The new chemokine Prokineticin 2 (PROK2) and its receptors (PKR1 and PKR2) have a role in inflammatory pain and immunomodulation. Here we identified PROK2 as a critical mediator of neuropathic pain in the chronic constriction injury (CCI) of the sciatic nerve in mice and demonstrated that blocking the prokineticin receptors with two PKR1-preferring antagonists (PC1 and PC7) reduces pain and nerve damage. PROK2 mRNA expression was upregulated in the injured nerve since day 3 post injury (dpi) and in the ipsilateral DRG since 6 dpi. PROK2 protein overexpression was evident in Schwann Cells, infiltrating macrophages and axons in the peripheral nerve and in the neuronal bodies and some satellite cells in the DRG. Therapeutic treatment of neuropathic mice with the PKR-antagonist, PC1, impaired the PROK2 upregulation and signalling. This fact, besides alleviating pain, brought down the burden of proinflammatory cytokines in the damaged nerve and prompted an anti-inflammatory repair program. Such a treatment also reduced intraneural oedema and axon degeneration as demonstrated by the physiological skin innervation and thickness conserved in CCI-PC1 mice. These findings suggest that PROK2 plays a crucial role in neuropathic pain and might represent a novel target of treatment for this disease.
Identification of the neurobiological processes engaged in the pathological state that occurs during neuropathic pain may provide future therapeutic targets. Chemokines and their receptors are receiving growing interest as modulators of neuronal plasticity and for their ability to enhance nociceptive transmission under conditions of neuropathic pain [
In an animal model of CFA-induced paw inflammation, we brought evidence that Bv8/PROK2, upregulated in granulocyte invading the inflamed tissue is a major determinant in triggering and maintaining inflammatory pain [
The typical immune cell response to tissue injury is largely conserved in the lesioned peripheral nervous system: many neutrophils from the circulation invade the area immediately around the nerve injury site within 8–24 hours and haematogenous macrophages by 3-4 days post injury, whereas lymphocyte accumulation in the injured nerve is delayed by a week or more [
We can reasonably presume that PROK2 released by haematogenous neutrophils invading the damaged nerve triggers inflammation and contributes to pain.
Besides, in the immune system, PROK2 is also expressed in discrete nuclei into the brain and is constitutively expressed, at very low levels, in some DRG neurons also expressing the vanilloid receptor TRPV1 [
We have recently demonstrated in an animal model of neuropathic pain, the chronic constriction injury of the sciatic nerve (CCI), that 10 days after nerve injury an important activation of the prokineticin system is evident at peripheral and central level and that pharmacological blocking of the prokineticin receptors with the antagonist PC1 abolishes pain and controls some pathophysiological processes underlying the neuropathy.
Here we analysed the time-course of PROK2 upregulation in the DRG and in the injured sciatic nerve of mice repeatedly treated with saline or with PC1 for 7 days and demonstrated that the antihyperalgesic effect of PC1 temporally correlates with its ability to reduce the neuropathy-induced increase of PROK2 expression. The recent availability of a new prokineticin receptor antagonist, PC7, endowed with higher affinity and selectivity for the PKR1 pushed us to evaluate its antihyperalgesic effect in comparison with that of PC1 [
Experiments were carried out in male CD1 mice (25–30 g, Harlan Laboratories, Italy) according to protocols approved by the Animal Care and Use Committee of the Italian Ministry of Health and in compliance with the IASP and European Community (E.C.L358/118/12/86) guidelines. All efforts were made to minimize animal suffering and to reduce the number of animals used. Animals were housed individually in cages, under conditions of optimum light, temperature, and humidity (12 : 12 h light/dark cycles, 22± 2°C, 50–60%) with food and water ad libitum and acclimatized to the environment for 4-5 days before surgery or pharmacologic treatment.
Mononeuropathy was induced by the CCI of the sciatic nerve [
Behavioural experiments were carried out by researchers blind to treatments, between 10 am and 2 pm, in a reserved quiet temperature-controlled room. Mice were habituated to the testing environment and were handled daily two times for at least three days before baseline testing.
For testing heat sensitivity, animals were put in plastic boxes and allowed 30 min for habituation before examination. Heat sensitivity was tested by radiant heat using Hargreaves apparatus (Ugo Basile, Italy) and expressed as paw withdrawal latency (PWL). The radiant heat intensity is adjusted so that basal PWL is between 10 and 12 s with a cutoff of 20 s to prevent tissue damage. For testing mechanical sensitivity, animals were put in boxes on an elevated metal mesh floor and allowed 30 min for habituation before examination. Mechanical allodynia was assessed using the Dynamic Plantar Aesthesiometer (Ugo Basile Italy). The filament was applied to the skin of the midplantar area of the hind paw, and it began to exert an increasing upward force, reaching a maximum of 30 g in 10 s, until the paw was withdrawn. The paw withdrawal threshold (PWT) was defined as the force, in grams, at which the mouse withdrew its paw. PWL and PWT of ipsilateral and contralateral paws were measured thrice, and the reported value is the mean of the three evaluations.
The PKR antagonists PC1 [
PC1 was administrated chronically at the dose of 150
Increasing doses of PC7 (5, 15, and 45
PC7 (2-(5-(4-fluorobenzyl)-1-(4-methoxybenzyl)-1,4,5,6-tetrahydro-4,6-dioxo-1,3,5-triazin-2-ylamino)-ethyl)-guanidine) is a triazinic compound which displays higher affinity for the PKR1 (IC50 =
Mice were divided as follows: (i) CCI mice treated with saline (CCI-saline,
In sham, CCI-saline, and CCI-PC1 or CCI-PC7 animals, mechanical allodynia and thermal hyperalgesia were assessed before and from days 1 to 12 after CCI.
Total RNA was extracted from L4-L5 DRG and sciatic nerve, pooled from two mice, using the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction. RNA yield and purity were determined by spectrophotometry absorption at 260 and 280 nm. To obtain cDNA, 1
Ten days after injury nerve samples were homogenized in ice-cold phosphate-buffered saline containing a protease inhibitor cocktail (Roche Diagnostics, Monza, Italy). IL-6, IL-1
At 10 dpi DRG, sciatic nerve and plantar skin were dissected from transcardially perfused (PBS followed by 4% paraformaldehyde) mice embedded in cryostat medium and frozen. Serial sections (20
The specificity of the anti-PROK2 antibody was tested preadsorbing it with the protein PROK2 (500 ng) overnight at 4°C before incubation with tissue.
At 10 dpi the skin samples from plantar surface of sham, CCI-saline, and CCI-PC1 mice were embedded in paraffin in a correct orientation, so that they could be sectioned perpendicular to the skin surface.Plantar skin sections (5
The permeability of blood nerve barrier (BNB) was determined by Evans Blue dye extravasation in sciatic nerve, 3 dpi. Evans Blue dye (4%, 5 mL/kg) was injected into the tail vein of anaesthetized mice. After 30 min animals were perfused with PBS. The sciatic nerves were removed and incubated in 1 mL formamide (Sigma-Aldrich) at 60°C for 24 h. Evan’s Blue concentration was determined using a spectrophotometer (Shimadzu UV-160A) at a wavelength of 620 nm.
The data are expressed as
Results are expressed as mean ± SEM. When appropriate, One-way ANOVA followed by Tukey’s posttest for multiple comparisons or Two-way ANOVA followed by Bonferroni’s posttest repeated measures were performed using GraphPad Prism 5 for Windows version 5.4. Differences were considered significant at *°
Figure
Repeated systemic injections of PC1 (150 μg kg−1, twice a day) from 3 to 9 dpi reverted the CCI-induced thermal hyperalgesia (a) and mechanical allodynia (b) in two days. The antihyperalgesic effect lasted after treatment withdrawal, for all the observation period. Data represent means ± SEM of 6–9 mice. Two-way ANOVA was used for statistical evaluation, followed by Bonferroni’s test. °°°
Therapeutic treatment of CCI mice with PC1 from day 3 (when hyperalgesia peaks) to day 9 post injury (dpi) abolished thermal and also mechanical hyperalgesia so that from 6 up to 12 dpi PWL and PWT of injured paw did not differ from those of sham mice (Figure
Availability of a new Bv8 antagonist, named PC7, able to antagonize the Bv8-induced hyperalgesia at doses ten times lower than PC1 and endowed with higher selectivity for the PKR1 [
Antihyperalgesic effect of PC7. A single bolus s.c. injection of PC7 (5, 15, and 45 μg kg−1) on day 3 after CCI dose-dependently reverted the established CCI-induced thermal hyperalgesia (a) and mechanical allodynia (b). The highest dose of PC7 (45 μg kg−1) abolished hyperalgesia for about 3 h. Repeated systemic injections of PC7 (45 μg kg−1, twice a day) from 3 to 7 dpi significantly reduced the CCI-induced thermal hyperalgesia (c) and mechanical allodynia (d) for all the observation period. Data represent means ± SEM of 5 mice. Two-way ANOVA was used for statistical evaluation, followed by Bonferroni’s test. °
The prompt reversal of pain after acute administration depends on blocking the PKRs on peripheral sensory neurons. Indeed, as we have already demonstrated both receptors mediate the Bv8/PROK2-induced decrease of nociceptive threshold: PKR1 in cooperation with TRPV1 is the receptor mainly responsible for thermal hyperalgesia and PKR2 mainly contributes to mechanical allodynia [
In a previous paper we have already demonstrated that at 10 dpi PROK2 was overexpressed in the periphery and in the spinal cord of neuropathic mice but was maintained close to physiological levels in neuropathic mice treated with PC1 for 7 days.
Here we studied in detail the time-course (from day 1 to 17) of injury-induced PROK2 mRNA overexpression in the peripheral nervous system. PROK2 levels in the peripheral nervous system of healthy animals were negligible. RT-PCR gave
Time-course of PROK2 mRNA expression in injured sciatic nerve and ipsilateral DRG of CCI-saline mice and CCI-PC1 mice. PROK2 levels in the peripheral nervous system of healthy animals were negligible (
Then we performed a detailed analysis of the cellular localization and modulation of PROK2 in the peripheral nervous system at 10 dpi, the time of its maximal expression.
At 10 dpi, immunohistochemical analysis demonstrated a strong increase of immunoreactive protein PROK2 in the ipsilateral DRG of neuropathic mice respect to DRG of sham-operated mice, where the PROK2 signal was very faint (Figures
Representative sections of mouse L4-L5 ipsilateral DRG, at 10 dpi, from sham (a), CCI-saline (b), and CCI-PC1 (c) mice. Immunofluorescence double-staining of PROK2 (green) with GFAP (marker for satellite cells, red). Cell nuclei were counterstained with DAPI (blue fluorescence). In DRG of sham-operated mice the PROK2 signal was very faint, localized only along cell membrane of some neurons, mainly small sized (arrowheads), and in few GFAP+ satellite cells (arrow) (a). In neurons of CCI-saline mice, PROK2 immunofluorescence was significantly increased and showed a vesicular cytoplasmatic pattern which is dense in proximity of the neuronal membrane (arrowheads). The number of PROK2+ satellite cells is increased (arrows). PROK2 signal in CCI-PC1 mice was comparable with that of sham mice (c). Scale bar, 30 μm. (d) Evaluation of PROK2 fluorescence intensity. One-way ANOVA was used for statistical evaluation, followed by Tukey test for multiple comparisons ***
In the sciatic nerve of sham-operated mice PROK2 immunoreactivity (green) was very faint and appeared colocalized in GFAP positive cells (yellow) (Figure
Representative images of sciatic nerve section in the immediate proximity of the injury, from sham (a), CCI-saline (b), and CCI-PC1 (c) mice at 10 dpi. (a) In the sciatic nerve of sham-operated mice PROK2 immunoreactivity (green) was very faint and colocalized with GFAP (red) in elongated SC. (b) A heavy infiltration of PROK2-positive cells (green) was evident in the nerve from CCI-saline mice. (c) PC1 treatment significantly reduced the PROK2 immunoreactivity in these cells. Scale bar, 30 μm. Immunofluorescence double-staining showing colocalization (yellow, arrowheads) of PROK2 (green) with CD11b (macrophage marker, red) (d), GFAP (Schwann cell marker, red) (e), and S100β (Schwann cell marker, red) (f) in the immediate proximity of the injury in the sciatic nerve of CCI-saline mice. (d′) CD11b (red), (e′) GFAP (red), (f′) S100β (red), and (d′′, e′′, f′′) PROK2 (green) shown in single channels. Scale bar, 10 μm. Cell nuclei were counterstained with DAPI (blue fluorescence).
Immunostaining of activated macrophages (CD11b+, red), S100β+ SC (red), and GFAP+ SC (red) in the neuroma from CCI-saline and CCI-PC1 mice ((a)–(h)). Repeated treatment with the PKR-antagonist significantly reduced the GFAP+ activated SC (i) but did not affect S100β+ SC or macrophage infiltration ((c), (f)).
In longitudinal sections of the sciatic nerve (Figures
Representative images of CCI-induced upregulation of PROK2 in the longitudinally sliced sciatic nerve proximal and distal to the lesion. At 10 dpi a dramatic increase of PROK2 signal (green, a and c) in fibres and in GFAP+ structures (yellow) was evident both proximal and distal to the lesion. The PROK2 signal was dramatically reduced in the nerve from CCI-PC1 mice (green, b and d). Scale bar: 30 μm. High-magnification images showed macrophages that infiltrate the nerve distal to the lesion (scale bar: 10 μm). (e) Double immunofluorescence labelling for PROK2 (green) and CD11b (red) showing that in the CCI-saline mice the infiltrating macrophages contain PROK2 (yellow). (f) PROK2 signal was absent in macrophages infiltrating the nerve from CCI-PC1 mice. (e′, f′) CD11b (red), and (e′′, f′′) PROK2 (green) shown in single channels. Cell nuclei were counterstained with DAPI (blue fluorescence).
Confocal images of representative sections of longitudinally sliced sciatic nerve, proximal and distal to the lesion, immunostained for PROK2 (green) and NF200 (red) from sham-operated, CCI-saline and CCI-PC1 mice at 10 dpi. Scale bar: 30 μm. PROK2-green signal was localized between NF200 positive fibers in CCI-saline mice but was not found in the nerve from CCI-PC1 mice.
Confocal images of representative sections of longitudinally sliced sciatic nerve proximal and distal to the lesion, immunostained for PROK2 (green) and CGRP (red) from sham-operated, CCI-saline and CCI-PC1 mice at 10 dpi. Scale bar: 30 μm.
Double staining with GFAP demonstrated PROK2 in SC (yellow) scattered between fibres in the proximal nerve (Figure
In the distal but not proximal injured nerve we found large CD11b+ cells many of which are also positive for PROK2 in neuropathic mice (Figure
In CCI-mice double-stained with NF200, which recognizes the heavy chain of neurofilaments
in myelinated fibres [
Double staining with CGRP, which recognizes peptidergic neurons, demonstrated the presence of PROK2 protein in CGRP-immunoreactive (IR) fibres in proximal and in distal injured nerve of saline treated mice. PROK2 signal was very low in injured nerve of PC1-treated mice (Figures
These analyses clearly demonstrate that during nerve injury a large amount of PROK2 is expressed by almost all cell types present in the nerve, both resident and infiltrating, for a sustained period of time.
Bv8/PROK2-PKRs are therefore ligand/receptor pairs in the regulation of pain sensation [
Injury of the peripheral nervous system induces immune and nonimmune cells to produce cytokines at and distal to lesion sites. Proinflammatory cytokines contribute to axonal damage and they also stimulate spontaneous nociceptor activity [
We and others have analyzed the time-course of the production of pro- and anti-inflammatory cytokines in the nervous tissues after sciatic nerve injury [
Sciatic nerve cytokine levels ten days after CCI and after seven-day PC-1 treatment.
Cytokine |
Sham | CCI-Saline | CCI-PC1 |
---|---|---|---|
TNF |
0 | 6.4 ± 3.1 | 0.97 ± 1.33° |
IL-1 |
64.26 ± 13.59 | 557.7 ± 121** | 222.5 ± 110.31° |
IL-6 | 4.92 ± 3.37 | 13.35 ± 6.15* | 4.30 ± 2.88° |
IL-17 | 0.97 ± 0.13 | 13.9 ± 1* | 3.1 ± 1.8° |
IL-10 | 556.8 ± 50.35 | 326.8 ± 8.99* | 867.3 ± 95.2°°° |
Values are means ± SD of 5 nerves.
CCI of the sciatic nerve produces partial denervation of the paw skin and results in a significant reduction in epidermal thickness of the plantar surface of the injured paw [
(a) Histological examination of the plantar skin from sham, CCI-saline, and CCI-PC1 mice stained with hematoxylin-eosin at 10 dpi. (b) Quantification of epidermal thickness of the sham, CCI/saline, and CCI/PC1 mice (3 section/animal). Data are expressed as mean ± SEM of 4-5 animals. One-way ANOVA was used for statistical evaluation, followed by Tukey test for multiple comparisons: *
According to data reported by Peleshok and Ribeiro-da-Silva [
Confocal images of representative skin sections immunostained for CGRP and NF200 from sham-operated, CCI-saline, and CCI-PC1 mice at 10 dpi. (a) In sham-operated mice the CGRP positive fibers were distributed along the dermoepidermal junction. (b) In CCI-saline mice the CGRP positive fibers were absent. (c) In CCI-PC1 mice the CGRP positive fibers were present in dermis. (d) In sham-operated mice the NF200 positive fibers were distributed along the dermoepidermal junction and in dermis. (e) In CCI-saline mice very few NF200 positive fibers were observed. (f) In CCI-PC1 mice the NF200 positive fibers were present in dermis. Dashed line represents the dermoepidermal junction. Scale bar 20 μm.
The constrictive ligatures around the sciatic nerve evoke intraneural edema that causes the nerve to strangulate beneath the ligatures and induce axotomy of mostly large-diameter myelinated axons, sparing mainly C-fibres. Peripheral nerve injury and C-fibres activation increase the blood-nerve barrier (BNB) permeabilization in 24–48 h [
Evans Blue extravasation was measured at 3 dpi in injured and contralateral sciatic nerves. A significant increase in Evans Blue accumulation was evident in the injured nerve. The level of Evans Blue in the contralateral nerve was not significantly different from the level found in sham animals. Evans blue extravasation in the sciatic nerves of mice treated with PC1 (150 μg Kg−1, twice/day, at 1 and 2 dpi) did not differ from sham mice. Data are expressed as mean ± SEM of 4-5 animals. One-way ANOVA was used for statistical evaluation, followed by Tukey test for multiple comparisons: ***
Reduced intraneural oedema may result in sparing more axons from degeneration as demonstrated by the fact that skin innervation and skin thickness of neuropathic mice treated with the PKR-antagonist look like that of sham mice.
The results that we here present support an important role for PROK2 in the pathogenesis of neuroinflammation and neuropathic pain. In support of this hypothesis, we show that blocking the prokineticin receptors with two PKR1-preferring antagonists reduced pain and nerve damage. The major finding of this study is that the nerve damage induces an important PROK2-increase in resident and infiltrating cells but also in the sensory neurons and that impairment of PROK2 production is linked to amelioration of pathology indicating PROK2 as precocious determinant in the process of neuroinflammation.
A few studies focused on the mechanisms involved in regulating the expression of PROK2 in myeloid cells demonstrated that PROK2-upregulation occurs through G-CSF-induced activation of STAT3 that binds the enhancer site of its promoter [
Intracellular pathway regulating the expression of PROK2 in neurons has not yet been clarified but it may be well-funded to imagine a mechanism like that described in myeloid cells. Indeed, STAT3 activation by G-CSF, IL-6, and IL-1
Taken together, these considerations indicate that availability of molecules, like these PKR1-preferring antagonists, which in addition to direct modulating nociceptor excitability also control the PROK2 synthesis and release, improves the efficacy in reducing neuroinflammation and neuropathic pain.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work was supported by grants from the Italian Ministry of University and Scientific Research and from Sapienza University of Rome.