Differential effects of subcutaneous injection of formalin and bee venom on responses of wide-dynamic-range neurons in spinal dorsal horn of the rat

By using extracellular single unit recording technique, the effects of subcutaneous (s.c.) administration of formalin and bee venom on responses of wide-dynamic-range (WDR) neurons in the spinal dorsal horn were investigated in pentobarbital anaesthetised rats. Similar to the previous results, s.c. formalin injection into the cutaneous receptive field (RF) of WDR neurons produced a biphasic increase in spike response; however, s.c. bee venom injection into the RF produced a prolonged, monophasic increase in spike discharges over the background activity, suggesting that the two tonic pain models may have different underlying neural mechanisms.     

Peripheral inflammation induces tumor necrosis factor dependent AMPA receptor trafficking and Akt phosphorylation in spinal cord in addition to pain behavior

In the present study, intraplantar carrageenan induced increased mechanical allodynia, phosphorylation of PKB/Akt and GluR1 ser 845 (PKA site) as well as GluR1, but not GluR2 movement into neuronal membranes. This change in membrane GluR1/GluR2 ratio is indicative of Ca²⁺ permeable AMPA receptor insertion. Pain behavior was reduced and biochemical changes blocked by spinal pretreatment, but not post-treatment, with a tumor necrosis factor (TNF) antagonist, Etanercept (100 μg). Pain behavior was also reduced by spinal inhibition of phosphatidylinositol 3-kinase (PI-3K) (wortmannin; 1 and 5 μg) and LY294002; 50 and 100 μg) and Akt (Akt inhibitor IV; 3 μg). Phosphorylated Akt was found exclusively in neurons in grey matter and in oligodendrocytes in white matter. Interestingly, this increase was seen first in superficial dorsal horn and α-motor neurons (peak 45 min) and later (peak 2 h post-injection) in deep dorsal horn neurons. Akt and GluR1 phosphorylation, AMPA receptor trafficking and mechanical allodynia were all TNF dependent. Whether phosphorylation of Akt and of GluR1 are in series or in parallel or upstream of pain behavior remains to be determined. Certainly, TNF-mediated GluR1 trafficking appears to play a major role in inflammatory pain and TNF-mediated effects such as these could represent a path by which glia contribute to neuronal sensitization (spinal LTP) and pathological pain.     

Inflammation alters trafficking of extrasynaptic AMPA receptors in tonically firing lamina II neurons of the rat spinal dorsal horn

Peripheral inflammation alters AMPA receptor (AMPAR) subunit trafficking and increases AMPAR Ca²⁺ permeability at synapses of spinal dorsal horn neurons. However, it is unclear whether AMPAR trafficking at extrasynaptic sites of these neurons also changes under persistent inflammatory pain conditions. Using patch-clamp recording combined with Ca²⁺ imaging and cobalt staining, we found that, under normal conditions, an extrasynaptic pool of AMPARs in rat substantia gelatinosa (SG) neurons of spinal dorsal horn predominantly consists of GluR2-containing Ca²⁺-impermeable receptors. Maintenance of complete Freund’s adjuvant (CFA)-induced inflammation was associated with a marked enhancement of AMPA-induced currents and [Ca²⁺]_i transients in SG neurons, while, as we previously showed, the amplitude of synaptically evoked AMPAR-mediated currents was not changed 24 h after CFA. These findings indicate that extrasynaptic AMPARs are upregulated and their Ca²⁺ permeability increases dramatically. This increase occurred in SG neurons characterized by intrinsic tonic firing properties, but not in those exhibited strong adaptation. This increase was also accompanied by an inward rectification of AMPA-induced currents and enhancement of sensitivity to a highly selective Ca²⁺-permeable AMPAR blocker, IEM-1460. Electron microcopy and biochemical assays additionally showed an increase in the amount of GluR1 at extrasynaptic membranes in dorsal horn neurons 24 h post-CFA. Taken together, our findings indicate that CFA-induced inflammation increases functional expression and proportion of extrasynaptic GluR1-containing Ca²⁺-permeable AMPARs in tonically firing excitatory dorsal horn neurons, suggesting that the altered extrasynaptic AMPAR trafficking might participate in the maintenance of persistent inflammatory pain.     

过度通气对在体大鼠脊髓背角WDR神经元兴奋性影响的实验研究

目的:考察过度通气对大鼠脊髓背角广动力范围(Wide dynamic range,WDR)神经元自发和疼痛诱发放电频率的影响。方法:将25只SD大鼠随机分为N(正常通气)和H(过度通气)两组。两组均在吸入麻醉下行椎板切除,记录与大鼠后足掌部皮肤感觉对应的脊髓背角WDR神经元放电频率百分比。N组正常通气120分钟,旨在排除手术创伤对WDR神经元的影响;H组实施过度通气60分钟后,恢复正常通气继续观察60分钟,目的为考察过度通气对该神经元电活动的影响。结果:(1)N组各采样点间WDR神经元自发和诱发放电频率百分比均无显著性差异。(2)H组过度通气后WDR神经元自发和疼痛诱发放电频率百分比较对照值显著降低,并随正常通气的恢复而恢复。(3)H组过度通气期间WDR神经元自发及诱发放电频率百分比均较N组相应时间点显著降低。结论:过度通气可使大鼠脊髓背角WDR神经元兴奋性降低。     

Spinal R-phenyl-isopropyl adenosine inhibits spinal dorsal horn neurons responding to noxious heat stimulation in the absence and presence of sensitization

The effects of spinally administered R(−)N⁶-(2-phenylisopropyl) adenosine (R-PIA) on spinal dorsal horn neurons were investigated in anesthetized rats. Extracellular, single-unit recordings were measured during noxious heating of receptive fields on the hind paw. Three series of experiments were carried out to characterize the effects of R-PIA on spinal dorsal horn neuronal activity. In the first set of experiments, R-PIA dose-dependently suppressed noxiously evoked activity of spinal dorsal horn neurons. In the second set of experiments, R-PIA suppressed noxiously evoked activity in neurons sensitized by the topical application of mustard oil to a region of skin adjacent to their receptive fields. In the third set of experiments, R-PIA prevented mustard oil induced sensitization of dorsal horn neurons. In all cases, the adenosine receptor antagonist theophylline reversed the action of R-PIA. The results of these investigations indicate the involvement of spinal adenosine receptors in spinal pathways of central sensitization and in the modulation of somatically induced noxious pain.     

The scorpion toxin Amm VIII induces pain hypersensitivity through gain-of-function of TTX-sensitive Na channels

Voltage-gated Na⁺ channels (Nav) are the targets of a variety of scorpion toxins. Here, we investigated the effects of Amm VIII, a toxin isolated from the venom of the scorpion Androctonus mauretanicus mauretanicus, on pain-related behaviours in mice. The effects of Amm VIII were compared with the classic scorpion α-toxin AaH II from Androctonus australis. Contrary to AaH II, intraplantar injection of Amm VIII at relatively high concentrations caused little nocifensive behaviours. However, Amm VIII induced rapid mechanical and thermal pain hypersensitivities. We evaluated the toxins’ effects on Nav currents in nociceptive dorsal root ganglion (DRG) neurons and immortalized DRG neuron-derived F11 cells. Amm VIII and AaH II enhanced tetrodotoxin-sensitive (TTX-S) Nav currents in DRG and F11 cells. Both toxins impaired fast inactivation and negatively shifted activation. AaH II was more potent than Amm VIII at modulating TTX-S Nav currents with EC₅₀ of 5 nM and 1 μM, respectively. AaH II and Amm VIII also impaired fast inactivation of Nav1.7, with EC₅₀ of 6.8 nM and 1.76 μM, respectively. Neither Nav1.8 nor Nav1.9 was affected by the toxins. AaH II and Amm VIII reduced first spike latency and lowered action potential threshold. Amm VIII was less efficient than AaH II in increasing the gain of the firing frequency-stimulation relationship. In conclusion, our data show that Amm VIII, although less potent than AaH II, acts as a gating-modifier peptide reminiscent of classic α-toxins, and suggest that its hyperalgesic effects can be ascribed to gain-of-function of TTX-S Na⁺ channels in nociceptors.     

Reactive oxygen species contribute to neuropathic pain and locomotor dysfunction via activation of CamKII in remote segments following spinal cord contusion injury in rats

In this study, we examined whether blocking spinal cord injury (SCI)-induced increases in reactive oxygen species (ROS) by a ROS scavenger would attenuate below-level central neuropathic pain and promote recovery of locomotion. Rats with T10 SCI developed mechanical allodynia in both hind paws and overproduction of ROS, as assayed by Dhet intensity, in neurons in the lumbar 4/5 dorsal horn (^∗P < 0.05). To scavenge ROS, phenyl-N-tert-butylnitrone (PBN, a ROS scavenger) was administered immediately after SCI and for 7 consecutive days (early treatment) by either intrathecal (it; 1 and 3 mg) or systemic (ip; 10, 50 and 100 mg) injections. In addition, the high doses of it (3 mg) or ip (100 mg) injections were performed at 35 days (delayed treatment) after SCI. High doses of PBN (ip, 100 mg, and it, 3 mg) significantly attenuated mechanical allodynia in both hind paws at both early and delayed treatments, respectively (^∗P < 0.05). The abnormal hyperexcitability of wide dynamic range neurons after SCI was significantly attenuated by both early and delayed PBN treatment (^∗P < 0.05). Early PBN treatment (100 mg, ip, and 3 mg, it) attenuated overproduction of ROS in neurons in the lumbar 4/5 dorsal horn. In addition, it and ip t-BOOH (ROS donor) treatment dose-dependently produced mechanical allodynia in both hind paws (^∗P < 0.05). Both SCI and t-BOOH treatment groups showed significantly increased phospho-CamKII (pCamKII) expression in neurons and KN-93 (an inhibitor of pCamKII) significantly attenuated mechanical allodynia (^∗P < 0.05). In addition, high doses of PBN significantly promoted the recovery of locomotion (^∗P < 0.05). In conclusion, the present data suggest that overproduction of ROS contribute to sensory and motor abnormalities in remote segments below the lesion after thoracic SCI.     

Prevention of NKCC1 phosphorylation avoids downregulation of KCC2 in central sensory pathways and reduces neuropathic pain after peripheral nerve injury

Neuropathic pain after peripheral nerve injury is characterized by loss of inhibition in both peripheral and central pain pathways. In the adult nervous system, the Na⁺–K⁺–2Cl⁻ (NKCC1) and neuron-specific K⁺–Cl⁻ (KCC2) cotransporters are involved in setting the strength and polarity of GABAergic/glycinergic transmission. After nerve injury, the balance between these cotransporters changes, leading to a decrease in the inhibitory tone. However, the role that NKCC1 and KCC2 play in pain-processing brain areas is unknown. Our goal was to study the effects of peripheral nerve injury on NKCC1 and KCC2 expression in dorsal root ganglia (DRG), spinal cord, ventral posterolateral (VPL) nucleus of the thalamus, and primary somatosensory (S1) cortex. After sciatic nerve section and suture in adult rats, assessment of mechanical and thermal pain thresholds showed evidence of hyperalgesia during the following 2 months. We also found an increase in NKCC1 expression in the DRG and a downregulation of KCC2 in spinal cord after injury, accompanied by later decrease of KCC2 levels in higher projection areas (VPL and S1) from 2 weeks postinjury, correlating with neuropathic pain signs. Administration of bumetanide (30 mg/kg) during 2 weeks following sciatic nerve lesion prevented the previously observed changes in the spinothalamic tract projecting areas and the appearance of hyperalgesia. In conclusion, the present results indicate that changes in NKCC1 and KCC2 in DRG, spinal cord, and central pain areas may contribute to development of neuropathic pain.     

Effect of Spinal Manipulation Thrust Magnitude on Trunk Mechanical Activation Thresholds of Lateral Thalamic Neurons

Objectives

High-velocity low-amplitude spinal manipulation (HVLA-SM), as performed by doctors who use manual therapy (eg, doctors of chiropractic and osteopathy), results in mechanical hypoalgesia in clinical settings. This hypoalgesic effect has previously been attributed to alterations in peripheral and/or central pain processing. The objective of this study was to determine whether thrust magnitude of a simulated HVLA-SM alters mechanical trunk response thresholds in wide dynamic range (WDR) and/or nociceptive specific (NS) lateral thalamic neurons.

Methods

Extracellular recordings were carried out in the thalamus of 15 anesthetized Wistar rats. Lateral thalamic neurons having receptive fields, which included the lumbar dorsal-lateral trunk, were characterized as either WDR (n = 22) or NS (n = 25). Response thresholds to electronic von Frey (rigid tip) mechanical trunk stimuli were determined in 3 directions (dorsal-ventral, 45° caudalward, and 45° cranialward) before and immediately after the dorsal-ventral delivery of a 100-millisecond HVLA-SM at 3 thrust magnitudes (control, 55%, 85% body weight).

Results

There was a significant difference in mechanical threshold between 85% body weight manipulation and control thrust magnitudes in the dorsal-ventral direction in NS neurons (P = .01). No changes were found in WDR neurons at either HVLA-SM thrust magnitude.

Conclusions

This study is the first to investigate the effect of HVLA-SM thrust magnitude on WDR and NS lateral thalamic mechanical response threshold. Our data suggest that, at the single lateral thalamic neuron level, there may be a minimal spinal manipulative thrust magnitude required to elicit an increase in trunk mechanical response thresholds.     

Estrogen status and psychophysical stress modify temporomandibular joint input to medullary dorsal horn neurons in a lamina-specific manner in female rats

Estrogen status and psychological stress contribute to the expression of several chronic pain conditions including temporomandibular muscle and joint disorders (TMJD). Sensory neurons that supply the temporomandibular joint (TMJ) region terminate in laminae I and V of the spinal trigeminal nucleus (Vc/C_1–2 region); however, little is known about lamina-specificity and environmental influences on the encoding properties of TMJ brainstem neurons. To test the hypothesis that Vc/C_1–2 neurons integrate both interoceptive and exteroceptive signals relevant for TMJ nociception, we recorded TMJ-evoked activity in superficial and deep laminae of ovariectomized rats under high and low estradiol (E2) and stress conditions. Rats received daily injections of low (LE) or high (HE) dose E2 and were subjected to forced swim (FS) or sham swim conditioning for 3 days. The results revealed marked lamina-specificity in that HE rats displayed enhanced TMJ-evoked activity in superficial, but not deep, laminae independent of stress conditioning. By contrast, FS conditioned rats displayed increased background firing and TMJ-evoked activity of neurons in deep, but not superficial, laminae independent of E2 status. FS also enhanced TMJ-evoked masseter muscle activity and suggested the importance of deep dorsal horn neurons in mediating evoked jaw muscle activity. In conclusion, E2 status and psychophysical stress play a significant role in modifying the encoding properties of TMJ-responsive medullary dorsal horn neurons with a marked lamina-specificity.     

Dorsal horn NK1-expressing neurons control windup of downstream trigeminal nociceptive neurons

Windup is a progressive, frequency-dependent increase in the excitability of trigeminal and spinal dorsal horn wide dynamic range (WDR) nociceptive neurons to repetitive stimulation of primary afferent nociceptive C-fibers. Superficial dorsal horn neurokinin 1 receptor (NK1R)-expressing neurons were recently shown to regulate sensitization of WDR nociceptive neurons through activation of a defined spino-bulbo-spinal loop. However, the windup of WDR nociceptive neurons was not regulated through this loop. In the present study, we sought to identify the alternative circuit activated by dorsal horn NK1Rs that mediates WDR neuron windup. As a model we used the rat spinal trigeminal nucleus, in which the subnucleus oralis (Sp5O) contains a pool of WDR neurons that receive their nociceptive C-input indirectly via interneurons located in the medullary dorsal horn (MDH). First, we found that intravenous injection of NK1R antagonists (SR140333 and RP67580) produced a reversible inhibition of Sp5O WDR neuron windup. Second, we anatomically identified in the MDH lamina III a subpopulation of NK1R-expressing local interneurons that relay nociceptive information from the MDH to downstream Sp5O neurons. Third, using microinjections of NK1R antagonists during in vivo electrophysiological recordings from Sp5O WDR neurons, we showed that WDR neuron windup depends on activation of NK1Rs located in the MDH laminae I-III. We conclude that, in contrast to central sensitization that is controlled by a spino-bulbo-spinal loop, Sp5O WDR neuron windup is regulated through a local circuit activated by MDH lamina III NK1Rs.     

Spinal administration of MK-801 and NBQX demonstrates NMDA-independent dorsal horn sensitization in incisional pain

Surgery commonly causes pain and neural plasticity that are unique compared to other persistent pain problems. To more precisely study central sensitization and plasticity, we examined the role of ionotropic EAA receptors in dorsal horn neuron sensitization early after incision. Sensitization, in the form of increased background activity, increased mechanosensitivity or pinch receptive field expansion, was induced by plantar incision 1 h later in 30 neurons. (+)-5-Methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine (MK-801) or 1 mM 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo[f]quinoxaline-7-sulfonamide (NBQX) was administered through a microdialysis fiber to block NMDA and nonNMDA EAA receptors, respectively. Dorsal horn neuron sensitization was reexamined 1 h later. Spinal administration of NBQX blocked AMPA-induced excitation but did not affect excitation by NMDA. NBQX decreased background activity in the neurons that developed sustained increased activity after incision. The median decrease caused by NBQX was from 2.3 to 0.0 imp/s. Spinal administration of 5 mM MK-801 blocked NMDA-induced excitation but did not affect excitation by AMPA. The median change (from 2.6 to 1.1 imp/s) in background activity increased by incision was not significantly affected by MK-801. The responses to mechanical stimuli were enhanced after incision in wide dynamic range (WDR) neurons. NBQX eliminated these responses but MK-801 had no effect. The pinch receptive field (RF) expansion into uninjured areas of the paw and hindquarters occurred after incision. Only 1 of 13 neurons exhibited RF expansion after spinal NBQX administration; 9 of 12 neurons had RF expansion remaining after MK-801. Thus, nonNMDA receptors are critical and NMDA-independent factors influence the increased responsiveness of dorsal horn neurons that occur early after incision.     

Systemic dexmedetomidine augments inhibitory synaptic transmission in the superficial dorsal horn through activation of descending noradrenergic control: An in vivo patch-clamp analysis of analgesic mechanisms

α₂-Adrenoceptors are widely distributed throughout the central nervous system (CNS) and the systemic administration of α₂-agonists such as dexmedetomidine produces clinically useful, centrally mediated sedation and analgesia; however, these same actions also limit the utility of these agents (ie, unwanted sedative actions). Despite a wealth of data on cellular and synaptic actions of α₂-agonists in vitro, it is not known which neuronal circuits are modulated in vivo to produce the analgesic effect. To address this issue, we made in vivo recordings of membrane currents and synaptic activities in superficial spinal dorsal horn neurons and examined their responses to systemic dexmedetomidine. We found that dexmedetomidine at doses that produce analgesia (<10 μg/kg) enhanced inhibitory postsynaptic transmission within the superficial dorsal horn without altering excitatory synaptic transmission or evoking direct postsynaptic membrane currents. In contrast, higher doses of dexmedetomidine (>10 μg/kg) induced outward currents by a direct postsynaptic action. The dexmedetomidine-mediated inhibitory postsynaptic current facilitation was not mimicked by spinal application of dexmedetomidine and was absent in spinalized rats, suggesting that it acts at a supraspinal site. Furthermore, it was inhibited by spinal application of the α₁-antagonist prazosin. In the brainstem, low doses of systemic dexmedetomidine produced an excitation of locus coeruleus neurons. These results suggest that systemic α₂-adrenoceptor stimulation may facilitate inhibitory synaptic responses in the superficial dorsal horn to produce analgesia mediated by activation of the pontospinal noradrenergic inhibitory system. This novel mechanism may provide new targets for intervention, perhaps allowing analgesic actions to be dissociated from excessive sedation.     

Tolerance develops to the antiallodynic effects of the peripherally acting opioid loperamide hydrochloride in nerve-injured rats

Peripherally acting opioids are potentially attractive drugs for the clinical management of certain chronic pain states due to the lack of centrally mediated adverse effects. However, it remains unclear whether tolerance develops to peripheral opioid analgesic effects under neuropathic pain conditions. We subjected rats to L5 spinal nerve ligation (SNL) and examined the analgesic effects of repetitive systemic and local administration of loperamide hydrochloride, a peripherally acting opioid agonist. We found that the inhibition of mechanical hypersensitivity, an important manifestation of neuropathic pain, by systemic loperamide (1.5 mg/kg subcutaneously) decreased after repetitive drug treatment (tolerance-inducing dose: 0.75 to 6.0 mg/kg subcutaneously). Similarly, repeated intraplantar injection of loperamide (150 μg/50 μL intraplantarly) and D-Ala²-MePhe⁴-Glyol⁵ enkephalin (300 μg/50 μL), a highly selective mu-opioid receptor (MOR) agonist, also resulted in decreased inhibition of mechanical hypersensitivity. Pretreatment with naltrexone hydrochloride (5 mg/kg intraperitoneally) and MK-801 (0.2 mg/kg intraperitoneally) attenuated systemic loperamide tolerance. Western blot analysis showed that repetitive systemic administration of morphine (3 mg/kg subcutaneously), but not loperamide (3 mg/kg subcutaneously) or saline, significantly increased MOR phosphorylation in the spinal cord of SNL rats. In cultured rat dorsal root ganglion neurons, loperamide dose-dependently inhibited KCl-induced increases in [Ca²⁺]_i. However, this drug effect significantly decreased in cells pretreated with loperamide (3 μM, 72 hours). Intriguingly, in loperamide-tolerant cells, the delta-opioid receptor antagonist naltrindole restored loperamide’s inhibition of KCl-elicited [Ca²⁺]_i increase. Our findings indicate that animals with neuropathic pain may develop acute tolerance to the antiallodynic effects of peripherally acting opioids after repetitive systemic and local drug administration.     

The role of 5-hydroxytryptamine1A and 5-hydroxytryptamine1B receptors in modulating spinal nociceptive transmission in normal and carrageenan-injected rats

Single unit extracellular recordings from the dorsal horn neurons were obtained with glass micropipettes in pentobarbital-anesthetized rats. A total of 115 wide dynamic range (WDR) neurons were studied in 94 rats. In normal rats, the size of nociceptive receptive fields (RFs) of WDR neurons was approximately 123.3 +/- 8.21 mm2 (n = 88). Following carrageenan-induced inflammation, the RFs were markedly enlarged (332.4 +/- 30.1 mm2, n = 27, P < 0.001). The frequency of background activity of the WDR neurons in carrageenan-injected rats (11.3 +/- 2.1 imp/s, n = 27) was greater than that in normal rats (7.1 +/- 0.8 imp/s, n = 88, P < 0.05). In 82% of WDR neurons in normal rats, there was a separation between the A- and C-responses. In contrast, in 67% of the neurons in carrageenan-injected rats, the response to suprathreshold electrical stimuli was a long train with no separation between the A- and C-responses. In carrageenan-injected rats, the magnitude and duration of the nociceptive responses were significantly increased compared to those in normal rats, and the average C-response threshold (7.7 +/- 1.1 mA, n = 27) was lower than that in normal rats (10.4 +/- 0.7 mA, n = 88, P < 0.05). Intrathecal injection of the 5-hydroxytryptamine(1A) (5-HT1A) receptor agonist 8-hydroxy-DPAT hydroxybromide (8-OH-DPAT) (0.305, 1.525, 3.05, and 15.25 mM) dose-dependently increased Adelta- and C-responses and post-discharge in most of the WDR neurons. Following carrageenan-induced inflammation, the 8-OH-DPAT-induced facilitatory effect on Adelta- and C-responses and post-discharge was significantly enhanced (P < 0.05). Intrathecal injection of the 5-hydroxytryptamine1B (5-HT1) receptor agonist CGS12066A (0.222, 1.11, 2.22, and 11.1 mM) dose-dependently enhanced the C-response and post-discharge without influencing the Adelta-response. In carrageenan-injected rats, CGS12066A not only enhanced the facilitatory effect on the C-response and post-discharge, but also facilitated the Adelta-response. Intrathecal injection of the 5-HT(1A) receptor antagonist NAN-190 (0.2 mM) alone did not influence Adelta- and C-responses and post-discharge of WDR neurons in normal rats. When 0.2 mM NAN-190 was co-administered with 3.05 mM 8-OH-DPAT, the facilitatory effect of 8-OH-DPAT on Adelta- and C-responses and post-discharge was completely antagonized, whereas CGS12066A-induced facilitation on the C-response and post-discharge was not influenced by co-administration of 0.2 mM NAN-190 and CGS12066A. These data suggest that 5-HT1A and 5-HT1B receptor subtypes mediate the facilitatory effect of 5-HT on nociceptive processing in the spinal cord of rats. The excitability of dorsal horn WDR neurons and the sensitivity of the neurons to intrathecal 5-HT1A and 5-HT1B receptor agonists might increase following carrageenan-induced inflammation.     

The contribution of spinal neuronal changes to development of prolonged, tonic nociceptive responses of the cat induced by subcutaneous bee venom injection

To elucidate neurophysiological mechanisms of persistent pain induced by tissue injury, the present study was designed to investigate the effects of s.c. bee venom injection on responses of the dorsal horn nociceptive neurons and those of behavior in anesthetized and awake cats, respectively. A parallel comparative study was also performed to compare the effects of s.c. bee venom and formalin injections on neuronal responses by using an extracellular single-unit recording technique. The present results showed that s.c. bee venom injection into the peripheral cutaneous receptive field resulted in a protracted, tonic monophase of increase in spike responses of wide-dynamic-range (WDR) neurons for more than 1 h, while injection of the same volume of vehicle did not have such an effect. The mean number of spikes during the 60-min period after bee venom was 6.74±2.58 spikes/s (n=10), which showed a significant increase in firing rate over the background activity (2.23±0.96 spikes/s). Behavioral observations showed that s.c. bee venom injection into the dorsum of a hind paw also produced a prolonged, tonic single phase of response indicative of pain, suggesting that central neuronal changes may contribute to development of bee venom-induced prolonged, tonic pain in cats. The increased neuronal firing induced by s.c. bee venom could be suppressed by a single dose of i.v. morphine and resumed by naloxone. Blockade of the sciatic nerve with lidocaine resulted in a complete suppression of the bee venom-induced neuronal firing, suggesting that the central neuronal changes following s.c. bee venom are peripherally-dependent. Comparative studies showed that the duration and frequency of the bee venom-induced neuronal responses were comparable to those induced by s.c. formalin; however, responses of WDR neurons to mechanical stimuli applied to the injection site of the two chemical agents were quite different. Bee venom produced a significant enhancement of mechanical responses of WDR neurons, while, on the contrary, formalin produced a desensitization of sensory receptors in the injection site, suggesting that the two tonic pain models may have different underlying mechanisms.     

Noxious stimulation excites serotonergic neurons: A comparison between the lateral paragigantocellular reticular and the raphe magnus nuclei

The present study was designed to record electrophysiological responses to graded noxious thermal stimuli of serotonergic and nonserotonergic neurons in the lateral paragigantocellular reticular (LPGi) and the raphe magnus (RMg) nuclei in rats. All of the neurons recorded were juxtacellularly filled with neurobiotin and identified with double immunofluorescent labeling for both neurobiotin and serotonin. Under halothane anesthesia (0.75%), noxious thermal stimuli ?48°C activated almost all (88%) of the serotonergic neurons located within the LPGi (n = 16). The increase in firing was clear (3.4 ± 0.3 spike/s: mean of responses above the population median) and sustained during the whole application of strong thermal noxious stimuli, with a high mean threshold (48.3 ± 0.3°C) and large receptive fields. Recording of serotonergic neurons in the RMg (n = 21) demonstrated that the proportion of strongly activated (>2 spike/s) neurons (19% vs 59% for the LPGi) as well as the magnitude of the activation (2.1 ± 0.4 spike/s: mean of responses above the population median) to thermal noxious stimuli were significantly lower than in the LPGi (P < .05). Within the boundaries of both the LPGi and the RMg (B3 group), nonserotonergic neurons were also predominantly excited (75%) by noxious stimuli, and the resulting activation (7.9 ± 1.2 spike/s) was even greater than that of serotonergic neurons. Thermal noxious stimuli–induced activation of LPGi serotonergic cells probably plays a key role in serotonin-mediated modulations of cardiac baroreflex and transmission of nociceptive messages occurring under such intense noxious conditions.     

WDR response profiles of spinal dorsal horn neurons may be unmasked by barbiturate anesthesia

The number of WDR (convergent, multireceptive) neurons encountered in the spinal dorsal horn of physiologically intact, awake, drug-free cats has been much smaller than expected (9% in intact, drug-free animals). Control studies in barbiturate-anesthetized or spinal cord transected animals indicate that the dearth of WDR neurons was not just an artifact of the chronic recording technique. In those preparations WDR neurons represented 34% and 61% of the sample, respectively. Initial studies in which the effects of light barbiturate anesthesia on spinal dorsal horn neurons (n = 12) have been examined revealed that a 20 mg/kg dose of pentobarbital can, in some neurons (n = 4), unmask thermally evoked activity that was not present in the intact, drug-free animal. Responses to noxious mechanical stimuli were also enhanced following barbiturate administration. These changes resulted in a reclassification of neural type from low threshold in the intact, awake, drug-free animal to WDR in the anesthetized animal.     

Behavioral manifestations of neuropathic pain and mechanical allodynia, and changes in spinal dorsal horn neurons, following L4–L6 dorsal root constriction in rats

We investigated behavioral symptoms of neuropathic pain, and associated changes in dorsal horn neurons, in a rat model involving loose ligation of lumbar dorsal roots. The L4–L6 dorsal roots were exposed unilaterally and loosely constricted central to the respective ganglia with one (1-ligation) or two (2-ligation) silk 7-O ligatures. In control groups the dorsal roots were exposed but not ligated (sham-operated), or sutures were placed lengthwise between the dorsal roots (suture control). There was a significant reduction in mechanical withdrawal threshold on the operated side in both 1- and 2-ligation groups which began at 3 days, peaked at 2–5 week, and gradually recovered. A delayed threshold reduction was also seen on the non-operated side. Immediately post-surgery there was a significant increase (hypoalgesia) in thermal paw withdrawal latency (Hargreaves test) in 1- and 2-ligation groups on the operated (but not non-operated) side that recovered after 1 week. Significantly less weight was borne by the operated limb 1–5 weeks post-operatively in 1- and 2-ligation groups. The force of hind limb withdrawals elicited by graded noxious heat pulses (38–52°C) was significantly lower 1 week post-surgery on the operated side (1-ligation group) followed by recovery. Withdrawal forces were higher 5–9 week post-surgery on the non-operated side in 1- and 2-ligation groups. We found no evidence of cold allodynia. Neither sham-operated nor suture controls showed any signs of allodynia or hyperalgesia. Following behavioral testing, rats were anesthetized with halothane for single-unit recordings from lumbar wide dynamic range-type (WDR) neurons. At 22 week post-surgery, the mean area of mechanosensitive receptive fields was significantly larger for units on the operated side in 1- and 2-ligation groups compared with those on the non-operated side or with those from sham-operated rats. Mean stimulus-response functions to graded noxious heat pulses (38–52°C, 5 s) were not significantly different between operated and non-operated sides for 1- or 2-ligation groups, or compared with the 22-week sham-operated group. At 5 week post-surgery, the mean area of cutaneous receptive fields, and stimulus-response functions to graded noxious heat, were not significantly different between units recorded on operated versus non-operated sides, or compared with units from 5-week sham-operated rats. Spontaneous unit activity was significantly higher on the operated versus non-operated side in the 2-ligation (22-week) and sham (5-week) groups. Enlarged cutaneous receptive fields of dorsal horn neurons may contribute to mechanical allodynia associated with dorsal root constriction. However, the slow (>5 week) development of receptive field enlargement does not match the rapid development of allodynia. The lack of effect of dorsal root constriction on thermal sensitivity of dorsal horn units ipsilaterally corresponds to the lack of marked thermal hyperalgesia observed behaviorally.     

Suppression of WNK1-SPAK/OSR1 Attenuates Bone Cancer Pain by Regulating NKCC1 and KCC2

Our preliminary experiment indicated the activation of with-nolysine kinases 1 (WNK1) in bone cancer pain (BCP) rats. This study aimed to investigate the underlying mechanisms via which WNK1 contributed to BCP. A rat model of BCP was induced by Walker-256 tumor cell implantation. WNK1 expression and distribution in the lumbar spinal cord dorsal horn and dorsal root ganglion were examined. SPS1-related proline/alanine-rich kinase (SPAK), oxidative stress-responsive kinase 1 (OSR1), sodium-potassium-chloride cotransporter 1 (NKCC1), and potassium-chloride cotransporter 2 (KCC2) expression were assessed. Pain behaviors including mechanical allodynia and movement-evoked pain were measured. BCP rats exhibited significant mechanical allodynia, with increased WNK1 expression in the dorsal horn and dorsal root ganglion neurons, elevated SPAK/OSR1 and NKCC1 expression in the dorsal root ganglion, and decreased KCC2 expression in the dorsal horn. WNK1 knock-down by small interfering alleviated mechanical allodynia and movement-evoked pain, inhibited WNK1-SPAK/OSR1-NKCC1 activities, and restored KCC2 expression. In addition, closantel (a WNK1-SPAK/OSR1 inhibitor) improved pain behaviors, downregulated SPAK/OSR1 and NKCC1 expression, and upregulated KCC2 expression in BCP rats. Activation of WNK1-SPAK/OSR1 signaling contributed to BCP in rats by modulating NKCC1 and KCC2 expression. Therefore, suppression of WNK1-SPAK/OSR1 may serve as a potential target for BCP therapy.PerspectiveOur findings demonstrated that the WNK1-SPAK/OSR1 signaling contributed to BCP in rats via regulating NKCC1 and KCC2. Suppressing this pathway reduced pain behaviors. Based on these findings, the WNK1-SPAK/OSR1 signaling may be a potential target for BCP therapy.