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www.elsevier.com/locate/brainres
Brain Research 1019 (2004) 255–258
Research report
Increased substance P and tumor necrosis factor-a level in the paws
following formalin injection in rat tail
Mauro Bianchia,*, Cataldo Martuccia, Gabriele Biellab, Paolo Ferrarioa, Paola Sacerdotea
aDepartment of Pharmacology, University of Milano, Via Vanvitelli 32, Milan 20129, Italyb Istituto di Bioimmagini e Fisiologia Molecolare CNR, Via Fratelli Cervi 93, Segrate 20090, Italy
Accepted 7 June 2004
Available online 4 July 2004
Abstract
We previously described a rat model where the injection of formalin in the tail induced a facilitation of the hindpaw withdrawal
reflexes (hyperalgesia). In the present work, after injecting formalin in the tail, we measured the levels of pro-nociceptive mediators
tumor necrosis factor-a (TNF) and substance P (SP) in the rat paws. A significant increase of SP levels was evident in the hindpaw,
whereas no changes in SP were observed in the forepaw. Both in the hindpaw and in the forepaw the TNF levels were higher than
normal at each stage of measurement. Our results indicate that a prolonged neuronal activation induced by formalin injection is
associated with a change in nociceptive and inflammatory mediators in distal sites of the body. The fact that SP levels are changed in
the hindpaw but not in the forepaw might point to the activation of a mechanism of retrograde signaling from central synapses to paw
afferent nerves.
D 2004 Elsevier B.V. All rights reserved.
Theme: Sensory systems
Topic: Pain modulation: anatomy and physiology
Keywords: Cytokine; Hyperalgesia; Pain mediator; Retrograde signaling
1. Introduction
We have previously shown that the injection of
formalin in the rat tail produces a facilitation of the
responses to noxious thermal stimuli applied to the
hindpaw [2]. It has been demonstrated that this threshold
reduction (hyperalgesia) is due to the sensitization of the
postsynaptic neurons in the spinal segments, induced by
‘inappropriate’ projections from the tail on sciatic pro-
jection fields [6]. This model of centrally mediated
hyperalgesia has been used for evaluating the analgesic
effects of several drugs [3,4,1]. We showed also that,
after the formalin injection in the tail, the forepaws did
not show any threshold change to noxious stimuli, thus
confirming the somatotopic hypothesis [2]. Additional to
these evaluations, we now decided to explore possible
retrograde signaling mechanisms involved in the periph-
0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2004.06.007
* Corresponding author. Tel.: +39-02-50316930; fax: +39-02-
50316949.
E-mail address: [email protected] (M. Bianchi).
eral sensitization. Retrograde signaling in mature adult
neurons is a critical factor in the enhancement and
stabilization of synaptic activity generating LTP–LTD
phenomena [16]. These mechanisms are deeply involved
in the development of persistent pain. It has recently
been provided evidence of a widespread role of tumor
necrosis factor-a (TNF) in mediating hyperalgesia at
different levels [14], both facilitating neuronal excitability
and triggering the release of other pro-inflammatory
substances [15,17].
Substance P (SP) is another mediator that plays a key
role in the nociceptive input transmission in the spinal
cord [12]. It is also the major mediator of neurogenic
inflammation released by primary sensory afferent fibers
activated by noxious stimuli in the skin and in the joints
[19,22].
In most of the studies conducted on the involvement of
cytokines and SP in hyperalgesia, this latter is achieved by
the injection of inflammatory substances in the same site
where the hyperalgesia is to be evaluated. With this
approach, however, it is difficult to identify all the mech-
M. Bianchi et al. / Brain Research 1019 (2004) 255–258256
anisms involved in this phenomenon, and differentiate
between peripheral and central events.
In the rat, it has been demonstrated that the unilateral
stimulation of one paw can induce neurogenically mediated
changes also in the contralateral side [7,8].
In light of all the above cited observations, we thought
it might prove interesting to evaluate the levels of SP and
TNF in the paws following the injection of formalin in the
rat tail. We wanted to investigate with a designed bio-
chemical approach whether or not the persistent activation
of the nociceptive fibers in the tail was able to activate a
mechanism of retrograde signaling from the central syn-
apses to the peripheral afferent nerves in the paw, produc-
ing a modification of hyperalgesic mediators within the
paw.
2. Materials and methods
All the experiments were carried out on adult male
Sprague–Dawley rats (Charles River, Calco Italy), weigh-
ing 200–250 g, and housed in groups of four per cage, at
22F 2 jC with a light–dark cycle of 12:12 h and free
access to water and food. Eight rats were used in each
experimental group.
The ethical guidelines of the International Association
for the Study of Pain (IASP) were adhered to in the study
[23]. To induce a state of hindpaw hyperalgesia, a solution
of formalin (10% formaldehyde, 100 Al) was injected
intradermally in the distal part of the tail; 50 Al of this
solution was injected in the right side of the tail, and 50 Al inthe left side [2]. We chose to inject the solution in both sides
of the tail simply to obtain a more homogeneous distribution
of the formalin in the tail. The method of Hargreaves [9]
was used to assess the paw nociceptive thresholds to thermal
stimuli and hyperalgesia after formalin injection in the tail.
We used a Plantar Test apparatus (Ugo Basile, Comerio,
Italy). In brief, the rats were placed in a clear plastic
chamber and left to acclimatise for 5 min before testing.
Light from an 8 V–50 W halogen bulb (64607 OSRAM)
was delivered to the plantar skin of the middle part of the
rat’s hind and front paw through the base of the plastic box.
The beam was about 12 mm in diameter. The time taken for
the animal to withdraw its left paw was measured. We had
previously observed that, following formalin injection in the
tail, similar changes in nociceptive thresholds are induced in
both hindpaws. Hyperalgesic state was assessed by delta
reaction time (basal latency–test latency), positive results
thereby indicating hyperalgesia. The animals were tested
immediately before and 30 min, 1 and 24 h after formalin
injection. These points in time were chosen since we
previously demonstrated that between 30 and 60 min after
formalin injection the hyperalgesia is particularly evident
[2,1]. In order to avoid tissue damage and paw inflamma-
tion, only one measurement was taken at each time point.
Control animals did not receive any treatment.
Immediately after the evaluation of hyperalgesia, the
animals were killed by decapitation, and the entire hindpaw
and forepaw skin, both right and left, removed. The tissue
samples were weighed, frozen on dry ice and stored at � 70
jC until further processing for TNF and SP measurement.
Blood samples were collected, allowed to stand on ice for
2 h, centrifuged and serum was stored at � 70 jC.For TNF evaluation, skin samples of the right paws were
homogenized in 3 ml of phosphate-buffered saline (PBS)
containing 10 mM EDTA and 20 KIU/ml aprotinin (Sigma).
After centrifugation at 10,000� g, the supernatant were
frozen at � 70 jC for TNF assay.
For SP evaluation, skin samples of the left paws were
homogenized in 3 ml of 0.1 N acetic acid, centrifuged at
10,000� g and the supernatants frozen at � 70 for radio-
immunoassay.
The levels of TNF in blood and paw supernatants were
measured by means of an enzyme-linked immunosorbent
assay (ELISA) kit specific for rat TNF (Bender Medsystem,
Prodotti Gianni, Milano, Italy). The anti-TNF capture
monoclonal antibody (mAb) (5 Ag/ml) was absorbed on a
polystyrene 96-well plate and the TNF present in the sample
was bound to the antibody-coated wells. The biotinylated
anti-TNF detecting mAb (0.25 Ag/ml) was added to bind
TNF captured by the first antibody. After washing, strepta-
vidin-peroxidase was added to the wells to detect the
biotinylated detecting antibody and finally TMB substrate
was added. A colored product was formed in proportion to
the amount of TNF present in the sample, which was
measured at optical density 450 nm. The amount of cytokine
in each supernatant was extrapolated from the standard
curve. The standards were recombinant cytokine curves
generated in doubling dilutions from 2500 to 39 pg/ml.
SP was measured by radioimmunoassay (RIA), using
antiserum and methods previously described and validated
[5]. The antibody was raised in rabbit against synthetic SP,
and it is directed towards the C-terminal of the peptide;125I-SP was purchased from Amersham Biosciences
(Milano, Italy). Sensitivity of the radioimmunoassay is
10 pg/tube and intraassay and interassay variation coeffi-
cient are 8% and 11%.
Difference between controls and experimental groups
were analyzed by one-way analysis of variance (ANOVA),
followed by Bonferroni’s t-test for multiple comparisons. An
effect was determined to be significant if the P-value was less
than 0.05.
3. Results
As expected, the formalin injection in the tail induced a
significant hyperalgesic response in the hindpaw (Table 1).
This effect was evident 30 and 60 min after formalin
treatment, but had disappeared 24 h later. The values refer
to six different groups of eight rats, since the animals were
killed immediately after undergoing the plantar test. The
Fig. 2. SP levels in the hindpaw and forepaw as measured at different time
points after formalin injection in the rat tail. Controls (CTR) were naive,
untreated, animals. Values are meansF S.D. of eight rats. *P < 0.01 vs.
controls.
Table 1
Effect of formalin injection in the tail on forepaw (left paw) and hindpaw
(left paw) withdrawal reflexes
30 min 60 min 24 h
Forepaw
Controls 0.2F 0.3 0.4F 0.5 0.04F 0.3
Formalin 0.6F 0.2 0.5F 0.4 0.1F 0.1
Hindpaw
Controls 0.1F 0.1 0.05F 0.2 0.07F 0.2
Formalin 3.2F 0.6* 3.4F 0.5* 0.3F 0.2
Values are the meansF S.E.M. of the algebraic difference between basal
and test latencies. N= 8 rats
*P< 0.05 vs. controls.
M. Bianchi et al. / Brain Research 1019 (2004) 255–258 257
table also shows that the formalin injection in the tail did not
produce any change in the forepaw withdrawal latencies at
any time considered.
A significant change of TNF paw levels was observed
after formalin injection in the tail. The levels of this
cytokine were higher compared with those of control
animals both in the hindpaw and in the forepaw (Fig. 1).
TNF levels increased 30 min and 1 h after formalin
injection, and remained elevated even 24 h later.
Neither in control nor in formalin-treated animals did
this cytokine reach detectable serum levels (data not
shown).
The SP levels in rat paws of formalin-injected and
control animals are reported in Fig. 2. In the hindpaw,
no significant changes in SP concentration were present 30
min after formalin injection, whereas a significant increase
in this peptide was evident 1 h later. The hindpaw levels of
SP were higher than in controls even 24 h later. Interest-
ingly, we did not observe any modification of SP levels in
the forepaw (Fig. 2).
Fig. 1. TNF levels in the hindpaw and forepaw as measured at different time
points after formalin injection in the rat tail. Controls (CTR) were naive,
untreated, animals. Values are meansF S.D. of eight rats. *P< 0.01 vs.
controls.
4. Discussion
In this study, we have found that formalin injection in the
tail leads to changes in paw SP and TNF levels. It is worth
noting that these two mediators are affected differently by the
formalin injection. In fact, SP levels increased only in a body
area (the hindpaw) somatotopically related to that of the
injection site (the tail), while TNF showed a diffuse, non-
somatotopic, pattern of modification (both forepaw and
hindpaw). Peripheral sensory neurons release SP both ortho-
dromically, with laminar distribution into the dorsal horn of
the spinal cord, and antidromically at the other fiber terminals
of the afferent arch, in the peripheral tissue [10,19,21,22]. We
previously demonstrated that the formalin injection in the tail
leads to the hyperactivity of the spinal lumbar neurons both in
the proper and neighboring projection fields [6]. It is likely
that this mechanism is the background of the hyperalgesic
response in our behavioural test. In the light of these obser-
vations, we can now hypothesize that the ‘‘improperly’’
responding neurons of the lumbar sciatic projection field
activated by the tail tonic stimulation can transmit signals to
primary afferent neurons from the paw. This hypothesis
implies that the antidromic release of SP from hindpaw
primary afferents may occur without a direct stimulation of
peripheral proper nociceptors, thus providing biochemical
evidence that would suggest that the informational exchange
at the central synapses is bi-directional [7,8,16].
TNF is a cytokine produced by a variety of cell types,
including inflammatory cells (i.e. neutrophils, activated
lymphocytes, and macrophages) and tissue cells (endothelial
cells, smooth muscle fibers, keratinocytes, fibroblasts). In
recent studies, it has been shown that also glial and neuronal
cells are able to produce TNF [11,17]. In our model of
hyperalgesia, a significant elevation of TNF was present in
M. Bianchi et al. / Brain Research 1019 (2004) 255–258258
both the hindpaw and forepaw, suggesting a general, non-
specific spread of the cytokine after the induction of a
peripheral damage. This observation is consistent with the
results of other studies, where the inflammation induced in
the paw of one side by the injection of complete Freund’s
Adjuvant produced an increase of TNF levels also in the
contralateral, non-inflamed paw [20]. Thus, increased TNF
in both hindpaw and forepaw might represent a general
inflammatory response to formalin injection. As the TNF
level in the serum did not change, our results suggest that
the TNF increase in the paw may be the result of a local
production related to the perception of pain.
In our experimental conditions, the TNF and SP changes
do not correlate with the development of the hyperalgesic
state. In fact, a significant increase of TNF was present also
in the forepaw, while no hyperalgesia was detectable in this
body region. In addition, the change in SP levels show some
disparity with behavioural modifications. Indeed, the in-
crease in this mediator was confined to the hindpaw, but the
time course of the hyperalgesia was different from that of SP
tissue changes. While in fact the hyperalgesic state appeared
30 min after tail formalin injection, the SP levels started to
increase only 1 h after injection. Moreover, both TNF and
SP were still higher 24 h later, when no hyperalgesia was
present. Therefore, we can assume that peripheral TNF and
SP are not sufficient to sustain the hindpaw hyperalgesic
state in our experimental model. This observation is con-
sistent with the knowledge that inflammatory hyperalgesia
is a complex phenomenon produced by a considerable
number of mediators [13,18].
In conclusion, we have showed here that a persistent
stimulus applied to a peripheral site (the tail) is translated
into biochemical changes at distal sites.
The present data contribute to the identification of the
changes in the sensory neural pathways that take place in
this model of hyperalgesia.
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