Exogenous interleukin-6 increases cold allodynia in rats with a mononeuropathy

www.elsevier.com/locate/issn/10434666 Cytokine 30 (2005) 154e159

Exogenous interleukin-6 increases cold allodynia in rats with a mononeuropathy Kris C. Vissersa,*, Raf F. De Jongha, Vincent L. Hoffmannb, Theo F. Meertc a

Multidisciplinary Pain Center, Ziekenhuis Oost-Limburg, Campus Andre´ Dumont, Stalenstraat 2, 3600 Genk, Belgium b Department of Anesthesiology and Pain therapy, Universitair Ziekenhuis Antwerpen, Wilrijk, Belgium c J&J PRD, Beerse, Belgium Received 11 December 2003; received in revised form 9 November 2004; accepted 3 January 2005

Abstract Interleukin-6 (IL-6) is a pleiotropic cytokine, signaling intracellularly via its unique membrane-bound receptor IL-6R and gp130. In peripheral nerve injury models, IL-6 and IL-6R are increased at the injured nerve and the respective dorsal root ganglion. IL-6 is increased at the ipsilateral dorsal and ventral horn of the spinal cord. IL-6 is known to affect neuronal survival, differentiation and regeneration. It is involved in synaptic plasticity and hyperexitability and induces the synthesis or release of other substances with known neuroprotective or neuromodulatory effects. In this study, intrathecal administration of recombinant rat IL-6 to rats with a chronic constriction injury of the sciatic nerve, induced a logarithmic dose-dependent increase in cold allodynia with a threshold of 10 pg IL-6 and a maximal effect at 100 ng IL-6. Intrathecal administration of saline or denaturated IL-6 was without effect. In rats with a chronic constriction injury, systemic administered IL-6 did not induce a hyperalgesic effect, illustrating that IL-6 acts at the level of the dorsal root ganglion or the spinal cord. Intraplantar injection of 100 ng IL-6 in the operated hind paw resulted in an increased cold allodynia. This study demonstrates that the sensitivity to exogenous intrathecal or peripheral IL-6 increases in rats with a mononeuropathy. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Chronic constriction injury; Nerve injury; Neuropathic; Rat; rrIL-6

1. Introduction Patients with a neuropathic pain syndrome are difficult to treat, possibly because several neuro-immune and humoral factors, which play a role in this pain, are not well defined yet. The peripheral nerve, the dorsal root ganglion (DRG), the spinal cord or even the brain are locations where these factors might exert their role to initiate or maintain pain. In addition, spinal cord microglia and astrocytes were proposed as key cells during exaggerated pain states [1]. Once activated, they * Corresponding author. Tel.: C32 89 32 50 01; fax: C32 2 772 99 36. E-mail address: [email protected] (K.C. Vissers). 1043-4666/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2005.01.008

can release a variety of neuro-active substances including tumor necrosis factor a (TNFa), interleukin-1 (IL-1), interleukin-6 (IL-6), nitric oxide and ATP. These factors alter pain both by enhancing the primary afferent release of substance P and excitatory amino acids (EAA), and by increasing the excitability/reactivity of pain transmission neurons [2] . Interleukin-6 is a pleiotropic cytokine that signals intracellularly via binding to its unique membranebound receptor IL-6R [3]. This binding complex IL-6/ IL-6R induces homodimerisation of another membranebound glycoprotein gp130, which transduces the signal into the cell [3]. As gp130 is constitutively present in high amounts throughout the body, the effect of IL-6 crucially depends on the presence of membrane-bound

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80

g

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0 Pre

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T 60 T 120 T 180 T 240 T d1 T d3

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Fig. 1. Automatic Von Frey readings in CCI-operated animals for the left operated and the right non-operated hind paw in rats. Absolute data are represented in grams as means G SEM (N Z 14). Time points are: before CCI operation (Pre); the seventh post-operative day 1 h before saline injection (T 0), at the 100 ng IL-6 injection 1 h later (T 120), further at 1-h, 2-h, 1-day, 3-day and 5-day post-rrIL-6 injection.

systemic administration of 100 ng of rrIL-6, (3) the intrathecal injection of 100 ng of denaturated rrIL-6, or (4) the intrathecal injection of 0.001 and 0.1 pg of rrIL6, did not affect cold allodynia over time (see Fig. 2). In non-operated animals, the injection of rrIL-6 100 ng did not result in an increased pain behavior. At the screening on the cold plate, before intrathecal injections, the mean lifting time G SEM of the different groups was 67.77 G 10.61 s. No differences among the groups were observed before the intrathecal injections. After saline injection, the mean lifting time was

2000

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p 10 g 0 pg 1 ng 10 n 10 g 0 ng

pg

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1 0.

D

je c

tio n sa en l pr e at ur at ed ve hi cl e

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In this study, CCI-operated animals demonstrated a clear cold allodynia seven days post-operatively: for all animals there was at least a difference of 10 s in the lifting of the left versus the right hind paw on a cold plate of 0  C. Using the automatic Von Frey, a tactile allodynia was observed in the left CCI-operated hind paw versus the right non-operated hind paw for all time points during the testing (see Fig. 1). The magnitude of this tactile hypersensitivity was not influenced by intrathecal administration of different doses of rrIL-6 ( p O 0.05). In CCI-operated animals, the administration of (1) repetitive intrathecal injections of 10 ml of saline, (2) the

Left CCI hindpaw Right hindpaw

rrIL-6

% of change compared to initial lifting time

2. Results

saline 100

Pr e-

IL-6R or soluble IL-6R. In nervous tissue, IL-6 is known to affect neuronal survival, neuronal differentiation and nerve regeneration [4,5]. It is involved in synaptic plasticity and hyperexitability and it can induce the synthesis or release of other substances with neuroprotective or neuromodulatory effects [6]. Under normal conditions, both IL-6 and IL-6R concentrations are low on primary or secondary order nociceptors [7,8]. A peripheral nerve injury in animals induces an increase in IL-6 and IL-6R levels in the injured nerve [7,9,10], in the respective DRGs [8] and increased presence of IL-6 in the ipsilateral dorsal and ventral horn of the spinal cord [11]. Injection of IL-6 in the hind paw [12], in the DRG [13,14] or in the cerebrospinal fluid [11] resulted in mechanical- and/or thermal-allodynia. In non-operated rats, intrathecal injection of a high dose of IL-6 (100 ng) resulted in touch-evoked allodynia without thermal hyperalgesia, whereas the intrathecal injection of the cytokine in rats with previous sciatic cryolesion resulted in thermal hyperalgesia without mechanical allodynia [11]. The nociceptive role of IL-6 in DRG or spinal cord was also demonstrated by the prevention of allodynia with intrathecal injection of anti-rat-IL-6 antibodies both before and after surgical manipulation of the sciatic nerve [15]. In accordance with the increased heat allodynia after cutaneous IL-6 administration, which obligatorily depends on the presence of the IL6R [16], we hypothesize that IL-6R or gp130 are upregulated in DRG or the spinal cord after a chronic constriction injury (CCI) of the sciatic nerve, leading to an increased sensitivity to intrathecal IL-6 during this condition [17]. The aim of this study was (1) to identify the minimal dose of recombinant rat IL-6 (rrIL-6) inducing an increased pain behavior in CCI rats, (2) to look for a dose-dependent increased sensitivity of cold allodynia after intrathecal administration of rrIL-6 in CCI-operated SpragueeDawley rats and (3) to demonstrate an increased peripheral sensitivity to intraplantar administered rrIL-6 in CCI-operated animals.

Fig. 2. Dose-response curve of different dosages of IT rrIL-6 on cold allodynia in CCI-operated animals. Data are presented in relation to the initial lifting time, which is arbitrary set at 100 statistical significance ( p ! 0.001) of the different dosages is represented as * versus the initial pre-injection lifting on the cold plate (N Z 14).

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74.00 G 12.33 s. This value was 109% of the initial screening value and was without statistical significance ( p O 0.05) (see Fig. 2). Comparable data were observed with denaturated rrIL-6, as well as with IV injections of 100 ng of rrIL-6. Intrathecal injections of different doses of rrIL-6 from 0.01 pg to 100 ng gradually increased the paw lifting time of the CCI hind paw from 176.71 G31.88 s at 0.1 pg to 259.33 G 16.05 s at 100 ng (see Fig. 2). No increased lifting of the right hind paw was observed. On the cold plate, the peripheral administration of 100 ng rrIL-6 demonstrates no increased lifting behavior in non- or sham-operated animals, whereas a significant lifting was observed of the left hind paw in CCIoperated animals ( p ! 0.001) (see Fig. 3). No increased lifting of the right hind paw was observed.

3. Discussion The present study demonstrates an increased sensitivity for cold allodynia for both the peripheral and intrathecal exogenous administered rrIL-6 in rats with a chronic constriction injury of the sciatic nerve. Seven days after a CCI intervention, rats showed increased cold allodynia related to the dose of intrathecal injected IL-6, with a threshold of 10 pg or more than 250 million molecules of IL-6. In contrast to the bell-shaped doseresponse curve of body temperature to systemic injected IL-6 [18], the dose-response of intrathecal IL-6 on cold allodynia was found to be logarithmic. A maximal thermal allodynia was observed at 100 ng intrathecal rrIL-6 injection, as paw lifting was almost continuous

50

IL-6 Vehicle *

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Lifting time (sec)

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Fig. 3. Lifting time of the left CCI-operated hind paw on the cold plate after intraplantar injection of 100 ng of rrIL-6. Data are represented as absolute data in the different test conditions for a 5 min observation period. Injection of 100 ng of rrIL-6 in the left hind paw of nonoperated control animals, sham- and CCI-operated animals. Statistical significance of the different conditions is represented as * for p ! 0.05 and *** for p ! 0.001 (N Z 14).

during the 5 min observation period on the cold plate. Increased cold allodynia after intrathecal injections is not related to an increase of the cerebrospinal fluid pressure or to gradients in electrolytes because intrathecal administration of saline or denaturated IL-6 was without any significant allodynic effect. This study further indicates that under the present test conditions, IL-6 acts at the level of DRG or the spinal cord, because of the immediate effect on cold allodynia after intrathecal administration of IL-6, the absence of a hyperalgesic response to systemic administered IL-6 together with the fact that IL-6 only minimally penetrates the potent bloodebrain barrier in both directions [19,20]. However, a direct effect on the brain via transportation through the cerebrospinal fluid cannot be excluded. In the present study, intrathecal injection of rrIL-6 in normal rats did not induce thermal allodynia. This finding indicates that DRGs or the spinal cord show increased sensitivity to IL-6 seven days after CCI. IL-6 acts via its unique receptor IL-6R, and via gp130 [3]. Other studies showed that after sciatic nerve lesions both IL-6 and IL-6R are rapidly upregulated at the site of the lesion and the DRG [8,9]. At these locations, the disappearance of IL-6 within one week is in contrast with the longer-lasting presence of the IL-6R. It has been shown that IL-6 is also shortly upregulated in the corresponding dorsal and ventral horn of the spinal cord [11]. A histo-immunochemical study on the presence of IL-6R in the spinal cord after sciatic nerve lesion is, to the best of our knowledge, not yet published. The increased responsiveness to exogenous intrathecal rrIL-6 after CCI, found in the present study argues for a comparable behavior of both IL-6 and IL-6R in the DRGs or spinal cord compared with their behavior known at the injured sciatic nerve. Our findings are in contrast with an electrophysiological study, where the application of IL-6 directly on the spinal cord resulted in an inhibitory effect on the post-discharge response in spinal nerve ligation rats [21]. However, in this electrophysiological study, important methodological differences can be described compared to our behavioral study including the concentration of rrIL-6 used, the location of the rrIL-6 administration, a different time window of the experiments and the use of a different animal model to study neuropathic pain behavior. IL-6 exerts a beneficial effect in the nervous tissue, such as increasing neuronal survival, neurite outgrowth and nerve regeneration [4,22]. The knowledge of the mechanisms of upregulation, downregulation or shedding of the IL-6R is sparse, especially if one considers neurons or glial cells. Apparently, the signal from the nerve lesion leading to an increase of IL-6 in the central nervous system is weaker or shorter-lasting compared to the signal to increase the responsiveness to IL-6 via its receptors. There is still lack of an explanation for this phenomenon.

K.C. Vissers et al. / Cytokine 30 (2005) 154e159

The systemic administration of 100 ng of rrIL-6 after CCI did not alter cold- or mechanical allodynia. This indicates that IL-6 acts centrally and a potent bloode brain barrier prevents IL-6 diffusion to neuronal structures that transmit or transduce pain. This finding also contradicts the hypothesis of systemic spread of IL6 to explain the mechanical allodynia of a hind paw after contralateral intraplantar IL-6 injection [12]. In contrast, we found that the intraplantar administration of rrIL-6 causes an increase in cold allodynia only in CCIoperated animals. This increased reactivity on the peripheral administration of rrIL-6 in CCI animals demonstrates the upregulation of the cognate IL-6 receptor at the peripheral nerve endings of the ipsilateral sciatic nerve. The different response of intrathecal IL-6 administration on cold and tactile allodynia can be explained by the different sensitivity of sympathetic nerve fibers to the stimulation modalities. Additionally, IL-6 has no electrophysiological influence on the sensitivity of A-beta fibers which mainly transduce mechanical components of allodynic behavior [21]. In the CCI model, known as an inflammatory model of neuropathic pain, neurogenic inflammation is induced by loose ligatures of catgut around the sciatic nerve [23]. Firstly, this neurogenic inflammation causes a pain message to the spinal cord, leading to hypersensitivity. Secondly, the release of substances around and by the nerve terminals induces an increased sensitivity into their own receptive fields. The neuronal released substances, such as substance P, trigger all of the cardinal signs of inflammation: reddening of the area, swelling, and pain. In turn, substance P induces pro-inflammatory cytokine release from a variety of immune cells [24,25]. These proinflammatory cytokines induce pain by activating painresponsive sensory nerve terminals. The substance P-pro-inflammatory cytokine positive feedback loop induces spinal cord sensitization, which results in an upregulation of bradykinines. These bradykinines may further stimulate the proposed substance P-pro-inflammatory cytokine loop, as bradykinin increases IL-1, TNF, and IL-6 [26,27]. It suggests that preservative proinflammatory cytokine release could, by stimulation of sensory nerves, be a contributing factor to the maintenance of central sensitization observed in neuropathic pain in animals and Complex Regional Pain Syndrome patients [28]. In addition, physiological pathways of the increased sensitivity to IL-6 at several levels of pain transmission by upregulation of one or both IL-6 receptors needs further attention in order to consider IL-6, IL-6-R or gp130 as targets for treatments of several pain conditions. In conclusion, in CCI-operated rats, the increased sensitivity to cold after intrathecal or subcutaneous administration of rrIL-6 indicates that this cytokine may play a major role in the modulation of the

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hypersensitivity in neuropathic pain behavior. Further immuno-histochemical studies should consider the upregulation of both receptors IL-6R and gp130 receptor in the spinal cord and at the peripheral nerve endings in these conditions to confirm the role of IL-6 in neuropathic pain conditions.

4. Material and methods All studies were conducted following the ethical guidelines of the IASP [29] and were approved by the Local Animal Care Ethics Committee. Male Spraguee Dawley rats (Charles Rivers), weighing 250e280 g at the start of the experiment, were housed individually in standard rodent cages with sawdust bedding and soya free food and water ad libitum. The housing room was air-conditioned with a 12/12-h day/night cycle (lights on at 7:00 a.m.). Background noise was presented during the light period by playing a conventional radio station. The same surrounding conditions were used in the laboratory. 4.1. Sciatic nerve ligation Before surgery, all rats were pre-tested on the cold plate; those animals that were lifting their hind paws without an operation were withdrawn from this study. The rats were anesthetized with 1 ml/rat subcutaneous ThalamonalÒ (fentanyl/droperidol) and 40 mg/kg intraperitoneal sodiumpentobarbital. According to the model of Bennett and Xie, the sciatic nerve of the left hind paw was exposed at the level of the middle of the thigh by blunt dissection through the biceps femoris. Proximal to the sciatic’s trifurcation, 7 mm of nerve was freed and four loose ligatures of 4-0 chromic catgut were placed around the sciatic nerve. Great care was taken to tie the ligatures in such a way that the diameter of the nerve was seen to be just barely constricted when viewed with a microscope using a 40! magnification. After surgery all animals received 10 mg/kg naloxone IP [30]. In sham-operated animals, the same surgical procedure was followed until the nerve was exposed, the connective tissue was freed, and no ligatures were applied. 4.2. Lumbar spinal catheter placement During the same operation an intrathecal cannula was placed at the level L4/L5 via the lumbar route described earlier by Boersma et al. [31]. The dorsal aspect of the L3eL6 vertebrae was exposed by skin incision and a small cut through the paravertebral muscles. The spinal process of the L5 vertebra was carefully removed and the ligamentum flavum and the dura mater were penetrated at the base of L4 vertebra by a spinal catheter. This catheter was 5 mm inserted into the subarachnoideal space, parallel to the cord and fixed with a drop of

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HistoacrylÒ glue (B.Braun Surgical GmbH, Melsungen, Germany) and a suture with silk. The wound was closed in layers. The external part of the catheter was tunneled subcutaneously to the neck of the animals. Approximately 5 cm of the catheter was left outside and the external tip of the catheter was closed by melting with a soldering iron. Sham rats, without CCI, were anesthetized following the same protocol to receive the same catheter placement. After completion of the experiment, all animals were sacrificed and the catheter position was checked, using methylene blue injected through the intrathecal catheter for macroscopic correct positioning in the intrathecal space. Data of animals with failed intrathecal catheter placement were withdrawn.

4.5.2. Intrathecal administration On the seventh post-operative day, all animals were evaluated on the cold plate. Repetitive, remote intrathecal injections in free moving animals of vehicle solution were performed with an interval of 60 min in order to observe potential increased sensitivity for cold after repetitive intrathecal injections and to habituate the animals to intrathecal injections. Rats were randomly allocated to receive one of the following injections in a 10 ml volume: (1) intrathecal rrIL-6 (Biosource e Belgium lyophilized recombinant Rat interleukin-6): 0.001, 0.1, 10 pg, 1 or 100 ng, (2) vehicle (saline), (3) denaturated IL-6 (by boiling at 94  C for 5 min). The intravenous or intraperitoneal administered 100 ng IL-6 was diluted in a 2 ml volume for correct reabsorption purposes. All substances were dissolved in saline.

4.3. Cold plate testing

4.6. Study design

Cold plate testing was performed on a metal plate of 30 ! 30 cm with transparent acrylic walls (height 30 cm). The surface of the cold plate was cooled by a flow-through cooling apparatus which holds the surface temperature stable at ÿ0.5 G 0.5  C (Julabo F 25Ò, Julabo Labortechnik, Seelback, Germany). For testing, the animal was placed on the cold plate and the duration of lifting of the left and right hind paw was measured during an initial period of 5 min. For the sciatic nerve ligated (CCI) animals, only rats with a difference in lifting time O10 s between the ligated and non-ligated paw were used for further testing.

For each condition and dosage a total of 14 rats per group was used. A first intrathecal injection was performed with isotonic saline. One hour later, a second injection was performed with the study substance. Immediately after each injection the animals were reevaluated on the cold plate and the automatic Von Frey test was performed. Animal behavior and side effects were noted, by an independent observer, blinded to the study substances.

4.4. Automatic Von Frey testing Mechanical hypersensitivity was assessed by placing the animal in cages with a metal mesh platform. The plantar surface of the paw was touched with a metal probe (diameter of 1 mm) connected to a pressure transducer (SomedicÒ, Sales AB, Ho¨rby, Sweden). The applied force was increased until the animal withdrew the paw. The animals were allowed to habituate for 30 min in plastic cages before the first measurement. The average of three consecutive readings on both hindlimbs in each animal was used for analysis [32].

4.7. Statistics Results from the cold plate testing were compared to the baseline values and expressed as a percentage of the pre-injection value. For Von Frey testing all data were expressed as absolute data. All data are expressed as means G SEM. Differences between experimental conditions were evaluated using the ManneWhitney U-test (two tailed) (Sigmastat version 2.0, SPSS Chicago 1997). Statistical significance was considered from p ! 0.05.

Acknowledgements We thank Ria Biermans, Frank Geenen and Pierre Ottevaere for their technical assistance.

4.5. IL-6 experiments 4.5.1. Peripheral administration Recombinant rat IL-6 100 ng (Biosource e Belgium lyophilized recombinant Rat interleukin-6), or vehicle in a volume of 50 ml was injected intraplantarly seven days post-CCI surgery in the different conditions: CCIoperated, sham-operated and non-operated animals. Immediately after injection, animals were tested on the cold plate for 5 min.

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