Intern. J. Neuroscience, 117:617–625, 2007Copyright C© 2007 Informa HealthcareISSN: 0020-7454 / 1543-5245 onlineDOI: 10.1080/00207450600773806

Brief Communication

EFFECTS OF INTERLEUKIN-1β ON SPINAL CORDNOCICEPTIVE TRANSMISSION IN INTACT ANDPROPENTOFYLLINE-TREATED RATS

OSVALDO ARRIAGADALUIS CONSTANDILALEJANDRO HERNANDEZRAFAEL BARRA

Laboratory of Neurobiology, Department of BiologyFaculty of Chemistry and BiologyUniversity of Santiago of ChileSantiago, Chile

RUBEN SOTO-MOYANO

Laboratory of Hormones and ReceptorsInstitute of Nutrition and Food Technology (INTA)University of ChileSantiago, Chile

CLAUDIO LAURIDO

Laboratory of Neurobiology, Department of BiologyFaculty of Chemistry and BiologyUniversity of Santiago of ChileSantiago, Chile

Received 9 February 2006.This research was supported by grants 020343HK from Dicyt and 1050099 from Fondecyt.Address correspondence to Prof. Claudio Laurido, Laboratory of Neurobiology, Department

of Biology, Faculty of Chemistry and Biology, University of Santiago of Chile, Ave. Alameda B.O’Higgins 3363, Casilla 40 correo 33, Santiago, Chile. E-mail: claurido@lauca.usach.cl

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To investigate the contribution of glial cells in the spinal cord nociceptivetransmission, the effect of intrathecally administered interleukin-1β (IL-1β) wasstudied in rats treated with the glial cell inactivator propentofylline and submittedto a C-fiber-mediated reflex paradigm evoked by single and repetitive (wind-up)electric stimulation. Intrathecal IL-1β did not modify the C reflex integratedactivity in either group of animals, while producing increased wind-up in intactand decreased wind-up in propentofylline pre-treated rats. Results suggest that theexcitatory effect of IL-1β on spinal wind-up activity in healthy rats is produced bya glial mediator, whereas the inhibitory effect resulted from a direct effect of thecytokine on dorsal horn neurons.

Keywords: C-fiber reflex, glia, interleukin-1β, pain, propentofylline, spinal cord,wind-up

When a direct traumatic injury is applied to the spinal cord, it has beenobserved that the spinal cord responds with robust glial reaction characterizedby decreased ramification, hypertrophy, proliferation, and the up-regulation ofimmunoregulatory molecules such as cytokines (Schnell et al., 1999). Similarly,glial cells of the spinal cord dorsal horn can produce cytokines in responseof a variety of conditions that produce hyperalgesia, including inflammation,infection, and trauma of the skin or viscera, of peripheral nerves, and of thecentral nervous system. Thus, spinal cytokines are thought to be involved incentral mechanisms underlying the maintenance and exaggeration of pain states(Watkins & Maier, 2000; Watkins et al., 2001).

Although the contribution of glia-neuron interactions in the spinalmechanisms subserving hyperalgesia is still not clarified, it has been reportedthat neurons of the central nervous system normally express receptors forinterleukin-1 (IL-1) and TNF and therefore they could serve as targets forthese spinally produced mediators (Vitkovic et al., 2000); besides, intrathecaladministration of IL-1 in normal mice (Tadano et al., 1999) and IL-6 in normalrats (DeLeo et al., 1996) induces hyperalgesia and allodynia; finally, intrathecaladministration of IL-1β and TNF in normal rats enhances both the acuteresponse and the wind-up activity of dorsal horn neurons (Reeve et al., 2000;Constandil et al., 2003).

In an effort to understand the contribution of glial activation and theassociated release of IL-1 on spinal cord nociceptive transmission in the rat, thecompound propentofylline [3,7-dihydro-3-methyl-1-(5-oxohexyl)-7-propyl-1H-purine-2,6-dione] was used in the present study. This compound is aethylxanthine derivative previously found to attenuate astrocytic activation ina rodent ischemia model (DeLeo et al., 1987). In ischemia, propentofylline has

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been shown to be neuroprotective through a multitude of actions, includinginhibition of glutamate release (Andine et al., 1990; Miyashita et al., 1992)and increased nerve growth factor secretion (Shinoda et al., 1990). In vitrostudies revealed that propentofylline maintains astrocytic glutamate uptakeand inhibits potentially neurotoxic functions adopted by microglia uponpathological activation (Schubert et al., 2000). In addition, systemic applicationof propentofylline has been found to inhibit lumbar spinal microglial activationfollowing middle cerebral artery occlusion (Wu et al., 1999). Thus, thecurrent study determined if propentofylline pretreatment (i) alters spinal cordnociceptive transmission in the rat, and (ii) modifies the effect of exogenousIL-1 administered into the spinal cord. This was carried out by comparing inpropentofylline-treated rats the effect of intrathecally administered IL-1β on theintegrated C-fiber nociceptive reflex responses as well as on the potentiationof the responses evoked by repetitive electric stimulation of the sural nervereceptive field (wind-up). As already reported, wind-up activity in dorsal hornneurons is a synaptic potentiation phenomenon of particular importance for thedevelopment and maintenance of chronic pain (Dickenson, 1990; Dickensonet al., 1997), but the role of glia and cytokines on wind-up activity has deservedlittle attention.

METHODS

During this study, the guidelines on ethical standards for investigations ofexperimental pain in animals of The Committee for Research on Ethical Issuesof the International Association for the Study of Pain (1980) were followed.Experiments were performed in Sprague-Dawley rats weighing 280–320 g.Rats were given one daily intrathecal injection of propentofylline (10 µg/10 µlsaline) during 10 days. Healthy rats receiving intrathecal injections of salineduring 10 days served as controls. Afterwards, the animals were submitted tothe electrophysiological study.

The C-fiber evoked flexor reflex, elicited in the right hindlimb ofurethane anaesthetized rats (1.2 g/kg i.p.), was recorded as describedpreviously (Strimbu-Gozariu et al., 1993). Briefly, rectangular electric pulsesof supramaximal strength and two ms duration were applied every 10 s to thesural nerve receptive field by means of 2 stainless steel needles inserted intothe skin of toes 4 and 5 (Grass S11 stimulator equipped with a Grass SIU 5stimulus isolation unit and a Grass CCU 1A constant current unit). The C-fiberevoked reflex response was recorded from the ipsilateral biceps femoris muscleby utilizing another pair of stainless steel needles. After amplification (Grass

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P511 preamplifier), the electromyographic responses were digitized at 100 KHzand integrated in a time-window from 150 to 450 ms after the stimulus by aPowerlab ML 820 instrument. Once stable C reflex responses were obtained, thestimulus strength was lowered and the current required for threshold activationof the C reflex determined (usually 6–7 mA). Integrated C reflex responses,evoked by single stimuli with two-fold the intensity of the threshold stimulatingcurrent, were then recorded. Afterwards, trains of 12 stimuli each at 1 Hz weredelivered to the toes in order to develop wind-up activity. In the C reflexparadigm, wind-up consists of a stimulus frequency-dependent remarkableincrement of the electromyographic integrated response (Laurido et al., 2001).All responses were stored on hard disk for later analysis. Least square regressionlines were fitted among experimental points showing only incremental trend(prior to wind-up saturation at the 6th or 7th stimulus), discarding the remainderpoints (Microcal Origin 5.0 software), as described elsewhere (Laurido et al.,2001). The slopes of the regression lines represent wind-up scores.

In both propentofylline- and saline- treated animals, the experiments beganwith the measurement of a basal recording of integrated C reflex responsesand wind-up activity prior to the administration of intrathecal saline (10 µl)and recombinant IL-1β (2 ng/10 µl). The intrathecal injections were madeby means of direct percutaneous injection (Mestre et al., 1994). 10–20 minafter administration of IL-1β, experimental series measuring integrated Creflex responses and wind-up scores were performed. Slopes of regressionlines (means ± SEM) obtained before (basal wind-up) and after the differentintrathecal doses of IL-1β were compared using one-way ANOVA followedby the Dunnett test for multiple comparisons (GraphPad Instat 3.0, GraphPadSoftware Inc., San Diego, California, USA). A probability of less than .05 wasconsidered to indicate statistically significant differences between means.

RESULTS

Application of 12 successive constant electric pulses at 1 Hz induced spinalwind-up in healthy intact and propentofylline-treated rats, as revealed by thegradual but remarkable increase of the integrated C reflex activity generated bythe repetitive stimuli. The stimulating current required for threshold activationof the C reflex in normal animals was 6.3 ± 0.4 mA, whereas a significantlygreater stimulating current of 8.2 ± 0.5 mA (p < .05, unpaired Student’s t-test)was necessary to evoke threshold C reflexes in propentofylline-treated rats.Intrathecal administration of a single dose of 2 ng of IL-1β to intact rats did notproduce significant changes in the integrated C reflex response, whereas the

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Figure 1. Change (in percentage) of C reflex integrated activity (A) and spinal cord wind-up(B) in normal and propentofylline-treated rats, 10 min after intrathecal saline and 10, 20, and 40min after 2 ng i.t. of interleukin-1β (IL-1β). Values are means ± S.E.M. Significant changes aftersaline or IL-1β administration are denoted by the asterisk (∗p < .05, one-way ANOVA followedby the Dunnett multiple comparisons test).

same i.t. dose resulted in about 80% increase of wind-up activity in the sameanimals 20 min after injection (Figure 1, A and B). In contrast, 2 ng of IL-1β

intrathecally administered to propentofylline-treated rats produced a significantreduction in wind-up scores 40 min after injection, without modifying theintegrated C reflex activity (Figure 1, A and B).

DISCUSSION

As a whole, the earlier results showed that in the rat, a 10-day period of treatmentwith propentofylline i.t. did not block the ability of dorsal horn neurons torespond to C-fiber nociceptive stimulation and to develop wind-up activityduring repetitive input. However, this treatment resulted in increased thresholdfor the triggering of C-fiber-dependent nociceptive reflexes, thus indicating thatglial cells of the spinal cord dorsal horn play some role in pain transmissionconveyed by the C fiber population even in absence of injury in peripheralsensitive nerves and/or in central spinal cord neurons. Apparently, this findingargues against the current point of view that glial cells are inactive in healthy,normal spinal cord (Watkins & Maier, 2000; Watkins et al., 2001). Nevertheless,at present it seems unclear if propentofylline may produce some subtle changesin neuronal function, in addition to its well-known inhibitory effects uponactivated glia (Sweitzer et al., 2001). Although intrathecal administration ofIL-1β did not affect the spinal cord transmission of single C-fiber input in

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normal rat, the cytokine markedly increased wind-up activity during repetitivestimulation in the same rats. This observation confirms previous results byConstandil et al. (2003), suggesting that IL-1 of glial origin could play animportant role in the maintenance of chronic pain by increasing wind-up activityin dorsal horn nociceptive neurons via excitation of IL-1 receptors existing inthese cells. However, alternative explanations involving activation of spinalglia by IL-1β and subsequent release of a glial mediator that may excite dorsalhorn nociceptive neurons are also possible. In this respect, there is a varietyof potential glial mediators that can fulfill an excitatory role on dorsal hornnociceptive neurons (reviewed by Watkins & Maier, 2000; Watkins et al., 2001):firstly, the excitatory aminoacid glutamate, which is known to be releasedfrom spinal cord glia and play a major role in wind-up elicitation; second,the ubiquitous molecule nitric oxide, which has been directly implicated inglutamate release from primary nociceptive afferent terminals; third, othercytokines such as IL-6 and tumour necrosis factor, which have been describedas having excitatory activity in dorsal horn cells. All these mediators canpotentially be released from glia after glial cell stimulation with IL-1β, providedglial cells are intact.

Unexpectedly, i.t. administration of IL-1β in propentofylline-treatedanimals resulted in depression of wind-up activity, that is, the opposed effectobserved in healthy untreated rats. This is suggestive that exogenous IL-1β

did not act directly on IL-1 receptors of dorsal horn neurons to enhancewind-up activity, but probably upon glial IL-1 receptors, thereby inducingthe release of a glial mediator responsible for the excitatory effects observedin intact rats. In these conditions, dorsal horn cells of animals with alteredglial function due to the propentofylline pretreatment would be able todevelop normal wind-up via the excitatory effect of neurotransmitters releasedby the primary nociceptive afferents (although enhanced threshold for thetriggering of C-reflexes was found), but unable to increase wind-up activityafter IL-1β administration because the hypothetical glial mediator is probablyreleased at low rate or even not released from altered glia. This explanation,nevertheless, can only account for ineffectiveness of IL-1β to increase wind-upactivity, but not for the inhibitory effect actually produced by this cytokinein the neuronal response gain to repetitive stimulation (wind-up). Indeed,decreased wind-up response after intrathecal IL-1β in propentofylline-treatedrats necessarily implies an inhibitory effect of the cytokine on dorsal hornneuronal activity, together with glial cell inactivation. Thus, in intact animalsthe excitatory effect of the unidentified glial mediator upon dorsal horn neuronswould predominate over the inhibitory action of IL-1β, whereas in rats with

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INTERLEUKIN-1β, PROPENTOFYLLINE AND NOCICEPTION 623

lesioned glia the inhibitory effects of IL-1β would become dominant. In thisregard, inhibitory effect of IL-1β has been shown in warm-sensitive (Hori etal., 1988) and glucose-sensitive (Plata-Salaman et al., 1988) neurons of thehypothalamus, whereas both inhibitory and excitatory effects of IL-1β havebeen observed on neocortical neurons (Lukats et al., 2005). Using patch-clamptechniques, it has been demonstrated that IL-1β at picomolar physiologicalconcentrations excites central neurons by activating a nonselective cationiccurrent, whereas at nanomolar pharmacological concentrations IL-1β caninhibit central neurons by inducing membrane hyperpolarization (Desson& Ferguson, 2003). Other patch-clamp studies have demonstrated thatnanomolar concentrations of IL-1β decreases inward calcium currents inhippocampal neurons (Plata-Salaman & Ffrench-Mullen, 1992) and sodiuminward currents in retinal ganglion cells (Diem et al., 2003), thus providinga molecular support for the inhibitory effect of the nanomolar intrathecaldose of IL-1β on wind-up activity in the nociceptive C reflex reportedherein.

In summary, the present results showed that propentofylline pre-treatmentin the rat results in impaired ability of IL-1β to increase spinal cord nociceptivetransmission to repetitive stimulation, thus providing a mechanistic explanationfor the already reported beneficial effect of glial cell inhibitors in the treatmentof experimental chronic pain.

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