Inhibition of Spinal Interleukin-33 Attenuates Peripheral Inflammation and Hyperalgesia in Experimental Arthritis.

Accumulating evidence indicates that the continuous and intense nociceptive from inflamed tissue may increase the excitability of spinal dorsal horn neurons, which can signal back and modulate peripheral inflammation. Previous studies have demonstrated that spinal interleukin (IL)-33 contributes to the hyperexcitability of spinal dorsal horn neurons. This study was undertaken to investigate whether spinal IL-33 can also influence a peripheral inflammatory response in a rat model of arthritis. Lentivirus-delivered short hairpin RNA targeting IL-33 (LV-shIL-33) was constructed for gene silencing. Rats with adjuvant-induced arthritis (AIA) were injected intrathecally with LV-shIL-33 3 days before the complete Freund's adjuvant (CFA) injection. During an observation period of 21 days, pain-related behavior and inflammation were assessed. In addition, the expression of spinal proinflammatory cytokines and the activation of spinal extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB) pathways were evaluated on 9 days after CFA treatment. The existence of tissue injury or inflammation in rats with AIA resulted in the upregulation of spinal IL-33, which is predominantly expressed in neurons, astrocytes, and oligodendrocytes. Intrathecal administration of LV-shIL-33 significantly alleviated hyperalgesia, paw swelling, and joint destruction, and attenuated the expression of proinflammatory cytokines [IL-6, IL-1ß, and tumor necrosis factor-α (TNF-α)], as well as the activation of ERK and NF-κB/p65 in the spinal cord. Our data suggest that spinal IL-33 contributes to the development of both peripheral inflammation and hyperalgesia. Thus, interference with IL-33 at the spinal level might represent a novel therapeutic target for painful inflammatory disorders.

Interleukin-33 modulates lipopolysaccharide-mediated inflammatory response in rat primary astrocytes.

Astrocytes have a crucial role in the modulation of the neuroinflammatory response. However, the underlying mechanisms have yet to be fully defined. Interleukin-33 (IL-33) is constitutively expressed in astrocytes, which has been found to orchestrate inflammatory responses in a large variety of immune-mediated and inflammatory diseases of the nervous system. Thus, the purpose of this study was to elucidate the potential effect of IL-33 in the regulation of inflammatory response in primary cultured astrocytes. We investigated the role of IL-33 in the regulation of inflammatory responses in the lipopolysaccharide-stimulated astrocytes. This study utilized lentiviral short hairpin RNA vectors to target IL-33 (LV-shIL-33) for gene silencing. After lipopolysaccharide stimulation, the expression levels of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α), as well as the activation of nuclear factor-kappa B (NF-κB) and extracellular signal-regulated kinase (ERK) signaling pathways, were evaluated to elucidate the mechanisms related to the contributions of IL-33 to the inflammatory response in astrocytes. We found that the expression IL-33 has increased in rat primary cultured astrocytes after lipopolysaccharide stimulation. Administration of LV-shIL-33 knocked down the expression of IL-33 and markedly reduced the overexpression of spinal IL-1ß, IL-6, and TNF-α, and attenuated the activation of ERK and NF-κB/p65. This study shows that IL-33 participates in regulating inflammatory responses in primary cultured astrocytes, which might provide additional targets for controlling inflammatory responses following neurological diseases. See Video abstract, http://links.lww.com/WNR/A627.

Relationship between muscle spindles and myofascial trigger spots according to Hoffmann reflex pathway and tissue morphology characteristics in a rat model.

OBJECTIVES: To determine how muscle spindles are involved in the pathophysiology of chronic myofascial trigger spots (MTrSs, similar to myofascial trigger points) in a rat injury model according to the characteristics of the Hoffmann reflex (H-reflex) and the anatomical relationship between muscle spindles and MTrSs. METHODS: 16 male Sprague-Dawley rats (7 weeks old) were randomly divided into experimental and control groups. A blunt strike injury and eccentric exercise were applied to the gastrocnemius muscle of rats in the experimental group once a week for 8 weeks as a MTrS modelling intervention. Subsequently, the rats were reared normally and rested for 4 weeks. At the end of the 12th week, the rats were examined for the presence of MTrSs defined by the detection of a palpable taut band exhibiting both a local twitch response and spontaneous electrical activity. After modelling, evocation of the H-reflex and morphological examination of muscle spindles and MTrSs were conducted. RESULTS: The threshold (0.35±0.04 mA) of the H-reflex and latency (1.24±0.18 ms) of the M wave recorded at MTrSs were not significantly different to those at non-MTrSs (P>0.05). Compared with non-MTrSs, a lower Mmax (4.28±1.27 mV), higher Hmax (median (IQR) 0.95 (0.80-1.08) mV) and Hmax/Mmax (median (IQR) 0.21 (0.16-0.40)), and shorter H wave latency (4.60±0.89 ms) were recorded at MTrSs (P<0.05). Morphologically, there was a close anatomical relationship between the MTrS cells and the muscle spindles. DISCUSSION: Compared with normal muscles, the H-reflex myoelectrical activity was enhanced and some muscle spindles might have been influenced by active MTrSs. Thus, muscle spindles may play an important role in the pathological mechanism underlying myofascial trigger points.

IL-33/ST2 signaling contributes to radicular pain by modulating MAPK and NF-κB activation and inflammatory mediator expression in the spinal cord in rat models of noncompressive lumber disk herniation.

BACKGROUND: Immune and inflammatory responses occurring in the spinal cord play a pivotal role in the progression of radicular pain caused by intervertebral disk herniation. Interleukin-33 (IL-33) orchestrates inflammatory responses in a wide range of inflammatory and autoimmune disorders of the nervous system. Thus, the purpose of this study is to investigate the expression of IL-33 and its receptor ST2 in the dorsal spinal cord and to elucidate whether the inhibition of spinal IL-33 expression significantly attenuates pain-related behaviors in rat models of noncompressive lumbar disc herniation. METHODS: Lentiviral vectors encoding short hairpin RNAs that target IL-33 (LV-shIL-33) were constructed for gene silencing. Rat models of noncompressive lumber disk herniation were established, and the spines of rats were injected with LV-shIL-33 (5 or 10 µl) on the first day after the operation. Mechanical thresholds were evaluated during an observation period of 21 days. Moreover, the expression levels of spinal tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and cyclooxygenase 2 (COX-2) and the activation of the mitogen-activated protein kinases (MAPK) and nuclear factor-κB (NF-κB) pathways were evaluated to gain insight into the mechanisms related to the contribution of IL-33/ST2 signaling to radicular pain. RESULTS: The application of nucleus pulposus (NP) to the dorsal root ganglion (DRG) induced an increase in IL-33 and ST2 expression in the spinal cord, mainly in the dorsal horn neurons, astrocytes, and oligodendrocytes. Spinally delivered LV-shIL-33 knocked down the expression of IL-33 and markedly attenuated mechanical allodynia. In addition, spinal administration of LV-shIL-33 reduced the overexpression of spinal IL-1ß, TNF-α, and COX-2 and attenuated the activation of C-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and NF-κB/p65 but not p38. CONCLUSIONS: This study indicates that spinal IL-33/ST2 signaling plays an important role in the development and progression of radicular pain in rat models of noncompressive lumber disk herniation. Thus, the inhibition of spinal IL-33 expression may provide a potential treatment to manage radicular pain caused by intervertebral disk herniation.

Intrathecal 5-azacytidine inhibits global DNA methylation and methyl- CpG-binding protein 2 expression and alleviates neuropathic pain in rats following chronic constriction injury.

The pathogenesis of neuropathic pain remains largely unknown. Epigenetic mechanisms may play a major role in regulating expression of pro- or antinociceptive genes. DNA methylation is a major epigenetic mechanism in vertebrates, and methyl- CpG-binding protein 2 (MeCP2) is directly involved in methylation-mediated gene silencing. To determine how changes in global DNA methylation and MeCP2 expression occur following chronic constriction injury (CCI) and how repression of DNA methylation affects these changes and attenuates neuropathic pain, we used intrathecal 5-azacytidine, a DNA methyltransferase inhibitor, in CCI rats. Rats received 0.9% saline or 5-azacytidine (10µmol·d(-1)) via spinal injection once daily from day 3 to day 14 after CCI surgery. Global DNA methylation and MeCP2 expression increased in the spinal cord in CCI rats on day 14 after CCI surgery. Mechanical allodynia and thermal hyperalgesia induced by CCI were attenuated by intrathecal 5-azacytidine from day 5 to day 14 after CCI surgery. The increases in global DNA methylation and MeCP2 expression in the spinal cord in CCI rats were also significantly inhibited by intrathecal 5-azacytidine. These results demonstrate that increased global DNA methylation and MeCP2 expression in the spinal cord after nerve damage may play an important role in neuropathic pain. 5-azacytidine shows potential for treating neuropathic pain.

[Effect of intrathecal ketamine injection on protein kinase C expression in the spinal dorsal horn of rats with formalin-induced pain].

OBJECTIVE: To investigate the expression of protein kinase C (PKC) in the spinal dorsal horn of rats with formalin-induced pain and the effect of intrathecal ketamine on PKC expression. METHODS: Thirty-two SD rats were randomly divided into 4 equal groups, namely the control group, intrathecal saline group (NS), 50 µg ketamine group (K1) and 100 µg ketamine group (K2). The rats were anesthetized with 10% chloral hydrate, and a microspinal catheter was inserted intrathecally into the lumbar region. Five days later, the rats in groups, K1 and K2 were subjected to intrathecal administration of 50 and 100 µg ketamine (10 µl), respectively, followed by 10 µl saline, and those in NS group received 20 µl saline only. Thirty minutes later, 5% formalin (50 µl) was subcutaneously injected into the left hindpaw. The pain intensity score (PIS) was utilized to assess antinociceptive behavior within 1 h after formalin injection. Twenty-four hours later, the left hindpaw thickness was measured and the expression of PKC in the spinal dorsal horn in the L5 segment was assayed using immunohistochemistry. RESULTS: Compared to group NS, groups K1 and K2 showed significantly decreased PIS (P<0.01) in the second phase of formalin-induced pain; 24 h later, the left hindpaw thickness of group NS increased obviously in comparison with that in the control group (P<0.01), whereas the thickness was significantly reduced in group K1 and K2 as compared to that in group NS (P<0.05). The number of immunoreactive cells and the immunohistochemical score of PKC in the spinal dorsal horn were significantly higher in group NS than in group C (P<0.01), but significantly lower in groups K1 and K2 than in group NS (P<0.05). CONCLUSION: Intrathecal ketamine produces obvious antinociception against formalin-induced pain in rats and inhibits the enhanced PKC expression in the spinal dorsal horn in response to formalin-induced pain, suggesting the important role of PKC in nociceptive signal transmission and modulation in the spinal cord.

Cyclooxygenase inhibitors suppress the expression of P2X(3) receptors in the DRG and attenuate hyperalgesia following chronic constriction injury in rats.

Recent evidence suggests that P2X(3) receptors express abundantly in nociceptive sensory neurons and play an important role in neuropathic pain. Upregulation of prostaglandin E2 (PGE2) after nerve injure is involved in the pathogenesis of neuropathic pain. An increase of P2X(3) receptors after chronic constriction injury (CCI) to the sciatic nerve has also been reported, the mechanisms are not known clearly. In this study, we examined the effects of systemic administration of cyclooxygenase (COX) inhibitors on analgesia and the expression of P2X(3) receptors in the dorsal root ganglia (DRG) in CCI rats. Rats received 0.9% saline, the nonselective COX inhibitor ibuprofen (40mgkg(-1)day(-1)) or the selective COX-2 inhibitor celecoxib (30mgkg(-1)day(-1)) by gavage twice daily from 3 to 14 days after surgery. Mechanical allodynia and thermal hyperalgesia induced by CCI were markedly attenuated by celecoxib from 5 to 14 days after surgery, and relieved by ibuprofen treatment from 7 to 10 days after surgery. The increase of P2X(3) receptors in the DRG in CCI rats on day 14 after surgery was also significantly inhibited; the effect of ibuprofen was stronger than that of celecoxib. These results demonstrate that up-regulated COX/PGE2 after nerve damage may play an important role in neuropathic pain. They are highly involved in the expression of P2X(3) receptors in the DRG in CCI rats.

[Isobaric and hyperbaric local anesthetic used in spinal anesthesia].

OBJECTIVE: To reveal the advantages and disadvantages of the application of isobaric and hyperbaric local anesthetic in spinal anesthesia so as to provide reference for clinical practice. METHODS: One hundred and sixty ASA patients (physical status I - II) undergoing lower abdominal surgery within 3 hours under spinal anesthesia (using CSEA technique via spinal needle in epidural needle) were allocated to 2 groups with 80 cases each. In lateral decubitus, patients randomly received a subarachnoid injection of 3.0 mL (15 mg) isobaric (Group I) or hyperbaric (Group H) bupivacaine and then turned supine. Hemodynamic changes and patients' responses were perioperatively observed. After subarachnoid injection, we recorded the time of onset and motor block, the peak sensory blocked level, the time of regression of 2 dermatomes, the time of the first administration of analgesics for a significant pain of the incision, the time of the regression of motor block to modified Bromage scale 2, and the time of recuperating the function of urination. RESULTS: Both isobaric and hyperbaric 0.5% bupivacaine solutions in a volume of 3.0 mL provided effective sensory and motor block for the operations. The time of onset and complete motor block were similar in the two groups. Compared with Group I, the time of peak sensory block in Group H was shorter, the peak sensory block level was higher (more maximal dermatomes of blocked sensory nerves), the time of regression of sensory and motor block were shorter, the time of recuperating the function of urination was longer, and the incidence of feeling sick, nausea, vomiting and hypotension was higher. CONCLUSION: Isobaric solution is superior to hyperbaric solution in spinal anesthesia.