Endogenous descending modulation: spatiotemporal effect of dynamic imbalance between descending facilitation and inhibition of nociception

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1 J Physiol (2010) pp Endogenous descending modulation: spatiotemporal effect of dynamic imbalance between descending facilitation and inhibition of nociception Hao-Jun You 1,2,JingLei 1,3,Mei-YuSui 1,2, Li Huang 1,2, Yong-Xiang Tan 1,2,ArneTjølsen 4 and Lars Arendt-Nielsen 3 1 Center for Biomedical Research on Pain (CBRP), College of Medicine, Xi an Jiaotong University, Xi an , P.R. China 2 Department of Physiology, College of Medicine, Xi an Jiaotong University, Xi an , P.R. China 3 Center for Sensory Motor Interaction (SMI), Laboratory for Experimental Pain Research, Aalborg University, Fredrik Bajers Vej 7 D-3, DK-9220 Aalborg, Denmark 4 Department of Biomedicine, University of Bergen, N-5009 Bergen, Norway In conscious rats, we investigated the change of nociceptive paw withdrawal reflexes elicited by mechanical and heat stimuli during intramuscular (i.m.) 5.8% hypertonic (HT) saline elicited muscle nociception. i.m. injection of HT saline caused rapid onset, long lasting (around 7 days), bilateral mechanical hyperalgesia, while it induced bilateral, slower onset (1 day after the HT saline injection), long-term (about 1 2 weeks) heat hypoalgesia. Ipsilateral topical pre-treatment of the sciatic nerve with 1% capsaicin significantly prevented the occurrence of both the bilateral mechanical hyperalgesia and the contralateral heat hypoalgesia. Intrathecal administration of either 6-hydroxydopamine hydrobromide (6-OHDA) or 5,7-dihydroxytryptamine (5,7-DHT), and intraperitoneal injection of naloxone all markedly attenuated the HT saline induced bilateral heat hypoalgesia, but not the mechanical hyperalgesia. Combined with experiments with lesioning of the rostroventral medulla with kainic acid, the present data indicate that unilateral i.m. injection of HT saline elicits time-dependent bilateral long-term mechanical hyperalgesia and heat hypoalgesia, which were modulated by descending facilitatory and inhibitory controls, respectively. We hypothesize that supraspinal structures may function to discriminate between afferent noxious inputs mediated by Aδ- and C-fibres, either facilitating Aδ-fibre mediated responses or inhibiting C-fibre mediated activities. However, this discriminative function is physiologically silent or inactive, and can be triggered by stimulation of peripheral C-fibre afferents. Importantly, in contrast to the rapid onset of descending facilitation, the late occurrence of descending inhibition suggests a requirement of continuous C-fibre input and temporal summation. Thus, a reduction of C-fibre input using exogenous analgesic agents, i.e. opioids, may counteract the endogenous descending inhibition. (Resubmitted 25 July 2010; acceptedafter revision 7 September 2010; first published online 13 September 2010) Corresponding author H-J. You: Center for Biomedical Research on Pain (CBRP), College of Medicine, Xi an Jiaotong University, Xi an , China. yhj@mail.xjtu.edu.cn Abbreviations 5,7-DHT, 5,7-dihydroxytryptamine; 5-HT, serotonin; HT, hypertonic; IT, isotonic; KA, kainic acid; NA, noradrenaline; 6-OHDA, 6-hydroxydopamine hydrobromide; RVM, rostroventral medulla. Introduction Pain originating from deep somatic structures, which is often described as diffuse, dull pain, represents a major concern for many pain patients. It is well known that muscle pain can be felt not only at the site of the primary muscle injury, but also involves the soft tissues that surround muscles, including ligaments and tendons. In contrast to many other somatic pain conditions, muscle pain has been considered difficult to treat efficiently. The potentially involved neural mechanisms, in particular central mechanisms, of muscle pain are as yet sparsely known (Graven-Nielsen, 2006). Intramuscular (I.M.) injection of hypertonic (HT) saline (5.8%) is regarded as a valid experimental approach for the investigation of muscle pain/nociception in humans and animals. Since the 1930s, it has been shown in humans that I.M. injection of HT saline into muscle, i.e. the tibialis DOI: /jphysiol

2 4178 H-J. You and others J Physiol anterior muscle, can effectively elicit pain not only at the primary injection area, but also at referred areas, typically to the anterior aspect of the ankle (Kellgren, 1938). Using laser Doppler technique on human subjects, we recently showed a significantly enhanced blood flow and skin temperature in both legs after unilateral I.M. injection of 5.8% HT saline into the tibialis anterior muscle (Lei et al. 2008). These results suggested a bilateral spinal and/or supraspinal regulation, i.e. descending modulation, caused by unilateral nociceptive stimulation of deep somatic structures. It is also well known that peripheral tissue injury and inflammation may lead to pain associated with enhanced responsiveness to noxious heat and mechanical stimuli, i.e. hyperalgesia and allodynia, in animals as well as in humans. It is generally considered that the secondary hyperalgesia occurring outside the injured area is mechanical, but not heat sensitive, and related to dynamic changes in central neural mechanisms. Several studies have demonstrated that central and peripheral mechanisms involving complex signalling cascades underlie the induction and maintenance of primary and secondary hyperalgesia in a number of models of pain (Treede et al. 1992; Urban & Gebhart, 1999). However, it is still unclear which components of the central nervous system, i.e. the spinal cord or supraspinal structures, contribute predominantly to secondary hyperalgesia, and why secondary heat hyperalgesia rarely is described in the literature. Thus, we here systematically investigated the variation of the withdrawal reflex evoked by noxious mechanical and heat stimuli in conscious rats, in a condition of muscle pain/nociception caused by I.M. injection of 5.8% saline. We demonstrated a time-dependent dynamic imbalance of descending modulations: early onset of descending facilitation and late occurrence of descending inhibition, giving mechanical hyperalgesia and heat hypoalgesia during HT saline induced muscle nociception. With respect to the evidence for the late occurrence of descending inhibition, a new concept of a silent supraspinal discriminator with different triggering thresholds for governing Aδ- and C-fibre mediated nociception has been further put forward. Methods Ethical approval and animals Male Sprague Dawley rats weighing g (10 weeks age) were provided by the Animal Center of the College of Medicine, Xi an JiaoTong University, and housed pairwise in plastic boxes under a 12:12 h light dark cycle (lights on at h) at C with food and water available ad libitum. All experiments were approved by the Xi an JiaoTong University Animal Care Committee in accordance with the Committee s guidelines for pain research in conscious animals, and comply with the policies and regulations of The Journal of Physiology (Drummond, 2009). The animals were acclimatized to the laboratory and habituated to the test boxes for at least 1 h each day 5 days prior to testing. The rats were used only once and killed at the end of the experiment by intraperitoneal injection of an overdose of sodium pentobarbital (200 mg kg 1 ). All efforts were made to minimize the number of animals used and their suffering. Surgery for RVM lesions and intrathecal catheterization The sodium pentobarbital anaesthetized (40 mg (kg b.w.) 1 ) rats were mounted in a stereotaxic frame with fixation of the head by ear bars and tooth plate (MP8003, RWD Life Science Co., Shenzhen, Guangdong Province, China). A mini craniotomy was conducted with a dental drill in order to perform the intracerebral microinjection. Before intracerebral microinjection, the rats were treated with diazepam (5 mg kg 1, I.P.) to prevent death due to kainic acid induced status epilepticus and distant brain damage (Ben-Ari et al. 1979). The rostroventral medulla of the brainstem (RVM: anteroposterior mm from Bregma, lateral ±0.5mm from midline, dorsoventral 10 mm from the cranium; Paxinos & Watson, 1998) was then bilaterally microinjected with 0.25 μl per side of either kainic acid (KA, 1 mg ml 1, Sigma-Aldrich Chemie Gmbh, Germany) or 0.9% saline. This intracerebral microinjection was slowly performed with a 0.5 μl microsyringe over a period of 2 min. After that, the microsyringe remained in place for 5 min, and was slowly withdrawn, and the skull was closed with dental cement. A recovery period of 4 days was allowed, during which animals behaviour and motor function were strictly monitored. Animals showing severe permanent neurological deficits and motor dysfunction were excluded from the remaining experiments. Under sodium pentobarbital anaesthesia (50 mg (kg b.w.) 1 ), the intrathecal (I.TH.) catheterization was performed using PE-10 polyethylene tubing (o.d. 0.5 mm, i.d mm). The catheter was passed through a slit cut in the spinal arachnoid of the T6 7 region, and advanced subarachnoidally to the area of the spinal lumbar enlargement. The length of the intrathecal section of the catheters was around 6 cm, and the total volume of each catheter was less than 4 μl. The outer end of the tubing was firmly fixed to the paravertebral muscles to prevent the inset tubing from moving. The wound was washed with sterile saline, treated with antibiotics, and the muscles and skin were sutured by layers. The whole operation was performed in strictly sterile conditions. After the catheterization, the animals were put back in the box for recovery. The total

3 J Physiol Descending modulation on pain 4179 recovery period after the I.TH. catheterization was 3 days, and animal showing significant signs of motor dysfunction were strictly excluded from the experiments. Intramuscular administration of hypertonic saline and other drugs administration As elsewhere (Ro & Capra, 2001; Lei et al. 2008), a volume of 0.2 ml hypertonic (HT, 5.8%) saline was intramuscularly injected into the gastrocnemius muscle of the left (ipsilateral) hind limb in order to establish muscle nociception. The injection site was in the middle part of the gastrocnemius muscle, and the depth of the injection was about 0.5 cm. The injection procedure was performed manually and lasted more than 30 s. A volume of 0.2 ml isotonic (IT, 0.9%) saline served as control. Different doses ( μgkg 1 ) of naloxone (a non-specific antagonist to opioid receptors, Dupont Pharma, USA) were injected intraperitoneally in control rats and in rats receiving intramuscular injection of HT saline. I.TH. administration of 10 μg 6-hydroxydopamine hydrobromide (6-OHDA, Sigma-Aldrich) or 20 μg 5,7-dihydroxytryptamine (5,7-DHT, Sigma-Aldrich) was performed via the intrathecal catheter 4 days prior to the I.M. injection of 5.8% saline. Both neurotoxins were administrated in a volume of 10 μl, and 0.9% NaCl with 0.2 mg ml 1 ascorbic acid served as vehicle. After the administration of either neurotoxins or vehicle, the catheters were flushed with 5 μl of 0.9% NaCl. All intrathecal injections were performed manually within 30 s. Experimental design Experimental study groups were randomized and blinded. According to the different experimental purposes, rats recruited in the current study were randomly divided into several individual groups; 8 10 rats randomly assigned in each group were included for the investigation. Measurement of mechanical and heat sensitivity. Withdrawal thresholds to mechanical and heat stimulation were measured for both ipsilateral and contralateral hind paws (heel part) 30 min prior to and 5 30 min, 1 4 h, and 1 7 days post the intramuscular injection of 5.8% HT saline. Some experiments involving muscle nociception elicited by I.M. injection of HT saline were performed after 4weeks. For the measurement of mechanically evoked behavioural responses, rats were placed in different individual Plexiglas chambers with mesh floors and transparent covers ( cm). An electronic von Frey device (2290 Electrovonfrey, IITC, Woodland Hills, CA, USA) was used to detect the mechanical paw withdrawal threshold. The filament was applied to the heel part of the hind paw according to the mapping of the withdrawal field of the gastrocnemius muscle (Schouenborg & Weng, 1994; You & Arendt-Nielsen, 2005). The filament that elicited a withdrawal response in 50% of trials was taken to be the mechanical threshold (g). A reduced or increased threshold for the withdrawal response compared with the threshold before the HT saline injection was defined as hyperalgesia or hypoalgesia, respectively. In addition to the von Frey evoked withdrawal reflex, the Randall Selitto test was performed in a part of the experiments for further investigation and identification (Randall & Selitto, 1957). Briefly, nociceptive thresholds were measured with a digital paw pressure meter (probe tip: 1 mm; cut-off pressure: 500 g; Model 2500, IITC) by applying increasing pressure to the animal s ipsilateral heel part of the hind paw until vocalization. Heat evoked paw withdrawal responses were determined using a 390G plantar stimulator Analgesia Meter (IITC). The rats were tested individually in a Plexiglas cubicle placed onto a constant temperature controlled transparent glass plate used to avoid temperature sink from the tested hind paws. The heat stimulus was a high-intensity beam (setting = 30 40% intensity of full power) aimed at the heel part of the hind paw. The withdrawal latency was defined as the time from the onset of noxious heat stimulation to withdrawal of the tested hind paw. The intensity of the beam was adjusted so that the latency of the paw withdrawal reflex was around s in untreated animals. A painful, but tolerable, sensation could be elicited using this s heat stimulation on the operator s hand. To avoid excessive tissue injury, manual cut-off of the heat stimulus was performed if no paw withdrawal reflex could be evoked during 20 s of heat stimulation. Assessment of motor function. Briefly, animals were placed on a Rota-Rod treadmill (Model 755, IITC) rotating at a gradually increasing speed from 5 to 30 r.p.m. for 30 s and maintained for another 120 s at 30 r.p.m. Rats with motor dysfunction after the chronic I.TH. catheterization and the neurotoxic lesion with either capsaicin or 6-OHDA/5,7-DHT were excluded from the remaining experiments. Topical treatment of sciatic nerve with capsaicin. Under sodium pentobarbital (50 mg (kg b.w.) 1, I.P.) and lidocaine (local application) anaesthesia, the sciatic nerve of the left hind limb was exposed, and a piece of cotton soaked with 0.25 ml of either vehicle or 1% capsaicin solution (Sigma-Aldrich vehicle: 10% ethanol, 10% Tween 80, and 80% saline) was gently wrapped around 1 cm of the sciatic nerve for 30 min as described elsewhere (Fitzgerald, 1983). One day after capsaicin treatment, the loss of sensitivity to graded radiant heat confirmed the effect of

4 4180 H-J. You and others J Physiol topical application of capsaicin. Following confirmation by the heat test, 5.8% HT saline was injected intramuscularly into the gastrocnemius muscle of the capsaicin treated hind limb to introduce muscle nociception. Histology for identification of KA lesion of RVM. At the end of the period of behavioural testing (about 7 days), the animals receiving KA lesion of RVM were deeply anaesthetized by an overdose of sodium pentobarbital (75 mg kg 1, I.P.) and transcardially perfused with 10% formalin. The brains were then isolated and stored in 30% sucrose for 2 days. Freezing serial sections (50 μm thickness) were cut in the coronal plane and stained with Nissl stain, and were screened under a microscope (Leica, Germany). Schematic reconstruction of the injection sites and lesion area was drawn according to the stereotaxic atlas of rats (Paxinos & Watson, 1998). Reported results are basedonobservationsmadeonratswithaccuratelocation of the lesions in the RVM. The histological analysis of cannula tip location and lesion area was performed without the knowledge of the behavioural results. Biochemical analyses by high-performance liquid chromatography (HPLC). As described elsewhere (Tjølsen et al. 1991), using HPLC associated with electrochemical approach (adjusted to 0.7 V verse the Ag AgCl electrodes) endogenous levels of noradrenaline (NA) and serotonin (5-HT) in the lumbar spinal cord were detected 1, 4 and 7 days after the administration of the two different neurotoxic drugs. Results were all calculated in nmol per g fresh spinal cord tissue. Statistical analysis All results were expressed as means ± S.E.M.The data were analysed using SigmaStat (Systat Software Inc., San Jose, CA, USA) and compared by means of one-way/two-way repeated measures ANOVA with post hoc Bonferroni s test for analysis of the differences in the observation time among different groups. P < 0.05 was considered statistically significant. Results Changes of the withdrawal reflex elicited by mechanical and heat stimuli during 5.8% saline-induced muscle nociception The bilateral paw withdrawal reflex to mechanical and heat stimuli was evaluated 30 min prior to, and 5 30 min, 1 4 h and 1 7 days after the intramuscular injection of 5.8% HT saline into the ipsilateral (left) gastrocnemius muscle (Fig. 1). During the first week after the HT saline injection, the mechanically evoked withdrawal reflex was significantly enhanced bilaterally (P < 0.05, one-way ANOVA, Fig. 1A). In contrast, no significant change of bilateral mechanically evoked withdrawal reflex was observed during the exposure to isotonic (0.9%) saline injection (P > 0.05, one-way ANOVA, Fig. 1A). A significant difference was found in the mechanical withdrawal reflex between isotonic saline and hypertonic saline treatments (ipsilateral: F (10,180) = 2.26, P < 0.05; contralateral: F (10,180) = 2.02, P < 0.05; Fig. 1A). In contrast to the bilateral enhanced mechanical responses, we did not find any significant changes of the heat evoked withdrawal reflex during the initial 4 h following the HT saline injection (P > 0.05, one-way ANOVA, Fig.1B). However, the latency of the heat evoked withdrawal reflex was significantly prolonged bilaterally, from 11.1 ± 0.8 s (ipsilateral) and 11.2 ± 0.8 s (contralateral) (baseline response) to 17.3 ± 0.9 s and 17.4 ± 0.9 s 1 day after the HT saline injection respectively, and lasted more than 7 days, indicating a HT saline-induced heat hypoalgesia but not hyperalgesia (Fig. 1B, P < 0.001, one-way ANOVA). A significant difference in heat evoked withdrawal reflex was found between isotonic saline and hypertonic saline injections (time effect: ipsilateral: F (6,108) = 25.37, P < 0.001; contralateral: F (6,108) = 26.62, P < 0.001; Fig. 1B). These decreased heat evoked responses declined to the control level gradually within 2 weeks (data not shown). We further tested the nociceptive vocalization threshold by applying increasing pressure to the heel part of the hind paws before and after the HT saline injection. Relative to controls that received 0.9% saline intramuscular injections, 5.8% HT saline injections caused bilaterally facilitated vocalization responses for more than the 1 week observation time (ipsilateral: F (12,216) = 2.14, P < 0.05; contralateral: F (12,216) = 2.23, P < 0.05; Fig. 1C). This suggested that the peripheral and central mechanisms underlying the modulation of the mechanical withdrawal reflex and vocalization threshold probably share similar neural modulating mechanisms. In additional experiments, we tested withdrawal reflexes evoked by stimulation of the middle part of the forepaws following unilateral muscle nociception elicited by injection of 5.8% HT saline into a hind limb. Similar to the results obtained in the hind paws, we observed significant bilateral, rapid onset, long-term (1 week) decrease of vocalization thresholds (241.2 ± 14.3g (baseline, ipsilateral) and ± 15.4 g (baseline, contralateral) vs ± 12.8 g (1 day, ipsilateral) and ± 12.2 g (1 day, contralateral), P < 0.05, n = 8, one-way ANOVA) and slower onset (1 day after the HT saline injection) long-lasting (>1 week) prolonged latency of heat evoked withdrawal reflex (10.5 ± 0.5 s (baseline, ipsilateral) and 10.1 ± 0.4 s (baseline, contralateral) vs ± 0.8 s (1 day, ipsilateral) and 16.2 ± 0.9 s (1 day, contralateral), P < 0.001, n = 8, one-way ANOVA).

5 J Physiol Descending modulation on pain 4181 Effects of different neurotoxic lesions on nociceptive behavioural responses during muscle nociception Histological analysis showed that the area of KA lesion in RVM usually had a distinct borderline, and was typically restricted to less than 1 mm (about ±0.5 mm from the cannula tip). Bilateral microinjection of KA, but not 0.9% saline, resulted in at least 70 80% gliosis and neuronal loss within the area of the RVM. Schematic reconstruction of RVM in rats receiving intracerebral KA lesions is presented in Fig. 2A. Four days after the microinjection with either KA or 0.9% saline, we tested the bilateral paw withdrawal thresholds to mechanical and heat stimuli. No significant influence of KA and saline on mechanical paw withdrawal threshold and heat paw withdrawal latency was found (P > 0.05) (Fig. 2B and C). Compared with vehicle Figure 1. Response thresholds of paw withdrawal reflexes elicited by mechanical (A and C) and heat (B) stimuli before and after the unilateral (left site) I.M. injection of either 0.9% (IT) saline or 5.8% (HT) saline Thirty minutes after the 5.8% HT saline injection a significant decreased paw withdrawal reflex was found, and lasted 6 days over time (P < 0.05, Fig. 1A). During the initial 4 h observation period following the HT saline injection heat evoked withdrawal reflex was not significantly influenced (P > 0.05). However, 1 day after the HT injection a long-term bilateral prolonged latency of heat evoked paw withdrawal reflex (hypoalgesia) was found and lasted around 7 14 days (Fig. 1B, data over 7 days are not shown). Similar to the withdrawal reflex elicited by von Frey filament, significant bilateral decreased vocalization thresholds were observed during muscle nociception, indicating that the vocalization responses evoked by painful mechanical stimulation and the paw withdrawal responses elicited by von Frey filament probably share similar neural modulating mechanisms. P < 0.05 and ##P < compared with IT saline injection. (B.: baseline response before I.M. injection of saline; L.: left site; R: right site.)

6 4182 H-J. You and others J Physiol (0.9% saline) treatment, after KA lesion of RVM neither mechanical hyperalgesia nor heat hypoalgesia was observed during HT saline induced muscle nociception (mechanical responses: ipsilateral: F (11,176) = 2.32, P < 0.05; contralateral: F (11,176) = 2.29, P < 0.05, Fig. 2B; heat responses: ipsilateral: F (6,96) = 33.36, P < 0.001; contralateral: F (6,96) = 35.41, P < 0.001, Fig. 2C). These data suggest that the bilateral mechanical hyperalgesia as well as heat hypoalgesia induced by unilateral intramuscular injection of HT saline are dependent on descending modulations from supraspinal structures. As shown in Fig. 3A, 1 day following the topical treatment of the left sciatic nerve with 1% capsaicin the bilateral mechanically evoked withdrawal reflexes were not significantly influenced by capsaicin treatment (P > 0.05, one-way ANOVA). However, during HT Figure 2. Schematic reconstruction showing the locations of injection sites (filled circles) and lesion regions (grey areas) in nine rats receiving bilateral kainic acid (KA) lesion Bilateral microinjection with KA resulted in at least 70 80% neuronal loss in the area of RVM. Effects of KA lesion of RVM on mechanically and heat elicited paw withdrawal reflex during muscle nociception are showed in B and C. No mechanical hyperalgesia and heat hypoalgesia were found following the RVM lesion during muscle nociception. P < 0.05 compared with KA lesion. ##P < compared with vehicle (0.9% saline) lesion. (BK: baseline response before intracerebral microinjection with KA/vehicle; BS: baseline responses before HT saline intramuscular injection; L. left site; R: right site.)

7 J Physiol Descending modulation on pain 4183 saline induced muscle nociception neither an ipsilateral nor a contralateral enhanced mechanically evoked withdrawal reflex was found. A significant difference of mechanical withdrawal reflex between capsaicin treatment and vehicle treatment during muscle nociception was found (ipsilateral: F (11,176) = 2.25, P < 0.05; contralateral: F (11,176) = 2.31, P < 0.05; Fig. 3A). After the same capsaicin lesion, ipsilateral heat-evoked withdrawal reflexes were significantly prolonged to a latency of 20 s (cut-off value of heat stimulation), whereas no significant influence of unilateral capsaicin treatment was found on contralateral heat-evoked response. Compared with topical treatment with vehicle, 1 day after the intramuscular injection of 5.8% saline, no contralateral heat hypoalgesia was observed (contralateral: F (6,96) = 30.23, P < 0.001, Fig. 3B). Figure 4A shows the spinal levels of NA and 5-HT during the first week after I.TH. administration of 6-OHDA and 5,7-DHT. One day after I.TH. neurotoxins the mean levels of NA and 5-HT were significantly reduced compared with the control treatment with vehicle (P < 0.05 and P < 0.001). These neurotoxin effects reached a maximum after 4 days. In addition, highly specific effects of I.TH. treatment with 6-OHDA and 5,7-DHT were observed, in that 6-OHDA did not affect the 5-HT level while the NA level was not significantly influenced by 5,7-DHT treatment. Four days after neurotoxic lesion with either 6-OHDA or 5,7-DHT, we observed a mechanically and heat elicited bilateral withdrawal reflex during 5.8% saline induced muscle nociception. Neither 6-OHDA nor 5,7-DHT showed any significant influence on the mechanically evoked behavioural responses (Fig. 4B). Long lasting (about 7 days) enhanced mechanical withdrawal reflexes were found bilaterally in both the 6-OHDA/5,7-DHT lesion groups and the vehicle group (6-OHDA: ipsilateral: F (12,168) = 1.19, P > 0.05; contralateral: F (12,168) = 1.16, P > 0.05; 5,7-DHT: ipsilateral: F (12,168) = 1.06, P > 0.05; contralateral: F (12,168) = 1.09, P > 0.05; Fig. 4B). In contrast, neurotoxic lesion with either 6-OHDA or 5,7-DHT markedly blocked the development of hypoalgesia to heat stimuli (Fig. 4C). Compared with vehicle treatment, in both neurotoxic lesion groups the responses to noxious heat were unchanged after I.M. HT saline (6-OHDA: ipsilateral: F (6,84) = 37.2, P < 0.001; contralateral: F (6,84) = 35.7, P < 0.001; 5,7-DHT: ipsilateral: F (6,84) = 40.1, P < 0.001; contralateral: F (6,84) = 39.8, P < 0.001; Fig. 4C). Figure 3. Involvement of capsaicin-sensitive peripheral afferents in mechanical hyperalgesia and heat hypoalgesia following the unilateral I.M. injection of 5.8% saline induced muscle nociception After the unilateral topic treatment of the left sciatic nerve with capsaicin (1%), no bilateral mechanical hyperalgesia was observed (Fig. 3A). Likewise, contralateral heat hypoalgesia was significantly blocked by the ipsilateral capsaicin treatment (Fig. 3B). This suggested that the bilateral descending facilitatory and inhibitory modulations on mechanical hyperalgesia and heat hypoalgesia was dependent on the ipsilateral noxious inputs conveyed by capsaicin-sensitive C afferents. P < 0.05 compared with capsaicin treatment. ##P < compared with vehicle treatment. (BC: baseline response before capsaicin/vehicle treatment; BS: baseline responses before HT saline intramuscular injection; L. left site; R: right site.)

8 4184 H-J. You and others J Physiol Effects of naloxone on mechanically and heat evoked withdrawal reflexes We investigated whether naloxone, a potent μ-opioid receptor antagonist, affects mechanically and heat evoked nociceptive withdrawal reflexes. During physiological conditions without muscle stimulation with HT saline, neither mechanically nor heat evoked withdrawal reflexes were significantly influenced by I.P. administration of naloxone at different doses ( μgkg 1 )(oneway ANOVA, P > 0.05), indicating that endogenous opioid mediated antinociceptive controls do not normally influence these nociceptive behavioural responses (Fig. 5A and B). One day after the 5.8% saline intramuscular injection, naloxone ( μgkg 1 ) was administrated, and potential effects were tested for 4h. No influence of naloxone on those enhanced mechanically evoked withdrawal reflexes was observed (ipsilateral: F (4,96) = 1.13, P > 0.05; contralateral: F (4,96) = 1.02, P > 0.05; Fig. 5C). By marked contrast, naloxone significantly reversed the inhibition of the heat evoked withdrawal reflex (ipsilateral: F (4,96) = 45.8, P < 0.001; contralateral: F (4,96) = 44.2, P < 0.001; Fig. 5D). Figure 4. Effects of I.TH. administration of 6-OHDA (10 μg) and 5,7-DHT (20 μg) on concentrations of noradrenaline (NA) and serotonin (5-HT) assessed by HPLC approach in the lumbar spinal cord (A, n = 10 per group), and mechanically and heat evoked paw withdrawal reflexes during unilateral I.M. injection of 5.8% saline induced muscle nociception (B and C) I.TH. treatment with both 6-OHDA and 5,7-DHT significantly reduced heat hypoalgesia, but not mechanical hyperalgesia, during muscle nociception. P < 0.001, #P < 0.05 and ##P < compared with vehicle (Vc) treatment. (BO: baseline response before I.TH. catheter operation; B: baseline responses 3 days after I.TH. catheter operation; BS: baseline responses 4 days after 6-OHDA and 5,7-DHT I.TH. administration, and before HT saline injection; L. left site; R: right site.)

9 J Physiol Descending modulation on pain 4185 Discussion This study shows that intramuscular injection of HT saline in rats elicits rapid onset long-term (1 week) mechanical hyperalgesia, both ipsilaterally and contralaterally. It also elicits bilateral long lasting (1 2 weeks) heat hypoalgesia with a slower onset 1 day after the HT saline injection. The bilateral mechanical hyperalgesia and heat hypoalgesia are modulated by descending facilitatory and inhibitory controls, respectively, and noradrenergic and serotonergic pathways are both involved in the action of descending inhibitory, but not facilitatory, control on heat hypoalgesia induced by C-fibres inputs. Intramuscular hypertonic saline-induced ipsilateral local muscle pain and contralateral mirror pain Studying potential mechanisms for hyperalgesia and referred pain, Lei et al. (2008) used an improved experimental design supplementing the model of inducing muscle pain in humans. They showed that intramuscular injection of 5.8% saline causes a significant increase in skin blood flow and skin temperature at the primary injection site (tibialis anterior muscle) and the referred areas, which occurred not only at the anterior aspect of the ipsilateral ankle, but also at the mirror area contralateral to the primary muscle injection site. Due to Figure 5. Effects of I.P. administration of naloxone ( μg kg 1 ) on mechanically and heat evoked paw withdrawal reflexes during the physiological condition (A and B) and 5.8% saline induced muscle nociception (C and D) No effects of different doses of naloxone on mechanically and heat induced withdrawal reflexes were found during physiological state. It is suggested that endogenous opioid-mediated antinociceptive effects have no role in acute nociception evoked by noxious mechanical and heat stimuli. During 5.8% saline intramuscularly induced muscle nociception, naloxone did not significantly influence mechanical hyperalgesia. By marked contrast, heat evoked hypoalgesia was significantly reversed by naloxone treatment, indicating opioid-regulated endogenous antinociceptive effects on heat evoked responses during muscle nociception. ##P < compared with vehicle (0.9% saline) treatment. (B.: baseline response before I.M. injection of HT saline; L: left site; R: right site.)

10 4186 H-J. You and others J Physiol experimental limitations, noxious heat, which no doubt influence the observation of blood flow and temperature, evoked responses were not observed (Lei et al. 2008). Our current animal experimental data are consistent with this previous report, and provide novel evidence that besides the enhanced mechanically evoked behavioural responses, a long-lasting(>7 days) secondary and contralateral heat hypoalgesia from 1 day after hypertonic saline injection was found. Although the mechanisms of contralateral responses following unilateral local muscle pain are not clearly understood, complementary evidence point to the involvement of central components in the contralateral hypersensitization (Levine et al. 1985; Sluka et al. 2001; Audette et al. 2004; Lei et al. 2008; Owen et al. 2010). Of particular importance, our experiments using KA lesions of the RVM indicate that supraspinal structures are involved in the phenomena of bilateral mechanical hyperalgesia and heat hypoalgesia during unilateral muscle nociception. It is interesting to note that in additional experiments we tested withdrawal reflexes in the forepaws following unilateral hind paw muscle nociception, and similar to the results obtained in the hind paws, significant, long lasting, mechanical hyperalgesia and heat hypoalgesia were observed bilaterally also in the forepaws. It is suggested that the descending modulation of spinally organized nociception is diffuse with regard to spatial characteristics (Fields et al. 2006). Accordingly, we may argue that experiments where the contralateral or other uninjured site throughout the body serve as a physiological, untreated control should be regarded with caution, as this may lead to incorrect interpretation and conclusions due to the diffuse character of the descending modulation. Valid nociceptive behavioural indices in assessment of nociception and descending modulation For pain research, nociceptive withdrawal reflexes and tail reflexes elicited by noxious mechanical and heat stimuli have widely been regarded as valid behavioural indices to assess pain responses in rodents (Le Bars et al. 2001). One may debate that in some cases mechanical stimulation could be able to elicit spinally organized withdrawal reflexes without being a pure noxious stimulus. In the current study, the Randall Selitto test showed a similar bilaterally decrease of vocalization thresholds after intramuscular injection of hypertonic saline as the reduction of withdrawal thresholds. These data indicate that withdrawal reflexes elicited by stimulation with von Frey filaments and vocalization activity evoked by paw squeezing may probably be mediated by similar nociceptive modulation pathways. Considering the opposite results of heat-evoked bilateral long-lasting hypoalgesia, the withdrawal reflex evoked by noxious mechanical and heat stimuli is not an unsophisticated spinal reflex but is modulated by descending facilitatory or inhibitory regulation, which involves activation of supraspinal structures (Pertovaara, 1998). We know that different populations of peripheral nociceptors can be excited by different modalities of stimuli, mechanical and heat stimuli preferentially activating A- versus C-nociceptors, respectively (Magerl et al. 2001). However, it is not clear whether the central responses to mechanical and heat stimuli are differentially modulated. Based on this behavioural study and other reports employing electrophysiological approach (Lumb, 2002; McMullan & Lumb, 2006), it is suggested that supraspinal structures have a function in discrimination between types of noxious afferent information. This discriminator exhibits either descending facilitatory effects on peripheral Aδ-fibre mediated responses or descending inhibitory actions on C-fibre mediated activities. If this is the case, pain research using mechanical withdrawal reflexes for investigation of descending inhibition may be associated with bias and may give rise to incorrect interpretation. Mechanical/heat hyperalgesia and hypoalgesia, time-dependent descending modulation, and inference on clinical pain Serious tissue injury may lead to hyperalgesia, which is referred to as an enhanced response to noxious stimuli. Primary hyperalgesia at the injury site is an increased pain response to noxious mechanical and heat stimulation, whereas secondary hyperalgesia is located in the uninjured area surrounding the primary injury site and has been described as enhanced pain to noxious mechanical, but not heat, stimulation (Lewis, 1936; Treede et al. 1992; Fuchs et al. 2000; Magerl et al. 2001). With regard to primary hyperalgesia, one might hypothesize that in addition to secondary or mirror heat hypoalgesia found in the current study, the descending inhibitory control upon C-fibre mediated responses may control heat evoked hyperactivity at the primary injury area as well. To our knowledge, peripheral, but not central, sensitization plays a dominating role in primary heat hyperalgesia (Treede et al. 1992). From this, it is likely that peripheral sensitization may mask heat hypoalgesia in the primary area of injury. Due to such local pronociceptive effects caused by peripheral sensitization, noxious stimulation applied to the uninjured area may give more information with respect to central neural modulation. As described above, supraspinal structures may function to discriminate responses evoked by activation of different subgroups of afferents, and thus determine the adequate descending effect: facilitation or inhibition.

11 J Physiol Descending modulation on pain 4187 In contrast to descending inhibition, which attenuates pain or nociception, descending facilitation could be considered as another protective function to warn against more noxious stimulation (Pertovaara, 1998; Tillu et al. 2008). We know that unilateral topical treatment of the nerve with capsaicin can block the noxious muscular type IV afferent fibres excited by hypertonic saline (Iggo 1961; Fitzgerald, 1983). One most important finding in the current study is that after the unilateral capsaicin treatment, neither contralateral mechanically evoked enhanced withdrawal reflexes nor contralateral heat-evoked decreased withdrawal reflexes occur during muscle nociception. Combined with the results showing no effects of the lesion of RVM with KA on mechanical/heat paw withdrawal thresholds, it is suggested that the descending facilitatory/inhibitory controls of the supraspinal discriminator are not tonically active, and are triggered by peripheral noxious input from C-afferents. However, in the present study noxious C-afferent inputs evoked by intramuscular HT saline evoked effects with a significant temporal separation: rapid and late onset of descending facilitation and inhibition respectively. We suggest that the supraspinal discriminator has different triggering thresholds that determine its actions: a lower threshold for the facilitation of Aδ-fibre mediated responses and a higher threshold for the induction of descending inhibition of C-fibre mediated activities. Accordingly, the late occurrence of descending inhibition resulting in heat hypoalgesia may result from temporal summation recruited by continuous C-fibre inputs following intramuscular stimulation by HT saline. After neurotoxic lesion with either 6-OHDA or 5,7-DHT, no significantly effects on mechanically and heat evoked withdrawal reflexes were found in the present study. Additionally, during the exposure to muscle nociception we did not find any significant influence of neurotoxic lesion on the enhanced mechanical withdrawal reflex over time, and no enhanced or decreased heat evoked behavioural responses was observed after the hypertonic saline injection. All these findings suggest that neither 5-HT nor NA is involved in the descending facilitatory controls on pain. By contrast, both endogenous 5-HT and NA participate in the descending inhibitory controls of nociception, supported by the lack of heat hypoalgesia in animals with 5,7-DHT or 6-OHDA lesions. This finding is consistent with others, but further does not support the hypothesis that NA and 5-HT pathways exert tonic inhibition on spinally organized nociception (Tjølsen et al. 1991). Importantly, our experiment with naloxone further supports that there is no endogenous opioid-mediated antinociceptive control of nociceptive responses to acute stimulation as naloxone only reversed the attenuation of heat-evoked responses during hypertonic saline induced muscle nociception, and did not affect responses in the physiological condition. However, the data seem to some extent at odds with others reporting a facilitatory role of descending 5-HT pathways on pain (Suzuki et al. 2004). Interestingly, in experiments with female rats in muscle nociception, we found a different role of descending 5-HT and NA pathways in bilateral mechanical hyperalgesia and heat hypoalgesia. In female rats, descending 5-HT pathways are important in bilateral mechanical hyperalgesia, whereas descending NA pathways are involved in bilateral heat hypoalgesia, suggesting a complex role of sex differences in endogenous pain modulation (our unpublished observation). Today, tolerance and addiction to opioids with reduced antinociceptive effects is a serious clinical challenge. In the current study, the late occurrence of heat hypoalgesia suggests that triggering or induction of descending inhibition depends on temporal summation of C-fibre mediated noxious afferent signals. Exogenous opioid agonists bind to corresponding peripheral and central receptors, and may robustly inactivate the endogenous descending inhibition due to less input through nociceptive C-fibres. We believe that the more active endogenous descending inhibitory modulation, the more pronounced antinociceptive effects would be expected. 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