异丙酚对正常大鼠脊髓背角感觉神经元反应性的抑制作用

脊髓背角感觉神经元不仅在感觉信息的传递和调节中起到重要作用,也是各种内源性和外源性药物的作用靶位.为了解静脉麻醉剂异丙酚是否对背角感觉神经元的反应性具有调节作用,本实验采用在体单细胞胞外记录技术,观察了脊髓背表面直接滴注0.5 μmol异丙酚对戊巴比妥钠麻醉大鼠脊髓背角广动力域(WDR)神经元和低阈值机械感受型(LTM)神经元反应性的影响.实验发现,异丙酚能抑制背角WDR神经元由施加于外周感受野伤害性热刺激(45、47、49和53℃,15 s)和夹捏机械刺激(10 s)诱发的反应性,与DMSO对照组比较具有显著性统计学差异(P＜0.05);同样,异丙酚对非伤害性机械刺激诱发的WDR或LTM神经元的反应性也具有显著的抑制作用(P＜0.05).本结果提示,异丙酚可直接作用于正常大鼠脊髓背角神经元,对由非伤害性和伤害性纤维介导的神经元反应性均产生抑制作用,因此异丙酚的脊髓抗伤害作用可能不是特异性的.     

Behavioral and Electrophysiological Evidence for the Differential Functions of TRPV1 at Early and Late Stages of Chronic Inflammatory Nociception in Rats

We previously reported that vanilloid receptor type 1 (VR1, or TRPV1) was up-regulated in dorsal root ganglion (DRG) and the spinal dorsal horn after chronic inflammatory pain produced by complete Freund’s adjuvant (CFA) injection into the plantar of rat hind paw. In the present study, we found that subcutaneous or intrathecal application of capsazepine (CPZ), a TRPV1 competitive antagonist, could inhibit thermal hyperalgesia on day 1 and on day 14 but not on day 28 after CFA injection. With extracellular electrophysiological recording, the effect of CPZ on noxious electrical or heat stimulation evoked responses of wide dynamic range (WDR) neurons in the deep layers of the spinal dorsal horn was evaluated. Under noxious electrical stimulation to sciatic nerve, CPZ applied to the spinal cord produced an inhibition on Aδ- and C-fiber evoked responses of WDR neurons on day 1 and 14, but not on day 28. Under radiant heat stimulation to the receptive field skin, subcutaneous application of CPZ significantly inhibited the background activity and extended the response latency of WDR neurons on day 14. These results provide new evidence for the functional significance of TRPV1 at the early stage, but not the late stage, in the rat model of CFA-induced inflammatory pain. Special issue article in honor of Dr. Ji-Sheng Han. Hao Luo, Isabella Shi Xu, Yi Chen are Co-first authors.     

Activation of extracellular signal-regulated protein kinases 5 in primary afferent neurons contributes to heat and cold hyperalgesia after inflammation

Heat and cold hyperalgesia is a common feature of inflammatory pain. To investigate whether activation of extracellular signal-regulated protein kinase 5 (ERK5), also known as big mitogen-activated protein kinase 1, in primary sensory neurons participates in inflammatory pain, we examined the phosphorylation of ERK5 in the dorsal root ganglion (DRG) after peripheral inflammation. Inflammation induced by complete Freund's adjuvant produced heat and cold hyperalgesia on the ipsilateral hind paw and induced an increase in the phosphorylation of ERK5, mainly in tyrosine kinase A-expressing small- and medium-size neurons. In contrast, there was no change in ERK5 phosphorylation in the spinal dorsal horn. ERK5 antisense, but not mismatch, oligodeoxynucleotide decreased the activation of ERK5 and suppressed inflammation-induced heat and cold hyperalgesia. Furthermore, the inhibition of ERK5 blocked the induction of transient receptor potential channel TRPV1 and TRPA1 expression in DRG neurons after peripheral inflammation. Our results show that ERK5 activated in DRG neurons contribute to the development of inflammatory pain. Thus, blocking ERK5 signaling in sensory neurons that has the potential for preventing pain after inflammation.     

TRPA1 Mediates Mechanical Sensitization in Nociceptors during Inflammation

Inflammation is a part of the body's natural response to tissue injury which initiates the healing process. Unfortunately, inflammation is frequently painful and leads to hypersensitivity to mechanical stimuli, which is difficult to treat clinically. While it is well established that altered sensory processing in the spinal cord contributes to mechanical hypersensitivity (central sensitization), it is still debated whether primary afferent neurons become sensitized to mechanical stimuli after tissue inflammation. We induced inflammation in C57BL/6 mice via intraplantar injection of Complete Freund's Adjuvant. Cutaneous C fibers exhibited increased action potential firing to suprathreshold mechanical stimuli. We found that abnormal responses to intense mechanical stimuli were completely suppressed by acute incubation of the receptive terminals with the TRPA1 inhibitor, HC-030031. Further, elevated responses were predominantly exhibited by a specific subgroup of C fibers, which we determined to be C-Mechano Cold sensitive fibers. Thus, in the presence of HC-030031, C fiber mechanical responses in inflamed mice were not different than responses in saline-injected controls. We also demonstrate that injection of the HC-030031 compound into the hind paw of inflamed mice alleviates behavioral mechanical hyperalgesia without affecting heat hyperalgesia. Further, we pharmacologically anesthetized the TRPA1-expressing fibers in vivo by co-injecting the membrane-impermeable sodium channel inhibitor QX-314 and the TRPA1 agonist cinnamaldehyde into the hind paw. This approach also alleviated behavioral mechanical hyperalgesia in inflamed mice but left heat hypersensitivity intact. Our findings indicate that C-Mechano Cold sensitive fibers exhibit enhanced firing to suprathreshold mechanical stimuli in a TRPA1-dependent manner during inflammation, and that input from these fibers drives mechanical hyperalgesia in inflamed mice.     

C-fos antisense oligodeoxynucleotide decreases subcutaneous bee venom injection-induced nociceptive behavior and fos expression in the rat

Oligodeoxynucleotide complementary to c-fos mRNA was applied to characterize its effect on the spinal cord Fos expression and relevant nociceptive behaviors challenged by subcutaneous injection of bee venom to the rat hind paw. Nociceptive behavioral responses (spontaneous pain and hyperalgesia) following bee venom (0.2 mg/50 microl) injection were assessed in adult male Sprague-Dawley rats receiving intrathecal administration of c-fos antisense oligodeoxynucleotide (ASO, 50 microg/10 microl), sense oligodeoxynucleotide (SO, 50 microg/10 microl) and saline (10 microl) 4 h prior to bee venom injection. The lumbar spinal cord expression of Fos protein 2 h after bee venom injection in the ASO-, SO- and saline-treated animals was observed by immunohistochemistry. The results showed that pretreatment of c-fos ASO markedly reduced the flinching response and primary thermal hyperalgesia, but without significant effects on mechanical hyperalgesia and secondary thermal hyperalgesia. At the same time, ASO treatment also significantly decreased the expression of Fos protein within the lumbar region of the spinal cord ipsilateral to the injection. The results provide further evidence that Fos protein contributes to the activation of the spinal dorsal horn neurons and the generation and/or maintenance of spontaneous pain and primary thermal hyperalgesia induced by subcutaneous injection of bee venom.     

Selective Inflammatory Pain Insensitivity in the African Naked Mole-Rat (Heterocephalus glaber)

In all mammals, tissue inflammation leads to pain and behavioral sensitization to thermal and mechanical stimuli called hyperalgesia. We studied pain mechanisms in the African naked mole-rat, an unusual rodent species that lacks pain-related neuropeptides (e.g., substance P) in cutaneous sensory fibers. Naked mole-rats show a unique and remarkable lack of pain-related behaviors to two potent algogens, acid and capsaicin. Furthermore, when exposed to inflammatory insults or known mediators, naked mole-rats do not display thermal hyperalgesia. In contrast, naked mole-rats do display nocifensive behaviors in the formalin test and show mechanical hyperalgesia after inflammation. Using electrophysiology, we showed that primary afferent nociceptors in naked mole-rats are insensitive to acid stimuli, consistent with the animal's lack of acid-induced behavior. Acid transduction by sensory neurons is observed in birds, amphibians, and fish, which suggests that this tranduction mechanism has been selectively disabled in the naked mole-rat in the course of its evolution. In contrast, nociceptors do respond vigorously to capsaicin, and we also show that sensory neurons express a transient receptor potential vanilloid channel-1 ion channel that is capsaicin sensitive. Nevertheless, the activation of capsaicin-sensitive sensory neurons in naked mole-rats does not produce pain-related behavior. We show that capsaicin-sensitive nociceptors in the naked mole-rat are functionally connected to superficial dorsal horn neurons as in mice. However, the same nociceptors are also functionally connected to deep dorsal horn neurons, a connectivity that is rare in mice. The pain biology of the naked mole-rat is unique among mammals, thus the study of pain mechanisms in this unusual species can provide major insights into what constitutes “normal” mammalian nociception.     

A fluorescence-immunohistochemical study on phosphorylation of ERK1/2, p38, and STAT3 in rat dorsal root ganglia following noxious stimulation of hind paw sensory neurons

A fluorescence-immunohistochemical investigation was performed in lumbar dorsal root ganglia (DRGs) neurons of the rat with regard to ERK1/2-, p38- and STAT3-phosphorylation in response to nociceptor activation in the rat. The stimuli applied were perineural capsaicin treatment of the sciatic nerve, mustard oil application to the hind paw and heat or cold stimulation of the hind paw. The time points of investigations were 15 min/30 min after perineural capsaicin, 30 min/2 h/4 h for mustard oil, 10 min/4 h for cold and 30 min/2 h/8 h for the heat stimulus. All four stimuli lead to a time-dependent, significant 2-3 fold increase in the number of small and medium size DRG cells displaying cytoplasmic staining for p-ERK1/2, but to no activation of satellite cells. Phosphorylated p38 immunoreactivity was increased in the cytoplasma of DRG cells at 2 h after the mustard oil treatment of the hind paw and 30 min after the perineural capsaicin application to the sciatic nerve axons, but not following heat or cold stimuli to the hind paws. Phospho-STAT3 staining was characteristically observed as nuclear and cytoplasmic staining. It was found increased after the perineural capsaicin application to the sciatic nerve axons, however, no marked increase was found with the other 3 noxious stimuli. The present results show that sensory neurons respond with a selective long-lasting increase in p-ERK1/2 in small and medium-size DRG cells, when their axons or axon terminals are stimulated by capsaicin, mustard oil, noxious heat or noxious cold.     

Selective inflammatory pain insensitivity in the African naked mole-rat (Heterocephalus glaber)

In all mammals, tissue inflammation leads to pain and behavioral sensitization to thermal and mechanical stimuli called hyperalgesia. We studied pain mechanisms in the African naked mole-rat, an unusual rodent species that lacks pain-related neuropeptides (e.g., substance P) in cutaneous sensory fibers. Naked mole-rats show a unique and remarkable lack of pain-related behaviors to two potent algogens, acid and capsaicin. Furthermore, when exposed to inflammatory insults or known mediators, naked mole-rats do not display thermal hyperalgesia. In contrast, naked mole-rats do display nocifensive behaviors in the formalin test and show mechanical hyperalgesia after inflammation. Using electrophysiology, we showed that primary afferent nociceptors in naked mole-rats are insensitive to acid stimuli, consistent with the animal's lack of acid-induced behavior. Acid transduction by sensory neurons is observed in birds, amphibians, and fish, which suggests that this tranduction mechanism has been selectively disabled in the naked mole-rat in the course of its evolution. In contrast, nociceptors do respond vigorously to capsaicin, and we also show that sensory neurons express a transient receptor potential vanilloid channel-1 ion channel that is capsaicin sensitive. Nevertheless, the activation of capsaicin-sensitive sensory neurons in naked mole-rats does not produce pain-related behavior. We show that capsaicin-sensitive nociceptors in the naked mole-rat are functionally connected to superficial dorsal horn neurons as in mice. However, the same nociceptors are also functionally connected to deep dorsal horn neurons, a connectivity that is rare in mice. The pain biology of the naked mole-rat is unique among mammals, thus the study of pain mechanisms in this unusual species can provide major insights into what constitutes “normal” mammalian nociception.     

脊髓细胞外调节激酶1/2在大鼠后足切割痛中的表达和作用

目的：探讨大鼠后足切割后脊髓ERK的表达情况。方法：以大鼠右后足切割作为急性疼痛模型;用免疫组织化学法测试脊髓磷酸化ERK（pERK）表达情况。ERK抑制剂U0126（1μg）在切割前20min或切割后20min鞘内注射。用von Frey纤维测试大鼠机械性痛敏。结果：大鼠后足切割后1min,在切割侧L4-L5脊髓浅层背侧角（板层Ⅰ和板层Ⅱ）ERK被迅速地激活,并在5min达到峰值,随后恢复到基础值。切割前鞘内给予U0126能显著减轻机械性痛敏,然而,切割后鞘内给予U0126对机械性痛敏的作用并不明显。结论：脊髓ERK在大鼠后足切割痛中产生机械性痛敏发挥了重要的作用。     

Chronic Compression of the Dorsal Root Ganglion Enhances Mechanically Evoked Pain Behavior and the Activity of Cutaneous Nociceptors in Mice

Radicular pain in humans is usually caused by intraforaminal stenosis and other diseases affecting the spinal nerve, root, or dorsal root ganglion (DRG). Previous studies discovered that a chronic compression of the DRG (CCD) induced mechanical allodynia in rats and mice, with enhanced excitability of DRG neurons. We investigated whether CCD altered the pain-like behavior and also the responses of cutaneous nociceptors with unmyelinated axons (C-fibers) to a normally aversive punctate mechanical stimulus delivered to the hairy skin of the hind limb of the mouse. The incidence of a foot shaking evoked by indentation of the dorsum of foot with an aversive von Frey filament (tip diameter 200 μm, bending force 20 mN) was significantly higher in the foot ipsilateral to the CCD surgery as compared to the contralateral side on post-operative days 2 to 8. Mechanically-evoked action potentials were electrophysiologically recorded from the L3 DRG, in vivo, from cell bodies visually identified as expressing a transgenically labeled fluorescent marker (neurons expressing either the receptor MrgprA3 or MrgprD). After CCD, 26.7% of MrgprA3⁺ and 32.1% MrgprD⁺ neurons exhibited spontaneous activity (SA), while none of the unoperated control neurons had SA. MrgprA3⁺ and MrgprD⁺ neurons in the compressed DRG exhibited, in comparison with neurons from unoperated control mice, an increased response to the punctate mechanical stimuli for each force applied (6, 20, 40, and 80 mN). We conclude that CCD produced both a behavioral hyperalgesia and an enhanced response of cutaneous C-nociceptors to aversive punctate mechanical stimuli.     

Effects of the neurotrophic factor artemin on sensory afferent development and sensitivity

Artemin is a neuronal survival and differentiation factor in the glial cell line-derived neurotrophic factor family.Its receptor GFRα3 is expressed by a subpopulation of nociceptor type sensory neurons in the dorsal root and trigeminal ganglia(DRG and TG).These neurons co-express the heat,capsaicin and proton-sensitive channel TRPV1 and the cold and chemical-sensitive channel TRPA1.To further investigate the effects of artemin on sensory neurons,we isolated transgenic mice(ARTN-OE mice) that overexpress art...     

MK—801降低炎性痛在鼠脊髓NOS表达和NO含量

用NADPH-d组织化学法,观察鞘内注射NMDA受体拮抗剂MK-801对大鼠右后掌皮下注射甲醛诱发的炎症性痛及痛过敏过程中脊髓后角一氧化氮合酶(NOS)表达的影响,同时测定一氧化氮(NO)代谢终产物     

Role of spinal cord alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in complete Freund's adjuvant-induced inflammatory pain

Background

Honeybee's sting on human skin can induce ongoing pain, hyperalgesia and inflammation. Injection of bee venom (BV) into the intraplantar surface of the rat hindpaw induces an early onset of spontaneous pain followed by a lasting thermal and mechanical hypersensitivity in the affected paw. The underlying mechanisms of BV-induced thermal and mechanical hypersensitivity are, however, poorly understood. In the present study, we investigated the role of mitogen-activated protein kinase (MAPK) in the generation of BV-induced pain hypersensitivity.

Results

We found that BV injection resulted in a quick activation of p38, predominantly in the L4/L5 spinal dorsal horn ipsilateral to the inflammation from 1 hr to 7 d post-injection. Phosphorylated p38 (p-p38) was expressed in both neurons and microglia, but not in astrocytes. Intrathecal administration of the p38 inhibitor, SB203580, prevented BV-induced thermal hypersensitivity from 1 hr to 3 d, but had no effect on mechanical hypersensitivity. Activated ERK1/2 was observed exclusively in neurons in the L4/L5 dorsal horn from 2 min to 1 d, peaking at 2 min after BV injection. Intrathecal administration of the MEK inhibitor, U0126, prevented both mechanical and thermal hypersensitivity from 1 hr to 2 d. p-ERK1/2 and p-p38 were expressed in neurons in distinct regions of the L4/L5 dorsal horn; p-ERK1/2 was mainly in lamina I, while p-p38 was mainly in lamina II of the dorsal horn.

Conclusion

The results indicate that differential activation of p38 and ERK1/2 in the dorsal horn may contribute to the generation and development of BV-induced pain hypersensitivity by different mechanisms.     

Pulsed Radiofrequency Reduced Complete Freund’s Adjuvant-induced Mechanical Hyperalgesia via the Spinal c-Jun N-terminal Kinase Pathway

Pulsed radiofrequency (PRF) treatment involves the pulsed application of a radiofrequency electric field to a nerve. The technology offers pain relief for patients suffering from chronic pain who do not respond well to conventional treatments. We tested whether PRF treatment attenuated complete Freund’s adjuvant (CFA) induced inflammatory pain. The profile of spinal c-Jun N-terminal kinases (JNKs) phosphorylation was evaluated to elucidate the potential mechanism. Injection of CFA into the unilateral hind paw of rats induced mechanical hyperalgesia in both the ipsilateral and contralateral hind paws. We administered 500-kHz PRF treatment in 20-ms pulses, at a rate of 2 Hz (2 pulses per second) either to the sciatic nerve in the mid-thigh, or to the L4 anterior primary ramus just distal to the intervertebral foramen in both the CFA group and no-PRF group rats. Tissue samples were examined at 1, 3, 7, and 14 days following PRF treatments. Behavioral studies showed that PRF applied close to the dorsal root ganglion (DRG) significantly attenuated CFA-induced mechanical hyperalgesia compared to no-PRF group (P < .05). And western blotting revealed significant attenuation of the activation of JNK in the spinal dorsal horn compared to no-PRF group animals (P < .05). Application of PRF close to DRG provides an effective treatment for CFA-induced persistent mechanical hyperalgesia by attenuating JNK activation in the spinal dorsal horn.     

The effect of galanin on wide-dynamic range neuron activity in the spinal dorsal horn of rats

The present study investigated the effect of galanin on wide-dynamic range (WDR) neuron activity in the dorsal horn of the spinal cord of rats. The evoked discharge of WDR neurons was elicited by transdermic electrical stimulation applied on the ipsilateral hindpaw of rats. Galanin was administered directly on the spinal dorsal surface of L3-L5. The evoked discharge frequency of the WDR neurons decreased significantly after the administration of galanin and the effect lasted for more than 30 min. Furthermore, the inhibitory effect of galanin on the evoked discharge frequency of WDR neurons was blocked by following administration of the galanin antagonist galantide, indicating that the inhibitory effect of galanin on the activity of WDR neurons was induced by activating galanin receptors in the dorsal horn of the spinal cord. The results suggest that galanin has an inhibitory role in the transmission of presumed nociceptive information in the dorsal horn of the spinal cord in rats.     

Calcitonin gene-related peptide 8-37 inhibits the evoked discharge frequency of wide dynamic range neurons in dorsal horn of the spinal cord in rats.

The present study was performed to explore the effect of calcitonin gene-related peptide 8-37 (CGRP8-37) on the electrical stimulation-evoked discharge frequency of wide dynamic range (WDR) neurons in the dorsal horn of the spinal cord in rats. The discharge frequencies of WDR neurons were evoked by transdermic electrical stimulation applied on the ipsilateral hindpaw. CGRP8-37 was applied directly on the dorsal surface of the L3 to L5 spinal cord. After the administration of 3 nmol of CGRP8-37, the evoked discharge frequency of WDR neurons decreased significantly, an effect lasting more than 30 min. The results indicate that CGRP receptors play an important role in the transmission of presumed nociceptive information in the dorsal horn of the spinal cord.     

A Novel and Selective Poly (ADP-Ribose) Polymerase Inhibitor Ameliorates Chemotherapy-Induced Painful Neuropathy

Background

Chemotherapy-induced neuropathy is the principle dose limiting factor requiring discontinuation of many chemotherapeutic agents, including cisplatin and oxaliplatin. About 30 to 40% of patients receiving chemotherapy develop pain and sensory changes. Given that poly (ADP-ribose) polymerase (PARP) inhibition has been shown to provide neuroprotection, the current study was developed to test whether the novel PARP inhibitor compound 4a (analog of ABT-888) would attenuate pain in cisplatin and oxaliplatin-induced neuropathy in mice.

Results

An established chemotherapy-induced painful neuropathy model of two weekly cycles of 10 intraperitoneal (i.p.) injections separated by 5 days rest was used to examine the therapeutic potential of the PARP inhibitor compound 4a. Behavioral testing using von Frey, paw radiant heat, cold plate, and exploratory behaviors were taken at baseline, and followed by testing at 3, 6, and 8 weeks from the beginning of drug treatment.

Conclusion

Cisplatin-treated mice developed heat hyperalgesia and mechanical allodynia while oxaliplatin-treated mice exhibited cold hyperalgesia and mechanical allodynia. Co-administration of 50 mg/kg or 25 mg/kg compound 4a with platinum regimen, attenuated cisplatin-induced heat hyperalgesia and mechanical allodynia in a dose dependent manner. Similarly, co-administration of 50 mg/kg compound 4a attenuated oxaliplatin-induced cold hyperalgesia and mechanical allodynia. These data indicate that administration of a novel PARP inhibitor may have important applications as a therapeutic agent for human chemotherapy-induced painful neuropathy.     

Increased Synaptophysin Is Involved in Inflammation-Induced Heat Hyperalgesia Mediated by Cyclin-Dependent Kinase 5 in Rats

Mechanisms associated with cyclin-dependent kinase 5 (Cdk5)-mediated heat hyperalgesia induced by inflammation remain undefined. This study was designed to examine whether Cdk5 mediates heat hyperalgesia resulting from peripheral injection of complete Freund''s adjuvant (CFA) in the spinal dorsal horns of rats by interacting with synaptophysin, a well known membrane protein mediating the endocytosis-exocytosis cycle of synaptic vesicles as a molecular marker associated with presynaptic vesicle membranes. The role of Cdk5 in mediating synaptophysin was examined through the combined use of behavioral approaches, imaging studies, and immunoprecipitation following CFA-induced inflammatory pain. Results showed that Cdk5 colocalized with both synaptophysin and soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs) consisting of VAMP-2, SNAP-25, and syntaxin 1A in spinal dorsal horn of rats. Increased synaptophysin expression of spinal cord horn neurons post intraplantar injection of CFA coincided with increased duration of heat hyperalgesia lasting from 6 h to 3 d. Intrathecal administration of roscovitine, a Cdk5 specific inhibitor, significantly depressed synaptophysin expression during peak heat hyperalgesia and heat hyperalgesia induced by peripheral injection of CFA. Data presented in this report indicated that calpain activity was transiently upregulated 6 h post CFA-treatment despite previous reports suggesting that calpain was capable of cleaving p35 into p25. Results from previous studies obtained by other laboratories demonstrated that significant changes in p35 expression levels within spinal cord horn neurons were not observed in the CFA-treated inflammatory pain model although significant upregulation of Cdk5 kinase was observed between 2 h to 7 d. Therefore, generation of p25 occurred in a calpain-independent fashion in a CFA-treated inflammatory pain model. Our results demonstrated that increased synaptophysin levels were involved in heat hyperalgesia mediated by Cdk5 in spinal cord dorsal horns of CFA-treated rats, suggesting that inhibiting abnormal activation of Cdk5-synaptophysin may present a novel target for diminishing inflammatory pain.