see supplementary information.
Primates that exhibited a comparatively higher severity of itching consistently displayed
an increased percentage of GRP expressing nerve fibers at the dermal-epidermal junction
of lichenified skin (p=0.03; Figure 1a & e). The mean percentage of GRP+/PGP9.5+ fibers
was 4.8 fold higher in primates exhibiting the highest itch intensity than in primates
with the lowest intensity. The frequency (Figure 1b) and duration (Figure 1c) of scratching
were significantly correlated with the percentage of GRP in lichenified skin (r=0.94,
p=0.02; for both frequency and duration), but not in non-lichenified skin (r=0.77,
p=0.10; for both frequency and duration). The innervation pattern of PGP9.5+ fibers
in the skin show no significant difference of fibers in animals with severe itch compared
to those with mild/moderate itch (Figure 1d).
Additionally, an increased of amount GRPR expressing cells was observed in the dorsal
horn of the spinal cord of primates exhibiting a higher itch severity compared to
animals with lower itch severity (p=0.002; Figure 2a & d). The frequency (Figure 2b)
and duration (Figure 2c) of scratching was also found to be significantly correlated
with the expression of GRPR in the superficial lamina I & II (r=0.94, p=0.02; for
both frequency and duration), but only trended toward significance in the deep lamina
III–V (r=0.83, p=0.06; for both frequency and duration) of the dorsal horn of the
thoracic spinal cord. The number of GRP+ fibers in lichenified skin also significantly
correlated to the amount of staining of GRPR in the superficial (r=0.94, p=0.01) and
deep (r=0.89, p=0.03) lamina of the dorsal horn of each primate (Fig. 2e).
GRPR and its ligand GRP are highly expressed in the spinal cord and skin of primates
with chronic itch, which to our knowledge is previously unreported. It is consistent
with previous finding in non-chronic itch mice models (Andoh et al., 2011; Sun and
Chen, 2007; Sun et al., 2009). Therefore, the GRP/GRPR system is a promising drug
target for the treatment of chronic pruritus in humans. Furthermore, this subset of
female Cynomolgus macaques offers a novel model of itch that could better represent
chronic itch in humans. This would allow for long term follow up studies assessing
other mediators involved in chronic itch.