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The Role of IL-6 and IL-1b in Painful Perineural Inflammatory Neuritis PDF
The Role of IL-6 and IL-1b in Painful Perineural Inflammatory Neuritis PDF
a r t i c l e i n f o a b s t r a c t
Article history: Inammation along a nerve trunk (perineural inammation), without detectable axonal damage, has
Received 8 October 2008 been shown to induce transient pain in the organ supplied by the nerve. The aims of the present study
Received in revised form 5 January 2009 were to study the role IL-6 and IL-1b, in pain induced by perineural inammation.
Accepted 15 January 2009
Methods: IL-6 and IL-1b secretion from rats sciatic nerves, L-5 Dorsal Root Ganglia (DRG), and the hind
Available online 29 January 2009
paw skin, 3 and 8 days following exposure of the nerve to Complete Freunds Adjuvant (CFA), were mea-
sured using ELISA method. Hind paw tactile-allodynia, mechano-hyperalgesia, heat-allodynia and electri-
Keywords:
cal detection thresholds were tested up to 8 days following the application of CFA, IL-6 or IL-1b adjacent
Neuritis
Neuropathic pain
to the sciatic nerve trunk. Employing electrophysiological recording, saphenous nerve spontaneous activ-
Inammation ity, nerve trunk mechano-sensitivity and paw tactile detection threshold (determined by recording action
Cytokines potential induced by the lowest mechanical stimulus) were assessed 3 and 8 days following exposure of
IL-6 the nerve trunk to CFA, IL-6, or IL-1b.
IL-1b Results: IL-6 and IL-1b secretion from the nerve was signicantly elevated on the 3rd day post-operation
(DPO). On the 8th DPO, IL-6 levels returned to baseline while IL-1b levels remained signicantly elevated.
The DRG cytokines level was increased on the 3rd and 8th DPOs, contralateral cytokines level was
increased on the 3rd DPO. The skin IL-6 level was increased bilaterally on the 3rd DPO and returned to
baseline on the 8th DPO. IL-1b levels increased in the affected side on the 3rd and bilaterally on the
8th DPO. Direct application of IL-6 or CFA on the sciatic nerve induced signicant hind paw tactile-allo-
dynia from the 1st to 5th DPOs, reduced electrical detection threshold from the 1st to 3rd DPOs, mechan-
o-hyperalgesia from 3rd to 5th DPOs and heat-allodynia on the 3rd DPO. Direct application of IL-1b
induced paw tactile and heat-allodynia on the 78th DPOs and mechano-hyperalgesia on the 58th DPOs.
Perineural inammation signicantly increased spontaneous activity myelinated bres 3 and 8 days fol-
lowing the application. Direct application of IL-6 induced elevation of spontaneous activity on the 3rd
while IL-1b on the 8th DPO.
Nerve mechano-sensitivity was signicantly increased on the 3rd day following exposure to CFA and IL-6
and on the 8th following CFA application. The rats paw lowest mechanical force necessary for induction
of action potential, was signicantly reduced 3 days following CFA application.
Conclusion: IL-6 and IL-1b play an important role in pain induced by perineural inammation. IL-6 activ-
ity is more prominent immediately following application (25th DPOs), while IL-1b, activity is more sig-
nicant in a later stage (58th DPOs).
2009 Elsevier Inc. All rights reserved.
0889-1591/$ - see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbi.2009.01.012
E. Eliav et al. / Brain, Behavior, and Immunity 23 (2009) 474484 475
type; Parke-Davis & Co., Detroit, MI) was implanted adjacent to the rank of the threshold of the control side from that of the treated
sciatic nerve. The Oxycel acts as a sponge and is placed loosely in side. Negative difference scores thus indicate the presence of tac-
the vicinity of the nerve without causing nerve constriction or tile-allodynia; the normal difference score is zero.
damage. The Oxycel was saturated with 200 ll of a solution con- Electrical detection threshold (allodynia) was assessed using a
taining one of the following: 0.2 lg of IL-6, 0.4 lg of IL-1b, 200 ll World Precision Instrument Electrical Stimulator A-365. With the
of CFA or 200 ll of saline. The quantity of cytokine applied was se- rat placed on a metal mesh, one electrode was attached to the grid
lected based on the levels detected in experiment 1 (i.e. the level and the other (spherical, 1 mm diameter) gently placed on the rats
that was secreted within 24 h). hind paw, when the rat was immobile a 100 Hz current was in-
creased slowly (0.1 milliamperes per second) until the rat with-
2.2.1. Behavioural assays drew the hind paw. Data is expressed as the ratio of the
The rats mid-plantar hind paw (sciatic nerve territory) was threshold in milliamperes from the treated side divided by that
tested for tactile and heat-allodynia, electrical detection threshold, from the contralateral side. Ratios lower than one, thus indicate
and for tactile-hyperalgesia, prior to surgery and every other day the presence of electrical allodynia; the normal ratio is one.
thereafter for 8 days, beginning the rst day post-operation
(DPO). Rats were habituated pre-operatively by allowing them 2.3. Experiment 3: Electrophysiology properties of cytokine application
1015 min in the sensory-testing apparatus for ve consecutive on the nerve
days. During this time the rats were tested in the hind paw area
with Von Frey hairs, a pin, heat and electrical stimuli and then re- Recordings were preformed 3 and 8 days following saphenous
turned to their cages. The examiner was unaware of the treatment nerve exposure either to CFA, Saline, IL-6, or IL-1b (n = 5 rats for
groups. time point). The saphenous nerve was chosen as it is entirely sen-
Heat-allodynia was assayed with the paw withdrawal test sory and therefore allows recordings with minimal background
(Bennett and Xie, 1988; Hargreaves et al., 1988). Rats are placed activity. The nerve was exposed from the mid-calf to the ischium
on the glass oor of an elevated platform. A high intensity, mova- and skin edges were used to form a pool that was lled with
ble radiant heat source, was placed underneath the glass and warmed parafn oil (34 C). The perineurium of the nerve was in-
aimed at the plantar surface of one hind paw. Care was taken cised at the site of recording about 35 mm proximal to the treat-
to initiate the test when the animal was at rest, not walking ment site. Fine axon bundles (microlaments) were teased from
and the hind paw was in contact with the glass oor of the test the nerve, disconnected centrally and placed on a single Ag/AgCl
apparatus. Stimulus onset activated a timer that was controlled recording electrode referenced to a nearby indifferent electrode.
by a photocell. The hind paw withdrawal reex interrupted the The bundles remained in continuity peripherally. This permitted
photocells light and automatically stopped the timer. Latencies detection of spontaneous discharge originating at the site of the
of the reex were measured from the onset of radiant heat until application or distally. Each strand was observed passively for
hind paw withdrawal to the nearest 0.1 s. Each hind paw was 2 min. If in this period any spontaneous action potentials were ob-
tested four times at intervals of 5 min. The light intensity was ad- served, the period of observation was extended. For each strand we
justed at the beginning of the experiment in order to produce monitored: the presence of spontaneous activity and spike re-
latencies of approximately 10 s and held constant thereafter. sponse to nerve inammation site tactile stimulation with
We express the data as difference scores, computed by subtract- 5.88 log g monolament. Percentage of bres spontaneously active
ing the latency of the control side from that of the treated side. and percentage of spike responses were calculated for each group,
Negative difference scores thus indicate the presence of hyper- 3 and 8 days following the operation.
sensitivity; the normal difference score is approximately zero Teased microlaments differ in axon content, therefore, in order
(Bennett and Xie, 1988). to count the number of axons sampled in teased microlaments we
Mechano-hyperalgesia was assayed with the pin-prick test (Tal used the method described by Liu et al. (2000). Briey, we gradu-
and Bennett, 1994). The rat was placed on an elevated, perforated ally increased the saphenous nerve stimulus intensity while noting
oor and the tip of a 0.2 mm diameter blunted acupuncture needle the number of all-or-non action potentials recruited to saturation.
(Needle No. 3, Seirin, Japan) was pushed against the mid-plantar Microlament counts were made from a random sample of 75
hind paw, until the needle slightly bends (the skin was dimpled teased microlaments in seven intact rats. These contained a mean
but not penetrated). Under these conditions the bended needle ex- of 5.2 6.8 A bres. The number of axons sampled per experiment
erts a mean force of 10.5 g as measured on a laboratory scale. The was estimated by multiplying these means by the number of
duration of the pin-prick evoked nocifensive withdrawal reex was microlaments observed in each experiment.
timed with a stopwatch. Normal responses are of very small dura- The conduction velocity of spontaneously active axons in each
tion, too quick to time accurately by hand and were therefore arbi- microlament was determined by dividing propagation distance
trarily assigned a duration of 0.5 s. The data are expressed as by the response latency to electrical stimulus pulses delivered to
difference scores, computed by subtracting the withdrawal dura- the nerve mechano-sensitive site. The stimuli were square wave
tion of the control side from that of the treated side. Positive pulses, 0.050.1 ms in duration, at 1 Hz at variable intensities up
difference scores thus indicate the presence of mechano-hypersen- to 20 mA.
sitivity; the normal difference score is zero. For the cutaneous detection threshold (paw mechanical detec-
Tactile-allodynia was tested with Von Frey hairs (Tal and Ben- tion threshold) we identied rst the laments receptive eld,
nett, 1994) utilizing Semmes-Weinstein monolaments (Stoelting by systematically applying supra-threshold tactile stimuli to the
Inc., Wood Dale, IL). This series includes monolaments sorted by rats paw; stimulus area that evoked recorded action potential
ranks expressing the log 10 of the force applied in milligrams. We was considered as the laments receptive eld.
employed laments from 2.36 to 5.88 that apply a force of 0.02 The force applied by lowest Semmes-Weinstein monolaments
60 g, respectively. With the rat placed on the perforated oor, the lament (0.0260 g) that produced action potential was desig-
monolaments were tested in order of increasing stiffness, with nated as the detection threshold.
each applied ve times at intervals of 14 s. to slightly different The stimuli were employed in order of increasing stiffness, with
loci of the mid-plantar hind paw. The rst hair to evoke at least each applied ve times at intervals of 14 s. CFA, IL-1b and IL-6
one withdrawal response was designated the threshold. We ex- groups were compared to rats that did not have any previous sur-
press the data as difference scores, computed by subtracting the gical procedure (Naive).
E. Eliav et al. / Brain, Behavior, and Immunity 23 (2009) 474484 477
2.4. Statistical analysis manipulated control nerves (n = 4; 5.53 2.89 ng/ml/mg/24 h).
IL-6 levels were not elevated in the distal (n = 4; 70.04 10.35 ng/
Alpha (two-tailed) for signicance in all analyses was set at ml/mg/24 h; P = 0.89) and the proximal (n = 4; 73.12 6.53 ng/ml/
0.05. Data was tabulated and analyzed using StatView 5 software mg/24; P = 0.87) segments or the contralateral nerves (n = 4;
(SAS Institute Inc., NC, USA). Behavioural statistical analyses were 45.47 2.96 ng/ml/mg/24 h; P = 0.97).
performed only on rats with data at all time points. Eight days following the procedure IL-6 levels had been reduced
For tactile-allodynia, mechano-hyperalgesia, electrical thresh- to normal levels in the segment adjacent to the CFA application
olds, and heat-allodynia, time points of relevance were analyzed (n = 10; 17.30 3.09 ng/ml/mg/24 h; P = 0.22 in comparison with
with a factorial analysis of variance (ANOVA). Fishers PLSD proce- non-manipulated control nerves; n = 4; 5.53 2.89 ng/ml/mg/
dure for pairwise comparisons was selected to examine differences 24 h).
between individual groups. Within group data were compared
using a repeated measures ANOVA (RANOVA). 3.1.1.2. IL-1b. Three days following the procedure the cytokine lev-
Percent of spontaneously active nerve bres was calculated at els in the nerve segment adjacent to the inammation (n = 10;
days 3 and 8 following the procedure. Differences amongst groups 473.55 99.87 ng/ml/mg/24 h; P = 0.006; ANOVA F6,23 = 4.10) was
at these time points were analyzed with a factorial ANOVA fol- signicantly elevated compared to the level assessed in the non-
lowed by Fishers PLSD comparisons. manipulated control nerves (n = 4; 0.19 0.09 ng/ml/mg/24 h).
Levels of IL-6 and IL-1b were calculated at days 3 and 8 follow- No signicant elevation was found, in the distal (n = 4; 272.55
ing the procedure. Differences amongst groups at these time points 60.26 ng/ml/mg/24 Hz; P = 0.092) the proximal (n = 4; 152.36
were analyzed with a factorial ANOVA followed by Scheffs pair- 29.96 ng/ml/mg/24 h; P = 0.333) portions nor in the contralateral
wise comparisons. nerves (n = 4; 131.66 3.48 ng/ml/mg/24 h; P = 0.40).
Eight days following the procedure the cytokine levels re-
mained signicantly elevated in the nerve portion adjacent to the
3. Results
inammation (n = 10; 356.70 55.78 ng/ml/mg/24 h; ANOVA
F4,18 = 5.85 P = 0.004) compare to the non-manipulated control
All results in graphs and text are expressed as means standard
nerves.
error of the mean (SEM).
3.1. Experiment 1: Cytokine levels following perineural inammation 3.1.2. DRG (Fig. 2)
3.1.2.1. IL-6. Three days following the CFA application, IL-6 was sig-
3.1.1. Nerve (Fig. 1) nicantly elevated in the ipsilateral DRG (n = 4; 156.64 19.62 ng/
3.1.1.1. IL-6. Three days following the surgical procedure a signi- ml/mg/24 h ANOVA F2,8 = 76.24) compared to the naive rats DRG
cant elevation in secreted IL-6 levels was found in the nerve seg- (n = 4; 3.18 0.18 ng/ml/mg/24 h, P < 0.001), and compared to
ment adjacent to the CFA application (n = 10; 173.58 36.14 ng/ the contralateral DRG (n = 4; 30.61 0.57 ng/ml/mg/24 h,
ml/mg/24 h; P = 0.04; ANOVA F4,18 = 3.95). compared to non- P < 0.001). The contralateral DRGs IL-6 level was signicantly ele-
Fig. 1. IL-6 (A) and IL-1b (B) 24 h secretion levels from sciatic nerves exposed to CFA induced neuritis was quantied employing ELISA. IL-6 and IL-1b secretion were
signicantly () elevated on the 3rd day post-operation at the site subjected to CFA. On the 8th day, IL-6 level returned to baseline while IL-1b level remained signicantly
elevated. Data are presented as means SEM.
478 E. Eliav et al. / Brain, Behavior, and Immunity 23 (2009) 474484
Fig. 2. IL-6 (A), and IL-1b (B) 24 h secretion level from L-5 DRG, 3 and 8 days following sciatic nerve CFA induced neuritis were quantied employing ELISA. In the ipsilateral
DRG, levels of both cytokines were signicantly () elevated on the 3rd and 8th days. In the contralateral DRGs, IL-6 and IL-1b levels were signicantly elevated on the 3rd
DPO.
vated compared to the naive rats DRG (P = 0.017). Eight days fol- The secretion from the proximal and distal portions of the af-
lowing the procedure, although IL-6 levels in the ipsilateral DRG fected nerve and from the contralateral nerves were elevated
decreased from the level of 3rd DPO, it remained signicantly ele- compared to the secretion from nave nerves but was not
vated (n = 4; 37.40 3.17 ng/ml/mg/24 h ANOVA F2,8 = 153.59) signicant.
compared to the naive rats DRG (3.18. 0.18 ng/ml/mg/24 h,
P < 0.001) and the contralateral DRG (n = 4; 6.33 0.16 ng/ml/mg/ 3.1.3.2. IL-1b. Three days following the application IL-1b level was
24 h, P < 0.001). signicantly elevated in the affected side (n = 6; 130.99 11.19 ng/
ml/mg/24 h ANOVA F2,8 = 5.98, P = 0.009) compared to the nave
3.1.2.2. IL-1b. Three days following CFA application, IL-1b was sig- rats paw (n = 6; 84.43 1.32 ng/ml/mg/24 h). On the 8th day the
nicantly elevated in the ipsilateral DRG (n = 4; 278.10 IL-1b level in the affected side (n = 6; 140.56 8.39 ng/ml/mg/
30.15 ng/ml/mg/24 h ANOVA F2,8 = 65.76, P < 0.001) compared to 24 h ANOVA F2,8 = 29.64, P < 0.001) and the contralateral (n = 6;
the naive rats DRG (7.30 2.93 ng/ml/mg/24 h), and compared 128.59 4.05 ng/ml/mg/24 h ANOVA F2,8 = 29.64, P < 0.001) side
to the contralateral DRG (69.11 1.04 ng/ml/mg/24 h P < 0.001). were signicantly elevated compared to the naive rats paw level.
The contralateral DRGs IL-1b level was signicantly elevated The secretion from the proximal and distal portions of the affected
compared to the level in the naive rats DRG (P = 0.034). Eight nerve and from the contralateral nerves were elevated compared
days following the procedure, IL-1b was signicantly elevated in to the secretion from nave nerves but was not signicant.
the ipsilateral DRG (n = 4; 64.84 15.00 ng/ml/mg/24 h ANOVA
F2,8 = 11.19) compared to the naive rats DRG (7.298 2.927 ng/ 3.2. Experiment 2: Cytokines application on the nerve (Fig. 4)
ml/mg/24 h, P = 0.001), and compared to the contralateral DRG
(22.88 1.95, P = 0.009). 3.2.1. Baseline measurements
The pre-treatment baseline scores were not signicantly differ-
3.1.3. Paw skin (Fig. 3) ent from the predicted normal values (difference scores of zero
Signs of inammation such as swelling or colour changes were for the mechanical and thermal behavioural tests and a ratio of
not observed in paws of rats that were exposed to perineural one for the electrical threshold). Moreover, there were no signif-
inammation, this is similar to our previous reports in the same icant between-group differences for the baseline scores in each
model (Eliav et al., 1999). behavioural modality. For all groups combined (with left and right
hind paws averaged together), baseline heat-evoked withdrawal
3.1.3.1. IL-6. Three days following the application IL-6 levels were latencies were 9.9 0.12 s. The baseline pin-prick evoked with-
signicantly increased in the affected side (n = 6; 156.47 6.65 ng/ drawal duration was 0.5 0.0 s. The baseline ratio of electrical-
ml/mg/24 h ANOVA F2,8 = 94.76, P < 0.001) and in the contralateral evoked withdrawal threshold was 32.5 3.1 mA. The baseline
side (n = 6; 103.37 5.78 ng/ml/mg/24 h ANOVA F2,8 = 94.76, median Von Frey hair threshold was 5.07 log 10 mg; (10 g force).
P < 0.001) compared to the nave rats paw (n = 6; 27.40 3.14 ng/ No signicant changes were observed in the contralateral hind
ml/mg/24 h). On the 8th day following the application the IL-6 level paw compared to baseline in any of experimental groups or
was not different from the nave rats secretion level. modalities.
E. Eliav et al. / Brain, Behavior, and Immunity 23 (2009) 474484 479
(ANOVA F4,25 = 11.93 for day 3, F4,25 = 15.49 for day 5, F4,25 = 3.96
for day 7 and F4,25 = 4.79 for day 8. Fishers PLSD test P < 0.05).
3.2.4. Tactile-allodynia
Results are expressed as difference between the surgery side
paw and the contralateral side (difference log 10 g thresholds
SEM).
In the rst day following the procedure, the CFA ( 0.08
0.12 log 10 g) and IL-6 ( 0.63 0.13 log 10 g) groups difference
scores were signicantly lower compared to the IL-1b group.
Three days following the procedure, the CFA ( 1.08 0.19 log
10 g) and the IL-6 ( 0.88 0.23 log 10 g) groups difference scores
were signicantly lower compared to the saline control group.
The CFA group difference scores were signicantly lower compared
to the IL-1b group as well.
Five days following the procedure only the CFA group
( 1.08 0.19 log 10 g) difference scores were signicantly lower
compared to the saline group. Seven days following the procedure,
only the IL-1b group ( 0.50 0.23 log 10 g) difference scores were
signicantly lower compared to the saline group.
Eight days following the procedure the IL-1b group difference
score ( 0.51 0.09 log 10 g) were signicantly lower compared to
the IL-6 and saline groups. (1st day ANOVA F4,25 = 7.34, 2nd day:
ANOVA F4,25 = 14.37, 3rd day: ANOVA F4,25 = 9.41, 5th day: ANOVA
F4,25 = 4.63, 7th day: ANOVA F4,25 = 5.57, 8th day: ANOVA F4,25 =
10.04, Fishers PLSD test P < 0.05).
Fig. 4. Hind paw heat-allodynia (A), mechano-hyperalgesia (B), tactile-alloynia (C) and electrical detection threshold (D) were assessed 1, 2, 3, 5, 7 and 8 days following
application of IL-6, IL-1b, CFA or Saline. IL-6 and CFA induced signicant () hind paw tactile-allodynia on days 15, electrical hypersensitivity on days 13, mechano-
hyperalgesia on days 35 and heat-allodynia only during day 3. Direct application of IL-1b induced paw tactile and heat-allodynia on days 7 and 8 and mechano-hyperalgesia
on days 58.
Fig. 5. Electrophysiological measurements from saphenous nerves exposed to CFA neuritis. (A) Spontaneous activity, (B) nerve trunk mechano-sensitivity, (C) paw tactile
detection threshold dened as the minimal force necessary to induce action potential. Signicantly increased activity was observed in the nerves exposed to CFA and IL-6 on
day 3, while in the nerves exposed to CFA and IL-1b on day 8. Signicantly increased mechano-sensitivity was observed in the nerves exposed to CFA and IL-6 on day 3, while
in the nerves exposed to CFA on day 8. The rats paw mechanical detection threshold was signicantly reduced 3 days following CFA application and 8 days following either
CFA or IL-6 application compared to naive rats.
E. Eliav et al. / Brain, Behavior, and Immunity 23 (2009) 474484 481
3.3.2. Nerve mechano-sensitivity (Fig. 5B) conduction velocity was slower than 5 m/s, 45% 615 m/s, 30% 16
Three days following the procedure, the percentage of the bres 24 m/s and 17% faster than 25 m/s. The mean conduction velocity
responding to mechanical stimulus in the CFA group (10.12 3.36% of the IL-6 exposed spontaneously active bres was 29.56
of 322 bres) was signicantly elevated compared to the saline ex- 1.23 m/s, where a total of 30 bres were observed: None of the -
posed group (1.33 0.09% of 324 bres (ANOVA F4,18 = 5.89, Fish- bres conduction velocity was slower than 5 m/s, 37% 1624 m/s
ers PLSD test P < 0.05). The percentage of the bres responding and 63% faster than 25 m/s. The average conduction velocity of
to mechanical stimulus in the IL-6 exposed group (6.98 1.11% the IL-1b exposed spontaneously active bres was 12.46
of 398 bres) was signicantly elevated compared to the saline 0.96 m/s, where a total of 22 bres were observed: None of the -
(P = 0.037) group. The percentage of the bres responding to bres conduction velocity was slower than 5 m/s or faster than
mechanical stimulus (5.12 0.83% of 255 bres) in the IL-1b group 24 m/s, 81% 615 m/s and 19% 1624 m/s.
was not signicantly elevated compared to the other groups. Eight days following the procedure the average conduction
Eight days following the procedure, the percentage of the bres velocity of the CFA exposed group spontaneously active bres
responding to mechanical stimulus in the CFA group: (10.32 was 15.86 1.69 m/s, a total of 22 bres were observed: 5% less
3.07% of 264 bres) was signicantly elevated compared to the sal- than 5 m/s, 41% 615 m/s, 36% 1624 m/s and 18% faster than
ine exposed group: (0.66 0.40% of 321 bres), and the IL-6 ex- 25 m/s. The average conduction velocity of the IL-6 exposed group
posed group (1.24 0.7% total of 231 bres; ANOVA F4,18 = 6.12, spontaneously active bres was signicantly (P < 0.0001) reduced
Fishers PLSD test P < 0.05). The percentage of the bres responding to 16.75 0.84 m/s, where a total of 21 bres were observed: None
to mechanical stimulus in the IL-1b group: (5.15 1.34% of 326 - of the bres conduction velocity was slower than 5 m/s, 33% were
bres) was elevated, but not signicant. 615 m/s, 62% 1624 m/s and only 5% faster than 25 m/s.
The average conduction velocity of the IL-1b exposed group spon-
3.3.3. Cutaneous mechano-sensitivity (Fig. 5C) taneously active bres was signicantly elevated (P < 0.0001) to
Three days following the procedure, CFA exposed nerves cutane- 25.80 1.01 m/s, where a total of 33 bres were observed: None of
ous detection threshold (0.92 0.153 g) was signicantly lower com- the bres conduction velocity was slower than 5 m/s, 6% were 6
pared to IL-1b (4.65 0.712 g) and naive rats detection threshold 15 m/s, 15% 1624 m/s and 79% faster than 25 m/s.
(3.71 0.334 g), ANOVA F4,11 = 11.66, Fishers PLSD test P < 0.05).
Eight days following the procedure CFA (1.66 0.194 g) and IL-6
4. Discussion
(1.63 0.324 g) exposed nerves cutaneous detection threshold
was signicantly lower compared to naive rats detection threshold
Previous studies have shown that perineural inammation with
(3.710 0.334 g).
no frank axonal damage is sufcient to produce pain and hypersen-
sitivity in the nerves target organ (Eliav et al., 1999, 2001; Chacur
3.3.4. Conduction velocity (Fig. 6)
et al., 2001; Gazda et al., 2001; Benoliel et al., 2002; Bove et al.,
Three days following the procedure the mean conduction veloc-
2003). A chronic, yet mild inammatory process that does not re-
ity of the CFA exposed group spontaneously active bres was
sult in nerve damage as well as severe inammation that does give
14.86 1.332 m/s. A total of 47 bres were studied, 8% of the bres
rise to such injury can result in central sensitization and chronic
neuropathic pain (Gazda et al., 2001; Eliav et al., 2004; Milligan
et al., 2004).
The present set of experiments studied the contribution of IL-6
and IL-1b to perineural inammation related pain (neuritis). We
measured the levels of secreted cytokines from related nerves,
DRG and skin paw following perineural inammation. The role of
these cytokines was further evaluated by direct application on the
sciatic nerve trunk of IL-6 and IL-1b, followed by measurement of
pain-related behaviour of the rats paw and assessment of the related
nerves electrophysiological properties. The dose applied to the
nerve corresponded to the secreted levels from a nerve exposed to
an inammatory adjuvant (CFA) on the 3rd DPO (200 ng of IL-6
and 400 ng of IL-1b). It has been shown in previous studies that neu-
ritis pain peaks between 2 and 5 days following CFA application and
resolves after 7 days (Eliav et al., 1999, 2001, 2004), therefore pain
behaviour was assessed up to 8 days following cytokine application.
For similar reasons, electrophysiological properties were recorded
on the 3rd and 8th days following the procedure.
DPO), but not in the contralateral nerves. Previous studies that in- lowing IL-6 and IL-1b application as well (Fig. 5A and B). IL-6, that
duced perineural inammation with a different inammatory induced pain on the 35th DPOs, induced both mechano-sensitiv-
adjuvant (zymozen) and nerve injury studies, have demonstrated ity and spontaneous activity on the 3rd DPO but not on the 8th. IL-
increases in the contralateral nerve cytokines levels (Gazda et al., 1b application that induced pain on the 78th DPOs induced signif-
2001; Kleinschnitz et al., 2005). This discrepancy may be associ- icant spontaneous activity on the 8th DPO but not on the 3rd. IL-1b
ated with the degree of injury or the severity of inammation; typ- did not induce signicant mechano-sensitivity.
ically CFA induced neuritis does not result in frank nerve damage, The percentage of mechano-sensitive and spontaneously active
while zymosan-induced neuritis results in peripheral demyelina- bres varies amongst studies, this may be related to different meth-
tion (Gazda et al., 2001; Kleinschnitz et al., 2005). Although the ods, the assessment time following the procedure and the equip-
CFA induced perineural inammation was not sufcient to induce ment utilized. Neuritis related neural spontaneous activity
contralateral nerve cytokines secretion, the induction of some de- comprises both C and A bres (Bove et al., 2003), most of the active
gree of central processing is supported by the elevation in contra- bres are thinly myelinated bres (Ad). Relying on the conduction
lateral DRG and skin cytokine levels (Wieseler-Frank et al., 2007). velocity to distinguish between the nerve types (Fig. 6), the sponta-
It is well known that IL-6 and IL-1b play an important role in neous activity following CFA application in the present study was
neuropathic pain (Arruda et al., 1998, 2000; Granados-Soto et al., not different. Three and 8 days following the application, 75% and
2001; Wolf et al., 2003; Bessler et al., 2006; Ma and Quirion, 77% of the active bres, respectively, were Ad bres. IL-6 application
2006), however the differential actions dependant upon the spe- induced mechano-sensitivity and ectopic discharges on the 3rd
cic timing of appearance of these cytokines has not been previ- DPO, similar to the CFA application, however surprisingly 63% of
ously reported. While IL-6 level followed the pain behaviour and the active bres conduction velocity was faster than 30 m/s. Similar
peaked on the 3rd DPO and returned to normal on the 8th DPO, patterns have been found following IL-1b application. IL-1b applica-
IL-1b level remained elevated on the 8th DPO in the absence of tion induced signicant pain and ectopic discharge 8 days following
detectable pain. This is true for all the three sites tested; the nerve, application, however 79% of the spontaneously active bres con-
DRG and related skin. This suggests a good association between duction velocity was faster than 30 m/s. The most painful phase fol-
pain behaviour and IL-6 levels (Murphy et al., 1999). The cells lowing cytokine application was characterized with myelinated
responsible for these cytokines were not investigated in this study, nerves spontaneous activity. This data can be compared to a previ-
this should be addressed in further research. ous study (Tal and Eliav, 1996) on sham operated rats, where only
54% of the nerves conduction velocity was faster than 30 m/s 2
4.2. Experiment 2: Cytokines application on the nerve 5 days following the procedure.
The cutaneous tactile detection threshold ndings may have
IL-6 induced pain arose shortly after its application to the nerve important clinical relevance (Fig. 5C). This experiment result shows
and lasted up to 5 days. In contrast, IL-1b application induced sig- that nerve trunk exposure to perineural inammation produces
nicant pain from 5 days after its application and the pain lasted hypersensitivity in the nerve distal end (the rats paw in the present
throughout the experimental period; at least 8 days following study): less mechanical force is required to produce action potential.
application (mechano-hyperalgesia 58 DOPs, heat and tactile- Several studies demonstrated reduced mechanical detection thresh-
allodynia 78 DPOs). A study that focused on IL-1b and TNFa dem- old in conditions that presumably involve perineural inammation.
onstrated that sciatic nerve intraneural injection of IL-1b induced Third molar extraction studies revealed that the rst days following
mechano-allodynia after 6 days and heat-allodynia only after the extraction are characterized by lingual and mental nerves der-
9 days. In the same study TNFa injection produced tactile and matomes showing reduced mechanical and electrical detection
heat-allodynia earlier, at 3 and 4 days following the procedure thresholds (Eliav and Gracely, 1998), and this hypersensitivity was
(Zelenka et al., 2005). Similar to the present study the cytokines reversed by steroid treatment (Barron et al., 2004).
were in contact with the sciatic nerve trunk at the mid-thigh level Joint-related temporomandibular disorders (TMD), probably
while the pain was assessed in the nerve target organ, the rats inducing auriculotemporal nerve perineural inammation revealed
paw. Consequently we can postulate that perineural IL-6 and TNFa, reduced electrical detection threshold (Eliav et al., 2003) while
initiate pain behaviour in the nerve distal end within 3 days while myogenic TMDs (either clinical or experimental) that does not in-
IL-1b is active in pain generation later (69 days). The clinical sig- volve inammation demonstrated mechanical, electrical and
nicance of this nding is not clear yet, however it may suggest vibrotactile elevated detection thresholds (Hollins et al., 1996;
that various cytokines although present in the inammatory milieu Stohler et al., 2001; Eliav et al., 2003). Mechanical allodynia and
at the same time may contribute to pain and sensitivity in different hypersensitivity have been shown to be present in patients suffer-
phases of the process. The immediate effect of IL-6 may suggest di- ing from osteoarthritis (Farrell et al., 2000a,b). Detection threshold
rect mechanisms in contrast to other cytokines that act indirectly assessment of early oral malignant lesions, presumably involving
or by activation of other, downstream events. perineural inammation (Anneroth et al., 1986) demonstrated re-
IL-6 produced only brief heat-allodynia (on DPO 3) while IL-1b duced electrical detection threshold in the nerves territory that
produced signicant heat-allodynia that started on DPO 7 and was were exposed to the malignant process (Eliav et al., 2002). Parana-
still present on the 8th DPO, at the end of the experiment. This can sal sinusitis sensory assessment demonstrated reduced electrical
point towards selective cytokine effects on pain modalities. IL-1b detection threshold of the skin overlying acute sinusitis (Benoliel
can signicantly affect heat-type pain while IL-6 may have only et al., 2006).
limited effects on heat pain. Perineural inammation induced with CFA, signicantly in-
creased IL-6 and IL-1b secretion from the affected nerves, their re-
4.3. Experiment 3: Electrophysiology properties of cytokines lated DRGs and the paw skin innervated by the nerve, induced pain
application on the nerve and hypersensitivity in the nerve target organ, spontaneous neural
activity, nerve trunk mechano-sensitivity and reduced the target
Perineural inammation (neuritis) induced by CFA application organ tactile detection threshold.
has been shown to produce nerve trunk mechano-sensitivity and IL-6 secretion correlated with the pain behaviour induced by per-
spontaneous activity, that typically correlates with pain behaviour ineural inammation: elevated when the pain peaked and reduced
(Kajander et al., 1992; Tal and Eliav, 1996; Eliav et al., 2001, 2004; as the pain resolved. Il-1b secretion remained elevated after the res-
Bove et al., 2003; Dilley et al., 2005). This correlation occurred fol- olution of the pain. Similar to perineural inammation induced by
E. Eliav et al. / Brain, Behavior, and Immunity 23 (2009) 474484 483
CFA application, application of IL-1b and IL-6 to the sciatic nerve Eliav, E., Tal, M., Benoliel, R., 2004. Experimental malignancy in the rat induces early
hypersensitivity indicative of neuritis. Pain 110 (3), 727737.
trunk vicinity produced pain in the nerve distal end (the rats paw),
Eliav, E., Teich, S., Benoliel, R., Nahlieli, O., Lewkowicz, A.A., Baruchin, A., Gracely, R.,
spontaneous activity and nerve trunk mechano-sensitivity. IL-6 2002. Large myelinated nerve ber hypersensitivity in oral malignancy. Oral
activity was more prominent immediately following application Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 94 (1), 4550.
(25 DPOs), while IL-1b, induced or maintained pain in a later stage Eliav, E., Teich, S., Nitzan, D., El Raziq, D.A., Nahlieli, O., Tal, M., Gracely, R.H.,
Benoliel, R., 2003. Facial arthralgia and myalgia: can they be differentiated by
(58 DPOs). Most of the spontaneously active bres following peri- trigeminal sensory assessment? Pain 104 (3), 481490.
neural inammation were thinly myelinated Ad bres. Heat-allo- Falchi, M., Ferrara, F., Gharib, C., Dib, B., 2001. Hyperalgesic effect of intrathecally
dynia induced by IL-1b was more robust than heat-allodynia administered interleukin-1 in rats. Drugs Exp. Clin. Res. 27 (3), 97101.
Farrell, M., Gibson, S., McMeeken, J., Helme, R., 2000a. Pain and hyperalgesia in
induced by IL-6. Both IL-1b and IL-6 contribute to neuropathic osteoarthritis of the hands. J. Rheumatol. 27 (2), 441447.
inammatory pain, but in different phases of the process. Farrell, M.J., Gibson, S.J., McMeeken, J.M., Helme, R.D., 2000b. Increased movement
pain in osteoarthritis of the hands is associated with A beta-mediated
cutaneous mechanical sensitivity. J. Pain 1 (3), 229242.
References Gazda, L.S., Milligan, E.D., Hansen, M.K., Twining, C.M., Poulos, N.M., Chacur, M.,
OConnor, K.A., Armstrong, C., Maier, S.F., Watkins, L.R., Myers, R.R., 2001. Sciatic
Anneroth, G., Hansen, L.S., Silverman Jr., S., 1986. Malignancy grading in oral inammatory neuritis (SIN): behavioral allodynia is paralleled by peri-sciatic
squamous cell carcinoma. I. Squamous cell carcinoma of the tongue and oor of proinammatory cytokine and superoxide production. J. Peripher. Nerv. Syst. 6
mouth: histologic grading in the clinical evaluation. J. Oral Pathol. 15 (3), 162 (3), 111129.
168. Govrin-Lippmann, R., Devor, M., 1978. Ongoing activity in severed nerves: source
Arruda, J.L., Colburn, R.W., Rickman, A.J., Rutkowski, M.D., DeLeo, J.A., 1998. Increase and variation with time. Brain Res. 159 (2), 406410.
of interleukin-6 mRNA in the spinal cord following peripheral nerve injury in Granados-Soto, V., Alonso-Lopez, R., Asomoza-Espinosa, R., Runo, M.O., Gomes-
the rat: potential role of IL-6 in neuropathic pain. Brain Res. Mol. Brain Res. 62 Lopes, L.D., Ferreira, S.H., 2001. Participation of COX, IL-1 beta and TNF alpha in
(2), 228235. formalin-induced inammatory pain. Proc. West. Pharmacol. Soc. 44, 1517.
Arruda, J.L., Sweitzer, S., Rutkowski, M.D., DeLeo, J.A., 2000. Intrathecal anti-IL-6 Hargreaves, K., Dubner, R., Brown, F., Flores, C., Joris, J., 1988. A new and sensitive
antibody and IgG attenuates peripheral nerve injury-induced mechanical method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32
allodynia in the rat: possible immune modulation in neuropathic pain. Brain (1), 7788.
Res. 879 (12), 216225. Hirano, T., 1991a. Cytokines and diseases. Abnormal expression of interleukin 6 and
Barron, R.P., Benoliel, R., Zeltser, R., Eliav, E., Nahlieli, O., Gracely, R.H., 2004. Effect of diseases. Nippon Naika Gakkai Zasshi 80 (9), 14011408.
dexamethasone and dipyrone on lingual and inferior alveolar nerve Hirano, T., 1991b. Interleukin 6 (IL-6) and its receptor: their role in plasma cell
hypersensitivity following third molar extractions: preliminary report. J. neoplasias. Int. J. Cell Cloning 9 (3), 166184.
Orofac. Pain 18 (1), 6268. Hoffmann, T., Sauer, S.K., Horch, R.E., Reeh, P.W., 2008. Sensory transduction in
Barton, B.E., 1997. IL-6: insights into novel biological activities. Clin. Immunol. peripheral nerve axons elicits ectopic action potentials. J. Neurosci. 28 (24),
Immunopathol. 85 (1), 1620. 62816284.
Barton, B.E., Shortall, J., Jackson, J.V., 1996. Interleukins 6 and 11 protect mice from Hollins, M., Sigurdsson, A., Fillingim, L., Goble, A.K., 1996. Vibrotactile threshold is
mortality in a staphylococcal enterotoxin-induced toxic shock model. Infect. elevated in temporomandibular disorders. Pain 67 (1), 8996.
Immun. 64 (3), 714718. Hu, S.J., Xing, J.L., 1998. An experimental model for chronic compression of dorsal
Bennett, G.J., Xie, Y.K., 1988. A peripheral mononeuropathy in rat that produces root ganglion produced by intervertebral foramen stenosis in the rat. Pain 77
disorders of pain sensation like those seen in man. Pain 33 (1), 87107. (1), 1523.
Benoliel, R., Biron, A., Quek, S.Y., Nahlieli, O., Eliav, E., 2006. Trigeminal neurosensory Ignatowski, T.A., Covey, W.C., Knight, P.R., Severin, C.M., Nickola, T.J., Spengler, R.N.,
changes following acute and chronic paranasal sinusitis. Quintessence Int. 37 1999. Brain-derived TNFalpha mediates neuropathic pain. Brain Res. 841 (12),
(6), 437443. 7077.
Benoliel, R., Wilensky, A., Tal, M., Eliav, E., 2002. Application of a pro-inammatory Junger, H., Sorkin, L.S., 2000. Nociceptive and inammatory effects of subcutaneous
agent to the orbital portion of the rat infraorbital nerve induces changes TNFalpha. Pain 85 (12), 145151.
indicative of ongoing trigeminal pain. Pain 99 (3), 567578. Kajander, K.C., Wakisaka, S., Bennett, G.J., 1992. Spontaneous discharge originates in
Bessler, H., Shavit, Y., Mayburd, E., Smirnov, G., Beilin, B., 2006. Postoperative pain, the dorsal root ganglion at the onset of a painful peripheral neuropathy in the
morphine consumption, and genetic polymorphism of IL-1beta and IL-1 rat. Neurosci. Lett. 138 (2), 225228.
receptor antagonist. Neurosci. Lett. 404 (12), 154158. Kleinschnitz, C., Brinkhoff, J., Sommer, C., Stoll, G., 2005. Contralateral cytokine gene
Boucher, T.J., McMahon, S.B., 2001. Neurotrophic factors and neuropathic pain. Curr. induction after peripheral nerve lesions: dependence on the mode of injury and
Opin. Pharmacol. 1 (1), 6672. NMDA receptor signaling. Brain Res. Mol. Brain Res. 136 (12), 2328.
Boucher, T.J., Okuse, K., Bennett, D.L., Munson, J.B., Wood, J.N., McMahon, S.B., 2000. Lewin, G.R., Rueff, A., Mendell, L.M., 1994. Peripheral and central mechanisms of
Potent analgesic effects of GDNF in neuropathic pain states. Science 290 (5489), NGF-induced hyperalgesia. Eur. J. Neurosci. 6 (12), 19031912.
124127. Liu, C.N., Wall, P.D., Ben-Dor, E., Michaelis, M., Amir, R., Devor, M., 2000. Tactile
Bove, G.M., Ransil, B.J., Lin, H.C., Leem, J.G., 2003. Inammation induces ectopic allodynia in the absence of C-ber activation: altered ring properties of DRG
mechanical sensitivity in axons of nociceptors innervating deep tissues. J. neurons following spinal nerve injury. Pain 85 (3), 503521.
Neurophysiol. 90 (3), 19491955. Ma, W., Quirion, R., 2006. Targeting invading macrophage-derived PGE2, IL-6 and
Chacur, M., Milligan, E.D., Gazda, L.S., Armstrong, C., Wang, H., Tracey, K.J., Maier, calcitonin gene-related peptide in injured nerve to treat neuropathic pain.
S.F., Watkins, L.R., 2001. A new model of sciatic inammatory neuritis (SIN): Expert Opin. Ther. Targets 10 (4), 533546.
induction of unilateral and bilateral mechanical allodynia following acute Milligan, E.D., Maier, S.F., Watkins, L.R., 2004. Sciatic inammatory neuropathy in
unilateral peri-sciatic immune activation in rats. Pain 94 (3), 231244. the rat: surgical procedures, induction of inammation, and behavioral testing.
Cunha, F.Q., Poole, S., Lorenzetti, B.B., Veiga, F.H., Ferreira, S.H., 1999. Cytokine- Methods Mol. Med. 99, 6789.
mediated inammatory hyperalgesia limited by interleukin-4. Br. J. Pharmacol. Milligan, E.D., Twining, C., Chacur, M., Biedenkapp, J., OConnor, K., Poole, S., Tracey,
126 (1), 4550. K., Martin, D., Maier, S.F., Watkins, L.R., 2003. Spinal glia and proinammatory
DeLeo, J.A., Colburn, R.W., Nichols, M., Malhotra, A., 1996. Interleukin-6-mediated cytokines mediate mirror-image neuropathic pain in rats. J. Neurosci. 23 (3),
hyperalgesia/allodynia and increased spinal IL-6 expression in a rat 10261040.
mononeuropathy model. J. Interferon Cytokine Res. 16 (9), 695700. Murphy, P.G., Ramer, M.S., Borthwick, L., Gauldie, J., Richardson, P.M., Bisby, M.A.,
DeLeo, J.A., Colburn, R.W., Rickman, A.J., 1997. Cytokine and growth factor 1999. Endogenous interleukin-6 contributes to hypersensitivity to cutaneous
immunohistochemical spinal proles in two animal models of stimuli and changes in neuropeptides associated with chronic nerve
mononeuropathy. Brain Res. 759 (1), 5057. constriction in mice. Eur. J. Neurosci. 11 (7), 22432253.
Devor, M., Janig, W., Michaelis, M., 1994. Modulation of activity in dorsal root Poole, S., Cunha, F.Q., Selkirk, S., Lorenzetti, B.B., Ferreira, S.H., 1995. Cytokine-
ganglion neurons by sympathetic activation in nerve-injured rats. J. mediated inammatory hyperalgesia limited by interleukin-10. Br. J.
Neurophysiol. 71 (1), 3847. Pharmacol. 115 (4), 684688.
Dilley, A., Bove, G.M., 2008. Resolution of inammation-induced axonal mechanical Reichert, F., Levitzky, R., Rotshenker, S., 1996. Interleukin 6 in intact and injured
sensitivity and conduction slowing in C-ber nociceptors. J. Pain 9 (2), 185192. mouse peripheral nerves. Eur. J. Neurosci. 8 (3), 530535.
Dilley, A., Lynn, B., Pang, S.J., 2005. Pressure and stretch mechanosensitivity of Ruzek, M.C., Miller, A.H., Opal, S.M., Pearce, B.D., Biron, C.A., 1997. Characterization
peripheral nerve bres following local inammation of the nerve trunk. Pain of early cytokine responses and an interleukin (IL)-6-dependent pathway of
117 (3), 462472. endogenous glucocorticoid induction during murine cytomegalovirus infection.
Eliav, E., Benoliel, R., Tal, M., 2001. Inammation with no axonal damage of the rat J. Exp. Med. 185 (7), 11851192.
saphenous nerve trunk induces ectopic discharge and mechanosensitivity in Sachs, D., Cunha, F.Q., Poole, S., Ferreira, S.H., 2002. Tumour necrosis factor-alpha,
myelinated axons. Neurosci. Lett. 311 (1), 4952. interleukin-1beta and interleukin-8 induce persistent mechanical nociceptor
Eliav, E., Gracely, R.H., 1998. Sensory changes in the territory of the lingual and hypersensitivity. Pain 96 (12), 8997.
inferior alveolar nerves following lower third molar extraction. Pain 77 (2), Saeh-Garabedian, B., Poole, S., Allchorne, A., Winter, J., Woolf, C.J., 1995.
191199. Contribution of interleukin-1 beta to the inammation-induced increase in
Eliav, E., Herzberg, U., Ruda, M.A., Bennett, G.J., 1999. Neuropathic pain from an nerve growth factor levels and inammatory hyperalgesia. Br. J. Pharmacol. 115
experimental neuritis of the rat sciatic nerve. Pain 83 (2), 169182. (7), 12651275.
484 E. Eliav et al. / Brain, Behavior, and Immunity 23 (2009) 474484
Samad, T.A., Moore, K.A., Sapirstein, A., Billet, S., Allchorne, A., Poole, S., Bonventre, Tal, M., Eliav, E., 1996. Abnormal discharge originates at the site of nerve injury in
J.V., Woolf, C.J., 2001. Interleukin-1beta-mediated induction of Cox-2 in the CNS experimental constriction neuropathy (CCI) in the rat. Pain 64 (3), 511518.
contributes to inammatory pain hypersensitivity. Nature 410 (6827), 471 Tilg, H., Trehu, E., Atkins, M.B., Dinarello, C.A., Mier, J.W., 1994. Interleukin-6 (IL-6) as
475. an anti-inammatory cytokine: induction of circulating IL-1 receptor antagonist
Schwartz, M., Sivron, T., Eitan, S., Hirschberg, D.L., Lotan, M., Elman-Faber, A., 1994. and soluble tumor necrosis factor receptor p55. Blood 83 (1), 113118.
Cytokines and cytokine-related substances regulating glial cell response to Wagner, R., Myers, R.R., 1996. Endoneurial injection of TNF-alpha produces
injury of the central nervous system. Prog. Brain Res. 103, 331341. neuropathic pain behaviors. Neuroreport 7 (18), 28972901.
Shamash, S., Reichert, F., Rotshenker, S., 2002. The cytokine network of Wallerian Watkins, L.R., Wiertelak, E.P., Goehler, L.E., Smith, K.P., Martin, D., Maier, S.F., 1994.
degeneration: tumor necrosis factor-alpha, interleukin-1alpha, and interleukin- Characterization of cytokine-induced hyperalgesia. Brain Res. 654 (1), 1526.
1beta. J. Neurosci. 22 (8), 30523060. Wewers, M.D., Dare, H.A., Winnard, A.V., Parker, J.M., Miller, D.K., 1997. IL-1 beta-
Sommer, C., Petrausch, S., Lindenlaub, T., Toyka, K.V., 1999. Neutralizing antibodies converting enzyme (ICE) is present and functional in human alveolar
to interleukin 1-receptor reduce pain associated behavior in mice with macrophages: macrophage IL-1 beta release limitation is ICE independent. J.
experimental neuropathy. Neurosci. Lett. 270 (1), 2528. Immunol. 159 (12), 59645972.
Song, M., Kellum, J.A., 2005. Interleukin-6. Crit. Care Med. 33 (12 Suppl.), S463 Wieseler-Frank, J., Jekich, B.M., Mahoney, J.H., Bland, S.T., Maier, S.F., Watkins, L.R.,
S465. 2007. A novel immune-to-CNS communication pathway: cells of the meninges
Sorkin, L.S., Xiao, W.H., Wagner, R., Myers, R.R., 1997. Tumour necrosis factor-alpha surrounding the spinal cord CSF space produce proinammatory cytokines in
induces ectopic activity in nociceptive primary afferent bres. Neuroscience 81 response to an inammatory stimulus. Brain Behav. Immun. 21 (5), 711718.
(1), 255262. Wolf, G., Yirmiya, R., Goshen, I., Iverfeldt, K., Holmlund, L., Takeda, K., Shavit, Y.,
Stohler, C.S., Kowalski, C.J., Lund, J.P., 2001. Muscle pain inhibits cutaneous touch 2003. Impairment of interleukin-1 (IL-1) signaling reduces basal pain sensitivity
perception. Pain 92 (3), 327333. in mice: genetic, pharmacological and developmental aspects. Pain 104 (3),
Sweitzer, S.M., Hickey, W.F., Rutkowski, M.D., Pahl, J.L., DeLeo, J.A., 2002. Focal 471480.
peripheral nerve injury induces leukocyte trafcking into the central nervous Woolf, C.J., Allchorne, A., Saeh-Garabedian, B., Poole, S., 1997. Cytokines, nerve
system: potential relationship to neuropathic pain. Pain 100 (12), 163170. growth factor and inammatory hyperalgesia: the contribution of tumour
Tadano, T., Namioka, M., Nakagawasai, O., Tan-No, K., Matsushima, K., Endo, Y., necrosis factor alpha. Br. J. Pharmacol. 121 (3), 417424.
Kisara, K., 1999. Induction of nociceptive responses by intrathecal injection of Zelenka, M., Schafers, M., Sommer, C., 2005. Intraneural injection of interleukin-
interleukin-1 in mice. Life Sci. 65 (3), 255261. 1beta and tumor necrosis factor-alpha into rat sciatic nerve at physiological
Tal, M., Bennett, G.J., 1994. Extra-territorial pain in rats with a peripheral doses induces signs of neuropathic pain. Pain 116 (3), 257263.
mononeuropathy: mechano-hyperalgesia and mechano-allodynia in the Zimmermann, M., 1983. Ethical guidelines for investigations of experimental pain
territory of an uninjured nerve. Pain 57 (3), 375382. in conscious animals. Pain 16 (2), 109110.