大鼠肌肉痛模型原始文献-2010年

J Physiol 588.21 (2010) pp 4177–4188 4177 Endogenous descending modulation: spatiotemporal effect of dynamic imbalance between descending facilitation and inhibition of nociception Hao-Jun You1,2, Jing Lei1,3, Mei-Yu Sui1,2, Li Huang1,2, Yong-Xiang Tan1,2, Arne Tjølsen4 and Lars Arendt-Nielsen3 1Center for Biomedical Research on Pain (CBRP), College of Medicine, Xi’an Jiaotong University, Xi’an 710061, P.R. China 2Department of Physiology, College of Medicine, Xi’an Jiaotong University, Xi’an 710061, P.R. China 3Center for Sensory–Motor Interaction (SMI), Laboratory for Experimental Pain Research, Aalborg University, Fredrik Bajers Vej 7 D-3, DK-9220 Aalborg, Denmark 4Department 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 injectionof naloxone allmarkedly attenuated theHTsaline inducedbilateral 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 weremodulated 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; accepted after revision 7 September 2010; first published online 13 September 2010) Corresponding authorH-J. You: Center for Biomedical Research on Pain (CBRP), College of Medicine, Xi’an Jiaotong University, Xi’an 710061, China. Email: 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.) injectionof 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 C© 2010 The Authors. Journal compilation C© 2010 The Physiological Society DOI: 10.1113/jphysiol.2010.196923 4178 H-J. You and others J Physiol 588.21 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 sub- jects, 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 under- lie 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, givingmechanical hyperalgesia and heat hypo- algesia during HT saline induced muscle nociception. With respect to the evidence for the late occurrence of descending inhibition, a new concept of a ‘silent’ supra- spinal 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 260–300 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 08.00 h) at 22–26◦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 pento- barbital (200mg 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 (40mg (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 (5mg 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 −10.30mm from Bregma, lateral ±0.5 mm from midline, dorso- ventral −10mm from the cranium; Paxinos & Watson, 1998) was then bilaterally microinjected with 0.25μl per side of either kainic acid (KA, 1 mgml−1, Sigma-Aldrich Chemie Gmbh, Germany) or 0.9% saline. This intra- cerebral microinjection was slowly performed with a 0.5μl 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 whichanimals’ behaviour andmotor functionwere strictly monitored. Animals showing severe permanent neuro- logical deficits andmotor dysfunctionwere excluded from the remaining experiments. Under sodium pentobarbital anaesthesia (50mg (kg b.w.)−1), the intrathecal (I.TH.) catheterization was performed using PE-10 polyethylene tubing (o.d. 0.5 mm, i.d. 0.25 mm). The catheter was passed through a slit cut in the spinal arachnoidof theT6–7 region, andadvanced sub- arachnoidally to the areaof 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 4μl. 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 C© 2010 The Authors. Journal compilation C© 2010 The Physiological Society J Physiol 588.21 Descending modulation on pain 4179 recovery period after the I.TH. catheterization was 3 days, and animal showing significant signs ofmotor 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 intra- muscularly 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 (50–450μg kg−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 10μg 6-hydroxydopamine hydrobromide (6-OHDA, Sigma-Aldrich) or 20μg 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 10μl, and 0.9% NaCl with 0.2 mgml−1 ascorbic acid served as vehicle. After the administration of either neurotoxins or vehicle, the catheters were flushed with 5μl of 0.9% NaCl. All intra- thecal 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 tomechanical andheat stimulation were measured for both ipsilateral and contralateral hind paws (heel part) 30min prior to and 5–30min, 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 4 weeks. For the measurement of mechanically evoked behavioural responses, rats were placed in different individual Plexiglas chambers withmesh floors and trans- parent covers (20× 20× 25 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 themechanical 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 10–11 s in untreated animals. A painful, but tolerable, sensation could be elicited using this 10–11 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 placedonaRota-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 (50mg (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, and80%saline)was gentlywrappedaround1 cmof the sciatic nerve for 30min as described elsewhere (Fitzgerald, 1983). One day after capsaicin treatment, the loss of sensitivity to graded radiant heat confirmed the effect of C© 2010 The Authors. Journal compilation C© 2010 The Physiological Society 4180 H-J. You and others J Physiol 588.21 topical application of capsaicin. Following confirmation by the heat test, 5.8% HT saline was injected intra- muscularly into the gastrocnemiusmuscle 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 (75mg 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 (50μm 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 based on observationsmade on rats with accurate location 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 30min prior to, and 5–30min, 1–4 h and 1–7 days after the intramuscular injection of 5.8% HT saline into the ipsilateral (left) gastro- cnemius 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 (contra- lateral) (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 iso- tonic 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 intra- muscular 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 centralmechanisms underlying the modulation of the mechanical withdrawal reflex and vocalization threshold probably share similar neural modulating mechanisms. In additional experiments,we testedwithdrawal reflexes evoked by stimulation of the middle part of the forepaws following unilateral muscle nocic