Pain 123 (2006) 98–105

www.elsevier.com/locate/pain

Vasoactive intestinal peptide (VIP) is a modulator of joint pain

in a rat model of osteoarthritis
Jason J. McDougall *, Lisa Watkins, Zongming Li
Department of Physiology and Biophysics, University of Calgary, 3330, Hospital Drive NW, Calgary, Alta., Canada T2N 4N1

Received 8 December 2005; received in revised form 6 February 2006; accepted 13 February 2006

Abstract

Osteoarthritis (OA) is a debilitating disease in which primarily weight-bearing joints undergo progressive degeneration. Despite
the widespread prevalence of OA in the adult population, very little is known about the factors responsible for the generation and
maintenance of OA pain. Vasoactive intestinal peptide (VIP) was identified in the synovial fluid of arthritis patients nearly 20 years
ago and the aim of this study was to examine whether VIP could be involved in the generation of OA pain. Hindlimb weight bearing
was used as a measure of joint pain, while von Frey hair algesiometry applied to the plantar surface of the ipsilateral hindpaw tested
for secondary mechanical hyperalgesia. Intra-articular injection of VIP into normal rat knee joints caused a significant shift in
weight bearing in favour of the contralateral non-injected hindlimb as well as causing a reduction in ipsilateral paw withdrawal
threshold. These pain responses were blocked by co-administration of the VPAC receptor antagonist VIP6–28. Induction of OA
by intra-articular sodium monoiodoacetate injection resulted in a reduction in weight bearing on the affected leg, but no evidence
of secondary hyperalgesia in the paw. Treatment of OA knees with a single injection of VIP6–28 diminished hindlimb incapacitance
while increasing paw withdrawal threshold. This study showed for the first time that peripheral application of VIP causes increased
knee joint allodynia and secondary hyperalgesia. Furthermore, antagonists that inhibit VIP activity may prove beneficial in the
alleviation of OA pain.
Ó 2006 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Keywords: Algesiometry; Incapacitance; Joint pain; Neuropeptides; Osteoarthritis; Monoiodoacetate

1. Introduction During arthritis, pro-inflammatory mediators are

Disorders of the musculoskeletal system are among
the most prevalent and debilitating ailments affecting
almost a third of the adult population (WHO, 2003).
Joint diseases such as osteoarthritis (OA) lead to a loss
of mobility and function, while quality of life is severely
diminished through suffering from chronic pain. Arthrit-
ic pain is generally not treated efficiently and the side
effects of commonly used analgesic agents often limit
the effectiveness of current therapies.
*
Corresponding author. Tel.: +1 403 220 4507; fax: +1 403 210
3949.
E-mail address: mcdougaj@ucalgary.ca (J.J. McDougall).
released into the joint (Scott et al., 1994) which sensitize
joint afferent nerves such that previously innocuous
physicochemical stimuli can activate these fibres leading
to the sensation of joint pain (Schaible and Schmidt,
1985; Schaible and Grubb, 1993). One important family
of agents known to be involved in the peripheral sensiti-
zation of joint afferents is the inflammatory neuropep-
tides. These potent neuromodulators are stored in the
terminal branches of Ad and C fibres where their release
into the joint lowers the activation threshold of nocicep-
tive nerve endings thereby promoting a pain response.
Local administration of substance P and nociceptin,
for example, sensitizes joint afferents leading to
increased neuronal firing in response to mechanical rota-
tion of the knee (Heppelmann and Pawlak, 1997;

0304-3959/$32.00 Ó 2006 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.pain.2006.02.015
 J.J. McDougall et al. / Pain 123 (2006) 98–105
McDougall et al., 2000, 2001; Pawlak et al., 2001). Thus,
pharmacological control of neuropeptide activity in
arthritic joints could attenuate sensory hyperactivity
thereby ameliorating pain perception.
Vasoactive intestinal polypeptide (VIP) is a 28-amino
acid neuropeptide which has been localized in peripheral
nerves (Larsson et al., 1976; Uddman et al., 1981). In
joints, VIP is contained in postganglionic sympathetic
as well as capsaicin-sensitive sensory nerve fibres inner-
vating the joint capsule (Abramovici et al., 1991; Ahmed
et al., 1995; Catre and Salo, 1999). The function of VIP
in joints is still unclear, but local administration of the
peptide has been shown to cause synovial hyperaemia
suggesting that VIP may be involved in the control of
joint inflammation (McDougall and Barin, 2005).
Indeed, serous and synovial fluid VIP levels are signifi-
cantly elevated in arthritis patients (Lygren et al.,
1986) supporting the idea that this peptide is involved
in the pathophysiology of inflammatory joint disease.
Two G-protein-coupled receptors (VPAC1 and
VPAC2) are known to bind VIP (Harmar et al., 1998).
VPAC receptors are expressed in the dorsal horn of
the spinal cord where they are involved in the central
modulation of sensory information (Dickinson and
Fleetwood-Walker, 1999). In neuropathic pain states,
VPAC2 receptors are upregulated in laminae III–IV of
the dorsal horn (Dickinson et al., 1999) while intrathecal
administration of VPAC receptor antagonists inhibited
mustard oil-evoked electrophysiological activity of dor-
sal horn neurones (Dickinson et al., 1999). In contrast,
VPAC receptor agonists were found to have an excitato-
ry effect on previously silent dorsal horn neurones sug-
gesting that VIP is involved in the neurotransmission
of pain.
The aim of the present study was to establish whether
VIP is involved in the generation of knee joint pain and
whether blockade of peripheral VPAC receptors by local
injection of the VPAC receptor antagonist VIP6–28 could
modulate joint pain in a rat model of OA.
2. Methods
2.1. Animals
Male Wistar rats (200–365 g of weight) were used in the
present study. Animals were kept in cages at room temperature
(22 °C) under a standard 12 h light–dark cycle with free access
to water and standard rodent food. The animal handling and
experimental procedures outlined in this study all adhered to
the guidelines set out by the Canadian Council for Animal
Care.
2.2. Experimental procedures for behavioural pain testing
On three consecutive days prior to behavioural testing, rats
were regularly handled and gradually habituated to the test
equipment. Pain assessments were carried out in a quiet, isolat-
99
ed room to minimize animal anxiety. The experimental room
was kept under constant illumination and environmental tem-
perature was maintained at 20 ± 1 °C.
2.3. Weight-bearing assessment protocol
Hindlimb weight bearing was determined using an incapac-
itance tester (Linton Instrumentation, Norfolk, UK) consist-
ing of a dual channel weight averager. Weight distribution
was measured between treated (intra-articular injection of
VIP: 0.1 ml bolus; 0.01, 0.1 and 1 nmol) and contralateral
non-treated hindlimbs. Rats were carefully placed in an angled
Plexiglas chamber positioned so that each hindpaw rested on a
separate force plate. Care was taken to ensure that the animal
weight was directed onto the force plates and not dissipated
through the walls of the chamber. The force exerted by each
hindlimb (measured in grams) was averaged over a 5 s period.
Each data point is the mean of three readings. The percent
weight distributed onto the treated (ipsilateral) hindlimb was
calculated by the following equation:
½weight on ipsilateral hindlimb=ðweight on ipsilateral
þ weight on contralateralÞ  100.
2.4. Mechanical secondary hyperalgesia protocol
A von Frey hair algesiometer (Ugo-Basile, Milan, Italy) was
employed to determine the latency and force required to elicit a
withdrawal response to a tactile mechanical stimulus applied to
the hindpaw. The rat was placed on a metal mesh surface in an
enclosed area and allowed to move about freely. With the animal
at rest (i.e., in the absence of any exploratory or grooming
behaviour) a touch stimulator unit was oriented under the ani-
mal and using an adjustable angled mirror the stimulating
microfilament (0.5 mm diameter) was positioned below the
plantar surface of the paw. Activation of the unit caused a fine
plastic monofilament to advance with constant speed and touch
the paw in the proximal metatarsal region. The filament exerts a
gradually increasing force to the plantar surface, starting below
the threshold of detection and increasing until the stimulus
becomes painful and the rat removes its paw. The force required
to elicit a paw withdrawal reflex is automatically recorded and
measured in grams. A maximum force of 50 g and a ramp speed
of 4.5 g/s were chosen for all of the algesiometry tests.
2.5. Pharmacological assessment of VPAC receptors in normal
joints
Normal untreated rats were placed in an anaesthetic cham-
ber and deeply anaesthetized by gaseous inhalation of 2–5%
isoflurane/100% O2 (1 L/min). Isoflurane was then continuous-
ly delivered at 2% via an inhalation mask. The right knee joint
was shaved and 0.1 ml of either VIP (0.01, 0.1 and 1 nmol) or
vehicle control (0.9% NaCl) was injected into the right knee
joint using a 27-gauge needle. Gaseous anaesthesia was then
discontinued and animals quickly recovered within 5 min. Pain
sensitivity (weight distribution and plantar algesiometry) was
measured before (0 min) and at 10, 30, 60, 180 and 360 min
after VIP injection. In a separate group of animals, VPAC
receptor involvement in VIP responses was further tested by
100 J.J. McDougall et al. / Pain 123 (2006) 98–105

co-injecting the highest concentration of VIP with the selective rats causing the animal to re-distribute its body weight
VPAC receptor antagonist VIP6–28 (1 nmol in a total volume in favour of the non-injected contralateral limb. While
of 0.1 ml). vehicle-injected normal animals distribute their body
weight evenly between the two hindlimbs, 1 nmol VIP
2.6. Osteoarthritis experiments
caused a significant (P < 0.0001, two-way ANOVA;
n = 10 animals/group) reduction in weight bearing on
A single intra-articular injection of sodium monoiodoace-
tate was introduced into the right knee (stifle) joint as the ipsilateral hindlimb (Fig. 1A). This shift in weight
described previously (Kalbhen, 1987). This treatment inhibits distribution was apparent within the first 10 min after
chondrocyte metabolism leading to cartilage degradation con- VIP injection reaching a maximal level of incapacitance
sistent with late stage OA. Briefly, rats were deeply anaesthe- at the 30 min time point with rats placing only 38% of
tized by isoflurane inhalation (2–5% isoflurane; 100% O2 at their body weight on the injected leg. Thereafter, joint
1 L/min). Following abolition of the hindpaw pinch withdraw- discomfort gradually subsided such that hindlimb
al reflex, a 27-gauge needle was introduced into the joint cavity weight distribution returned to control levels 6 h after
through the patellar ligament and between the tibial plateau VIP administration.
and femoral condyles. Once in place, 50 ll of 3 mg sodium
monoiodoacetate was injected into the joint and the animal
was allowed to recover for 14 days prior to pain assessment.
It has consistently been shown that this dose of sodium mono-
iodoacetate causes osteoarthritic-like focal lesions in articular
cartilage and subchondral bone thickening 14 days after
administration (Guingamp et al., 1997; Bove et al., 2003).
Pain assessments were subsequently carried out in sodium
monoiodoacetate treated animals by hindlimb incapacitance
and mechanical hyperalgesia using the same protocols as
described above. Osteoarthritic animals were randomly
assigned to two distinct groups. Group 1 animals were treated
with an intra-articular injection of VIP6–28 (0.1 ml bolus, 0.01,
0.1 and 1 nmol) into the ipsilateral osteoarthritic knee. The
other cohort of rats received an equal volume of 0.9% saline
and served as osteoarthritic controls. Pain behaviour measure-
ments were carried out at 10, 30, 60, 180 and 360 min after
VIP6–28 injection as before.

2.7. Statistical analysis

All data were normally distributed and were expressed as

means ± SEM. Differences between experimental groups were
analysed by two-way ANOVA with a Bonferroni post hoc test
to determine statistical differences between matching data
points. Concentration–response relationships were analysed
by one-way ANOVA with a Dunnett’s post hoc test. Area
under the curve analysis was used to compare time courses
associated with the mechanical hyperalgesia experiments. A
value of P < 0.05 was considered significant.

2.8. Drugs and reagents

Rat vasoactive intestinal peptide (VIP) was supplied by
Calbiochem–Novabiochem Corporation (La Jolla, CA,
USA); VIP6–28 and sodium monoiodoacetate were obtained
from Sigma–Aldrich (Ontario, Canada). All drugs were dis-
solved in 0.9% saline and stored at 20 °C until required.
3. Results
3.1. Effect of VIP on hindlimb weight distribution
Intra-articular injection of VIP caused a painful
response in the ipsilateral treated hindlimb of normal
Fig. 1. (A) Effect of intra-articular injection of 1 nmol VIP on
hindpaw weight distribution compared to vehicle (saline) control.
Following VIP injection, body weight was redistributed onto the non-
injected contralateral hindlimb while co-administration of the VPAC
receptor antagonist VIP6–28 (1 nmol) attenuated this VIP-induced
weight-bearing deficit. Compared to saline controls, the incapacitance
effect of VIP was consistent across the entire time course of assessment
(P < 0.0001 two-factor ANOVA; ***P < 0.001 Bonferroni post hoc
test; n = 10 animals/group). Data are means ± SEM. (B) Concentra-
tion–response relationship of VIP on hindlimb weight bearing taken
30 min after injection. Joint incapacitance produced by VIP was found
to be dose-dependent (P < 0.0001) with only the highest two concen-
trations of the neuropeptide causing a significant weight-bearing deficit
(**P < 0.01 Dunnett’s post hoc test; n = 8–10 animals/group). Data
are means ± SEM.
 J.J. McDougall et al. / Pain 123 (2006) 98–105
In order to determine whether VPAC receptors are
involved in VIP-induced incapacitance, the effect of
the VPAC receptor antagonist VIP6–28 on VIP responses
was tested. Co-injection of 1 nmol VIP6–28 and 1 nmol
VIP (0.1 ml total volume, intra-articular) completely
blocked VIP-mediated allodynia (P < 0.0001; Fig. 1A).
The weight-bearing deficit produced by VIP was
found to be dose-dependent (P < 0.0001, one-way
ANOVA; n = 8–10 animals/group; Fig. 1B).
3.2. Effect of VIP on mechanical secondary hyperalgesia
Fig. 2 shows the effect of intra-articular VIP injection
on mechanical hyperalgesia compared to saline vehicle
Fig. 2. (A) Time course of secondary hyperalgesia in the ipsilateral
paw following intra-articular injection of 1 nmol VIP. The force
required to elicit hindpaw withdrawal following VIP treatment was
significantly reduced compared to saline-injected animals and this
effect was blocked by co-administration of the VPAC receptor
antagonist VIP6–28. Maximal effect of VIP occurred 30 min after
injection (*P < 0.05 Bonferroni post hoc test; n = 10). Data are
means ± SEM. (B) Effect of different concentrations of VIP on
secondary hyperalgesia. The relationship was found to be dose-
dependent (P < 0.01, one-way ANOVA); however, only the highest
concentration of VIP caused a significant hyperalgesic effect
(**P < 0.01 Dunnett’s post hoc test; n = 10). Data are means ± SEM.
101
control. The force required for hindpaw withdrawal in
VIP treated rats (1 nmol) was significantly reduced com-
pared to saline-injected animals (P < 0.0001, two-way
ANOVA; n = 10 animals/group, Fig. 2A). The maximal
effect on mechanical hyperalgesia occurred 30 min after
VIP injection where paw withdrawal threshold was
found to be 25.4 ± 1.9 g compared to 33.5 ± 1.8 g in sal-
ine-injected animals.
The hyperalgesic effect of intra-articular VIP was
blocked by co-administration of the VPAC receptor
antagonist VIP6–28 (Fig. 2A). Two-factor ANOVA con-
firmed that the inhibitory effect of the antagonist was
consistent over the entire assessment period such that
the paw withdrawal threshold did not differ significantly
from saline-injected controls (P = 0.131). Compared to
pre-treatment controls, VIP caused an 11% decrease in
the area under the curve while only a 1% increase in
the area under the curve was observed in the saline
and VIP + VIP6–28 groups.
Concentration–response assessments found that VIP-
induced changes in hindpaw withdrawal threshold were
dose-dependent (P < 0.01; Fig. 2B).
3.3. Inhibition of osteoarthritis incapacitance by VIP6–28
In osteoarthritic animals treated with vehicle, the
amount of weight placed on the ipsilateral hindlimb
was reduced to about 40% (Fig. 3A). Intra-articular
injection of 1 nmol VIP6–28 caused a rapid (within
10 min) attenuation of this painful effect such that hind-
limb weight distribution was almost 50:50 between
osteoarthritic and contralateral non-arthritic joints.
The analgesic effect of VIP6–28 lasted for about 1 h after
injection although a slight, but statistically significant,
inhibition of incapacitance was observed 6 h after
administration of the VPAC receptor antagonist.
Treatment of OA knees with 0.01 and 0.1 nmol
VIP6–28 had no significant effect on joint incapaci-
tance while the 1 nmol dose did reduce the weight
bearing deficit (Fig. 3B) confirming that the effect
of the drug was dose-dependent (P < 0.0001).
3.4. Inhibition of hindpaw withdrawal thresholds in OA
rats by VIP6–28
Treatment of osteoarthritic knees with an intra-artic-
ular injection of 1 nmol VIP6–28 abrogated plantar
hyperalgesia. Force to withdrawal was significantly ele-
vated compared to saline-injected arthritic joints indica-
tive of an analgesic effect of the VPAC receptor
antagonist (Fig. 4A). Inhibition of pain behavioural
responses occurred 10 min after VIP6–28 treatment and
lasted for approximately 3 h. Compared to pre-treat-
ment control, saline injection caused a 5% decrease in
the area under the curve whereas VIP6–28 treatment pro-
duced a 58% increase in the area under the curve.
102 J.J. McDougall et al. / Pain 123 (2006) 98–105
Fig. 3. (A) Ipsilateral weight distribution in saline treated and VIP6–28
treated OA knee joints. In vehicle-treated animals, approximately 40%
of the animal’s body weight is placed on the arthritic hindlimb.
Treatment with a single intra-articular injection of VIP6–28 significantly
reduced the pain associated with the OA lesion (P < 0.0001 two-way
ANOVA, ***P < 0.001, **P < 0.01, *P < 0.05 Bonferroni post hoc
test; n = 10 animals/group). Data are shown as means ± SEM.
(B) Concentration–response of VIP6–28 on OA joint incapacitance.
Compared to saline treated controls, the analgesic effect of the
antagonist was only significant with the highest concentration of the
drug (P < 0.0001 one-way ANOVA, **P < 0.01 Dunnett’s post hoc
test). Data are means ± SEM.
Comparison of different concentrations of VIP6–28
revealed a dose-dependent effect of the VPAC receptor
blocker on paw withdrawal threshold in OA knees
(P < 0.0001; Fig. 4B).
4. Discussion
Epidemiological studies have revealed that over 70%
of people aged 65 years or older suffer from OA (Felson,
1995) with the knee joint being most commonly affected.
Despite the widespread prevalence of OA in the adult
population, very little is known about the causes of
OA pain or the chemical mediators involved in the initi-
ation of painful stimuli in OA joints. The discovery of
Fig. 4. (A) Effect of VIP6–28 treatment on hindpaw mechanical
hyperalgesia in OA rats. Force for hindpaw withdrawal was signifi-
cantly increased in VIP6–28-treated animals indicating an analgesic
effect (P < 0.0001 two-factor ANOVA; n = 10 animals/group).
**P < 0.01, *P < 0.05 Bonferroni post hoc test. Data are shown as
means ± SEM. (B) Changes in force required for hindpaw withdrawal
in OA joints in response to different doses of VIP6–28. The analgesic
effect of VIP6–28 was found to be dose-dependent across the tested dose
range (P < 0.0001 one-way ANOVA, **P < 0.01 Dunnett’s post hoc
test). Data are means ± SEM.
VIP in the synovial fluid of arthritis patients nearly 20
years ago prompted the suggestion that this polypeptide
may be involved in the pathophysiology of arthritis
(Lygren et al., 1986), although the role of VIP in joint
pain has never been tested. Here, we show for the first
time that intra-articular injection of VIP caused an acute
painful response in rat knees. Hindlimb incapacitance
measurements revealed that local injection of VIP
caused animals to re-distribute their weight in favour
of the non-injected contralateral hindlimb indicative of
hindlimb discomfort. This effect was not due to disten-
sion of the joint capsule nor the introduction of the
hypodermic needle, since an intra-articular injection of
an equal volume of vehicle had no effect on hindlimb
weight distribution. The algesic effect of VIP was found
to be rapid in onset (within 10 min after injection)
and lasted for 3 h. Blockade of the VIP response by
 J.J. McDougall et al. / Pain 123 (2006) 98–105
co-administration of the selective antagonist VIP6–28
confirmed that VIP was acting on VPAC receptors
located in the knee to elicit joint pain. Previous studies
have shown that VPAC receptors are expressed by
human fibroblast-like synoviocytes confirming a physio-
logical role for VIP in joint tissues (Takeba et al., 1999).
The precise location of VPAC receptors in joints is cur-
rently unknown, but the current data suggest that they
may be present on mechanosensory nociceptive nerve
terminals located within the joint capsule.
The present investigation also found that intra-artic-
ular injection of VIP caused secondary hyperalgesia in
ipsilateral hindpaws. Plantar algesiometry experiments
demonstrated that the force required to elicit hindpaw
withdrawal was significantly reduced following VIP
treatment. The inhibition of this response by VIP6–28
treatment again signifies VPAC receptor involvement
in this painful outcome. The precise neural pathways
involved in VIP-mediated secondary hyperalgesia are
outside the scope of this study but could involve cen-
tral sensitization of spinal cord neurones (Dickinson
et al., 1999). Primary afferents arising from the hind-
paw and the knee joint terminate in similar regions
of the dorsal horn. Peripheral sensitization of joint
afferents following VIP treatment would likely lead to
rapid central sensitization of second order neurones
in the spinal cord. Through a process of ‘‘wind-up’’,
neighbouring neurones that are coupled to the ipsilat-
eral hindpaw would also become sensitized rendering
them more sensitive to mechanosensory input from
the periphery precipitating the reduction in hindpaw
withdrawal threshold observed here. The fact that con-
tralateral paws were unaffected by VIP treatment (data
not shown) corroborates a localized wind-up phenom-
enon and disproves a general systemic effect of the
neuropeptide.
The mechanism responsible for VIP-induced joint
pain requires further investigation, but probably
involves the activation of protein kinase A. VPAC
receptors are known to be coupled to adenylyl-cyclase
activating G proteins whose stimulation leads to cAMP
generation and consequently protein kinase A activation
(Spengler et al., 1993). Central administration of protein
kinase A inhibitors dramatically reduces evoked activity
from L1 to L4 dorsal horn neurones in response to nox-
ious tactile stimuli (Young et al., 1995) indicating that
this particular second messenger system contributes to
the neurotransmission of mechanical pain. Future stud-
ies examining protein kinase A inhibition in the periph-
ery are required to determine whether similar second
messenger pathways are involved in VIP-mediated joint
nociception.
It has been known for some time that VIP can act on
mast cells to cause degranulation with subsequent
release of multiple pro-inflammatory and algogenic
agents into the extracellular space (Skofitsch et al.,
103
1983; Piotrowski and Foreman, 1985). One such agent
released by synovial mast cells is histamine whose levels
are elevated in the synovial fluid of arthritis patients
(Frewin et al., 1986; Malone et al., 1986). Electrophysi-
ological experiments have shown that local administra-
tion of histamine sensitizes knee joint afferents leading
to increased mechanosensitivity (Herbert et al., 2001).
This excitatory effect of histamine implies that VIP
may exert some of its nociceptive effects via a mast cell
dependent mechanism involving secondary release of
algogenic agents such as histamine. Although synovial
mast cells are thought to be not involved in VIP control
of joint blood flow (McDougall and Barin, 2005), future
studies should be considered to assess mast cell involve-
ment in VIP-mediated pain.
4.1. Effect of VPAC receptor blockade on OA pain
The present study also examined the effect of VPAC
receptor blockade with the selective VIP antagonist
VIP6–28 on hindlimb incapacitance and secondary
mechanical hyperalgesia in a model of OA. Fourteen
days after OA induction, rats re-distributed their body
weight 40:60% in favour of the non-arthritic contralat-
eral hindlimb. This finding is consistent with other
studies which found similar levels of hindlimb incapac-
itance following intra-articular injection of sodium
monoiodoacetate at this time point (Bove et al., 2003;
Combe et al., 2004; Fernihough et al., 2004; Pomonis
et al., 2005). The reduction in weight bearing associat-
ed with sodium monoiodoacetate treatment is known
to correlate with histologically determined OA scores
confirming that this is a viable model to assess OA
pain. Intra-articular injection of VIP6–28 significantly
attenuated hindlimb incapacitance and this analgesic
effect of the antagonist was still apparent 60 min after
treatment of the OA knee. Thus, blockade of VPAC
receptors in OA knee joints by VIP6–28 reduces articu-
lar discomfort probably by inhibiting the algesic effects
of locally released VIP.
Secondary mechanical hyperalgesia has been report-
ed in OA patients, although its occurrence is generally
rare (Bajaj et al., 2001; Kean et al., 2004). Comparison
of hindpaw withdrawal thresholds in monoiodoacetate
treated animals were comparable to normal saline con-
trols confirming a previously reported lack of second-
ary hyperalgesia in this model (Pomonis et al., 2005).
Nevertheless, VIP6–28 administration increased force
to withdrawal threshold in OA joints indicating that
VIP is having a sensitizing effect on peripheral nocicep-
tors. We may be sure that this phenomenon is due to
peripheral sensitization of joint afferents since VIP6–28
was given locally into the joint and the observed
changes in paw withdrawal threshold occurred very
quickly. Furthermore, VPAC receptor blockade had
no effect on contralateral paw withdrawal thresholds
104 J.J. McDougall et al. / Pain 123 (2006) 98–105
(data not shown) confirming that the observed
mechanosensitivity changes were confined to the inject-
ed hindlimb.
In conclusion, this is the first study to show that the
non-adrenergic/non-cholinergic neuropeptide VIP caus-
es a conspicuous pain response and secondary hyperalge-
sia when administered locally into the knee joint.
Antagonist experiments confirm that this effect is medi-
ated via VPAC receptors located in the knee. Further-
more, intra-articular injection of VIP6–28 into OA
joints alleviated joint discomfort and peripheral sensiti-
zation of knee joint afferents. Hence, control of VIP
activity by selective VPAC receptor antagonism may be
a favourable means of treating OA pain.
Acknowledgements
The financial support of the Alberta Heritage Foun-
dation for Medical Research (AHFMR), the Arthritis
Society of Canada, and the Canadian Institutes of
Health Research (CIHR) are gratefully acknowledged.
J.J.McD. is an AHFMR Scholar and a CIHR/Arthritis
Society of Canada New Investigator.
References
Abramovici A, Daizade I, Yosipovitch Z, Gibson SJ, Polak JM. The
distribution of peptide-containing nerves in the synovia of the cat
knee joint. Histol Histopathol 1991;6:469–76.
Ahmed M, Bjurholm A, Theodorsson E, Schultzberg M, Kreicbergs A.
Neuropeptide Y- and vasoactive intestinal polypeptide-like immu-
noreactivity in adjuvant arthritis: effects of capsaicin treatment.
Neuropeptides 1995;29:33–43.
Bajaj P, Graven-Nielsen T, Arendt-Nielsen L. Osteoarthritis and its
association with muscle hyperalgesia: an experimental controlled
study. Pain 2001;93:107–14.
Bove SE, Calcaterra SL, Brooker RM, Huber CM, Guzman RE,
Juneau PL, et al. Weight bearing as a measure of disease
progression and efficacy of anti-inflammatory compounds in a
model of monosodium iodoacetate-induced osteoarthritis. Osteo-
arthritis Cartilage 2003;11:821–30.
Catre MG, Salo PT. Quantitative analysis of the sympathetic
innervation of the rat knee joint. J Anat 1999;194:233–9.
Combe R, Bramwell S, Field MJ. The monosodium iodoacetate model
of osteoarthritis: a model of chronic nociceptive pain in rats?
Neurosci Lett 2004;370:236–40.
Dickinson T, Fleetwood-Walker SM. VIP and PACAP: very impor-
tant in pain? Trends Pharmacol Sci 1999;20:324–9.
Dickinson T, Mitchell R, Robberecht P, Fleetwood-Walker SM. The
role of VIP/PACAP receptor subtypes in spinal somatosensory
processing in rats with an experimental peripheral mononeuropa-
thy. Neuropharmacology 1999;38:167–80.
Felson DT. The epidemiology of osteoarthritis: prevalence and risk
factors. In: Kuettner KE, Goldberg CH, editors. Osteoarthritic
disorders. Rosemont (IL): American Academy of Orthopaedic
Surgeons; 1995. p. 13–24.
Fernihough J, Gentry C, Malcangio M, Fox A, Rediske J, Pellas T,
et al. Pain related behaviour in two models of osteoarthritis in the
rat knee. Pain 2004;112:83–93.
Frewin DB, Cleland LG, Jonsson JR, Robertson PW. Histamine levels
in human synovial fluid. J Rheumatol 1986;13:13–4.
Guingamp C, Gegout-Pottie P, Philippe L, Terlain B, Netter P, Gillet
P. Mono-iodoacetate-induced experimental osteoarthritis: a dose–
response study of loss of mobility, morphology, and biochemistry.
Arthritis Rheum 1997;40:1670–9.
Harmar AJ, Arimura A, Gozes I, Journot L, Laburthe M, Pisegna JR,
et al. International Union of Pharmacology. XVIII. Nomenclature
of receptors for vasoactive intestinal peptide and pituitary adenylate
cyclase-activating polypeptide. Pharmacol Rev 1998;50:265–70.
Heppelmann B, Pawlak M. Sensitisation of articular afferents in
normal and inflamed knee joints by substance P in the rat. Neurosci
Lett 1997;223:97–100.
Herbert MK, Just H, Schmidt RF. Histamine excites groups III and IV
afferents from the cat knee joint depending on their resting activity.
Neurosci Lett 2001;305:95–8.
Kalbhen DA. Chemical model of osteoarthritis – a pharmacological
evaluation. J Rheumatol 1987;14:130–1.
Kean WF, Kean R, Buchanan WW. Osteoarthritis: symptoms, signs
and source of pain. Inflammopharmacology 2004;12:3–31.
Larsson LI, Fahrenkrug J, Schaffalitzky De, Muckadell O, Sundler F,
Hakanson R, et al. Localization of vasoactive intestinal polypep-
tide (VIP) to central and peripheral neurons. Proc Natl Acad Sci
USA 1976;73:3197–200.
Lygren I, Østensen M, Burhol PG, Husby G. Gastrointestinal peptides
in serum and synovial fluid from patients with inflammatory joint
disease. Ann Rheum Dis 1986;45:637–40.
Malone DG, Irani AM, Schwartz LB, Barrett KE, Metcalfe DD. Mast
cell numbers and histamine levels in synovial fluids from patients
with diverse arthritides. Arthritis Rheum 1986;29:956–63.
McDougall JJ, Barin AK. The role of joint nerves and mast cells in the
alteration of vasoactive intestinal peptide (VIP) sensitivity during
inflammation progression in rats. Br J Pharmacol 2005;145:104–13.
McDougall JJ, Hanesch U, Pawlak M, Schmidt RF. Participation of
NK1 receptors in nociceptin-induced modulation of rat knee joint
mechanosensitivity. Exp Brain Res 2001;137:249–53.
McDougall JJ, Pawlak M, Hanesch U, Schmidt RF. Peripheral
modulation of rat knee joint afferent mechanosensitivity by
nociceptin/orphanin FQ. Neurosci Lett 2000;288:123–6.
Pawlak M, Schmidt RF, Heppelmann B, Hanesch U. The neurokinin-
1 receptor antagonist RP 67580 reduces the sensitization of primary
afferents by substance P in the rat. Eur J Pain 2001;5:69–79.
Piotrowski W, Foreman JC. On the actions of substance P, somato-
statin, and vasoactive intestinal polypeptide on rat peritoneal mast
cells and in human skin. Naunyn Schmiedebergs Arch Pharmacol
1985;331:364–8.
Pomonis JD, Boulet JM, Gottshall SL, Phillips S, Sellers R, Bunton T,
et al. Development and pharmacological characterization of a rat
model of osteoarthritis pain. Pain 2005;114:339–46.
Schaible H, Grubb BD. Afferent and spinal mechanisms of joint pain.
Pain 1993;55:5–54.
Schaible HG, Schmidt RF. Effects of an experimental arthritis on the
sensory properties of fine articular afferent units. J Neurophysiol
1985;54:1109–22.
Scott DT, Lam FY, Ferrell WR. Acute joint inflammation –
mechanisms and mediators. Gen Pharmacol 1994;7:1285–96.
Skofitsch G, Donnerer J, Petronijevic S, Saria A, Lembeck F. Release
of histamine by neuropeptides from the perfused rat hindquarter.
Naunyn Schmiedebergs Arch Pharmacol 1983;322:153–7.
Spengler D, Waeber C, Pantaloni C, Holsboer F, Bockaert J, Seeburg
PH, et al. Differential signal transduction by five splice variants of
the PACAP receptor. Nature 1993;365:170–5.
Takeba Y, Suzuki N, Kaneko A, Asai T, Sakane T. Evidence for
neural regulation of inflammatory synovial cell functions by
secreting calcitonin gene-related peptide and vasoactive intestinal
peptide in patients with rheumatoid arthritis. Arthritis Rheum
1999;42:2418–29.
 J.J. McDougall et al. / Pain 123 (2006) 98–105 105

Uddman R, Alumets J, Edvinsson L, Hakanson R, Sundler F. VIP
nerve fibres around peripheral blood vessels. Acta Physiol Scand
1981;112:65–70.
WHO, 2003. The burden of musculoskeletal conditions at the start of the
new millennium. World Health Organ Tech Rep Ser 2003;919:1–218.
Young MR, Fleetwood-Walker SM, Mitchell R, Dickinson T. The
involvement of metabotropic glutamate receptors and their
intracellular signalling pathways in sustained nociceptive trans-
mission in rat dorsal horn neurons. Neuropharmacology
1995;34:1033–41.