Purinergic signalling in spinal pain processing

Purinergic Signalling - Purinergic signalling plays important roles in somatosensory and nociceptive transmission in the dorsal horn of the spinal cord under physiological and pathophysiological...     

Effects of a metalloporphyrinic peroxynitrite decomposition catalyst,ww-85, in a mouse model of spinal cord injury

The aim of the present study was to assess the effect of a metalloporphyrinic peroxynitrite decomposition catalyst, ww-85, in the pathophysiology of spinal cord injury (SCI) in mice. Spinal cord trauma was induced by the application of vascular clips to the dura via a four-level T5–T8 laminectomy. SCI in mice resulted in severe trauma characterized by oedema, neutrophil infiltration, production of inflammatory mediators, tissue damage and apoptosis. ww-85 treatment (30–300 µg/kg, i.p. 1 h after the SCI) significantly reduced in a dose-dependent manner: (1) the degree of spinal cord inflammation and tissue injury, (2) neutrophil infiltration (myeloperoxidase activity), (3) nitrotyrosine formation and PARP activation, (4) pro-inflammatory cytokines expression, (5) NF-κB activation and (6) apoptosis. Moreover, ww-85 significantly ameliorated the recovery of limb function (evaluated by motor recovery score) in a dose-dependent manner. The results demonstrate that ww-85 treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma.     

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.     

Enhancement of thermal stability of chondroitinase ABC I by site-directed mutagenesis: An insight from Ramachandran plot

The application of chondroitinase ABC I (cABC I) in damaged nervous tissue is believed to prune glycosaminoglycan chains of proteoglycans, thereby facilitates axon regeneration. However, the utilization of cABC I as therapeutics is notably restricted due to its thermal instability. In the present study, we have explored the possibility of thermostabilization of cABC I through release of its conformational strain using Ramachandran plot information. In this regard, Gln140 with non-optimal φ and ψ values were replaced with Gly, Ala and Asn. The results indicated that Q140G and Q140A mutants were able to improve both activity and thermal stability of the enzyme while Q140N variant reduced the enzyme activity and destabilized it. Moreover, the two former variants displayed a remarkable resistance to trypsin degradation. Structural analysis of all mutants showed an increase in intrinsic fluorescence intensity and secondary structure content of Q140G and Q140A compared to the wild type which indicated more compact structure upon mutation. This investigation demonstrated that relief of conformational tension can be considered as a possible approach to increase the stability of the protein.     

Muscular torque generation during imposed joint rotation: torque-angle relationships when subjects' only goal is to make a constant effort

It is a reasonable expectation that voluntarily activated spinal motoneurons will be further excited by increases in spindle afferent activity produced by muscle stretch. Human motor behavior attributed to tonic stretch reflexes and to reflexes recruited by relatively slow joint rotation has been reported from several laboratories. We reinvestigated this issue by rotating the elbow joint over the central portion of its range while subjects focused on keeping their elbow flexion effort constant at one of three different levels and made no attempt to control the position, speed or direction of movement of their forearm. There is evidence that subjects' voluntary motor status is constant under these conditions so that any change in torque would be of involuntary origin. On average, torques rose somewhat and then fell as the elbow was flexed through a range of 80° at 10, 20 and 60°/s and a similar pattern occurred during elbow extension; i.e., both concentric and eccentric torque-angle profiles had roughly similar shapes and neither produced consistent stabilizing cross-range stiffness. The negative stiffness (rising torque) during the early part of a concentric movement and the negative stiffness (falling torque) during the later part of an eccentric movement would not have occurred if a stabilizing stretch reflex had been present. Positive stiffness rarely gave rise to torque changes greater than 20% in either individual or cross-subject averaged data. When angular regions of negative stiffness are combined with regions of low positive stiffness (torque change 10% or less), much of the range of motion was not well stabilized, especially during eccentric movements. The sum of the EMGs from biceps brachii, brachioradialis and brachialis showed a pattern opposite to that expected for a stretch reflex; there was an upward trend in the EMG as the elbow was flexed and a downward trend as the elbow was extended. There was little change in the shape of this EMG-angle relationship with either direction or velocity. The individual EMG-angle relationships were distinctive for each of these three elbow flexor muscles in four of the six subjects; in the remaining two, biceps was distinctive, but brachioradialis and brachialis appeared to be coupled. Although the EMGs of individual muscles were modulated over the angular range, no consistent stretch reflexes could be seen in the individual records. Thus, we could find no clear evidence for stretch reflex stabilization of human subjects maintaining a constant effort. Rather, muscle torque appears to be reflexly modulated across a much used portion of the elbow's angular range so that any appreciable stabilizing stiffness that is sustained for more than fractions of a second is associated with a change in effort.     

Toll-like Receptor 2 Mediates Peripheral Nerve Injury-induced NADPH Oxidase 2 Expression in Spinal Cord Microglia

We have previously reported that NADPH oxidase 2 (Nox2) is up-regulated in spinal cord microglia after spinal nerve injury, demonstrating that it is critical for microglia activation and subsequent pain hypersensitivity. However, the mechanisms and molecules involved in Nox2 induction have not been elucidated. Previous studies have shown that Toll-like receptors (TLRs) are involved in nerve injury-induced spinal cord microglia activation. In this study, we investigated the role of TLR in Nox2 expression in spinal cord microglia after peripheral nerve injury. Studies using TLR knock-out mice have shown that nerve injury-induced microglial Nox2 up-regulation is abrogated in TLR2 but not in TLR3 or -4 knock-out mice. Intrathecal injection of lipoteichoic acid, a TLR2 agonist, induced Nox2 expression in spinal cord microglia both at the mRNA and protein levels. Similarly, lipoteichoic acid stimulation induced Nox2 expression and reactive oxygen species production in primary spinal cord glial cells in vitro. Studies on intracellular signaling pathways indicate that NF-κB and p38 MAP kinase activation is required for TLR2-induced Nox2 expression in glial cells. Conclusively, our data show that TLR2 mediates nerve injury-induced Nox2 gene expression in spinal cord microglia via NF-κB and p38 activation and thereby may contribute to spinal cord microglia activation.     

LY2228820 Dimesylate,a Selective Inhibitor of p38 Mitogen-activated Protein Kinase,Reduces Angiogenic Endothelial Cord Formation in Vitro and in Vivo

LY2228820 dimesylate is a highly selective small molecule inhibitor of p38α and p38β mitogen-activated protein kinases (MAPKs) that is currently under clinical investigation for human malignancies. p38 MAPK is implicated in a wide range of biological processes, in particular those that support tumorigenesis. One such process, angiogenesis, is required for tumor growth and metastasis, and many new cancer therapies are therefore directed against the tumor vasculature. Using an in vitro co-culture endothelial cord formation assay, a surrogate of angiogenesis, we investigated the role of p38 MAPK in growth factor- and tumor-driven angiogenesis using LY2228820 dimesylate treatment and by shRNA gene knockdown. p38 MAPK was activated in endothelial cells upon growth factor stimulation, with inhibition by LY2228820 dimesylate treatment causing a significant decrease in VEGF-, bFGF-, EGF-, and IL-6-induced endothelial cord formation and an even more dramatic decrease in tumor-driven cord formation. In addition to involvement in downstream cytokine signaling, p38 MAPK was important for VEGF, bFGF, EGF, IL-6, and other proangiogenic cytokine secretion in stromal and tumor cells. LY2228820 dimesylate results were substantiated using p38α MAPK-specific shRNA and shRNA against the downstream p38 MAPK effectors MAPKAPK-2 and HSP27. Using in vivo models of functional neoangiogenesis, LY2228820 dimesylate treatment reduced hemoglobin content in a plug assay and decreased VEGF-A-stimulated vascularization in a mouse ear model. Thus, p38α MAPK is implicated in tumor angiogenesis through direct tumoral effects and through reduction of proangiogenic cytokine secretion via the microenvironment.     

后路减压内固定融合矫形治疗退变性腰椎侧凸伴椎管狭窄的临床研究

目的:探讨后路减压内固定融合矫形治疗退变性腰椎侧凸伴椎管狭窄的临床疗效。方法:将我院2009年1月~2015年1月收治的退变性腰椎侧凸伴椎管狭窄患者按照手术方法分为两组,实验组进行后路减压内固定融合矫形术治疗,对照组进行单纯后路减压固定矫形术治疗,对比两组患者术前、术后6个月和18个月的Cobb角、腰椎前凸角、日本骨科学会(JOA)评分、疼痛视觉模拟量表(VAS)情况,SRS-22国际标准量表评分情况以及出血量情况。结果:实验组术后18个月的Cobb角、腰椎前凸角分别为(16.8±5.16)°和(36.8±5.82)°,均分别低于对照组的(20.2±6.61)°和(41.2±5.67)°,且均低于术前(P0.05),而术前及术后6个月时两组比较无差异(P0.05);两组患者术后6个月和18个月的JOA评分、VAS评分比较均较术前明显改善(P0.05),且组间比较显示,术后18月两组比较均存在显著差异(P0.05);SRS-22国际标准评估量表显示,术后18个月两组患者的自理能力、自我评价、精神状态方面无显著差异(P0.05),而疼痛情况存在显著差异(P0.05)。两组术中出血量比较无统计学差异(P0.05)。结论:后路减压内固定融合矫形术治疗退变性腰椎侧凸伴椎管狭窄疗效显著,且后路减压内固定融合矫形术在改善患者的腰椎侧凸程度、功能障碍及疼痛程度方面优于单纯后路减压矫形内固定术,值得临床推广。     

椎弓根钉棒系统加植骨融合治疗胸腰椎爆裂性骨折并脊髓损伤的 临床研究

目的:探讨椎弓根钉棒系统加植骨融合治疗胸腰椎爆裂性骨折并脊髓损伤的疗效。方法:回顾性分析2010年3月至2014年12月,采用椎弓根钉棒系统加植骨融合治疗93例胸腰椎爆裂性骨折并脊髓损伤患者的资料,男56例,女37例。致伤原因:交通事故伤27例,高处坠落伤47例,重物压伤19例。骨折节段:L1骨折22例,L2骨折16例,L3骨折6例,T11骨折15例,T12骨折34例。结果:本研究共93例患者,所有患者经过一般12个月的随访,其中平均随访13.8月(10-16月)。与术前相比,患者术后6个月伤椎前缘高度比值明显增加,Cobb角值和椎管占位率明显降低(t=6.167,7.241,7.143,P0.05)。术后12个月伤椎前缘高度比值明显增加,Cobb角值和椎管占位率明显降低(t=9.345,11.541,11.263,P0.05)。且患者术后12个月与术后6个月在伤椎前缘高度比值、Cobb角、椎管占位率上比较,差异具有统计学意义(t=9.632,8.154,7.415,P0.05),根据Frankel神经分级,术后大部分患者神经功能有所恢复。其中,Frankel分级为A的患者有35例恢复,术后有效恢复率为87.5%;B级患者有25例恢复,术后有效恢复率89.3%。C级和D级患者术后有效恢复率均为100%,B,C,D级患者与A级患者有效恢复率相比,差异没有统计学意义(x~2=0.051,2.196,1.253,P0.05),随访12个月期间,2例患者术后5个月出现内固定物松动,1例术后12个月发生螺钉断裂,其余患者无伤口感染、肺部感染、深静脉血栓等并发症发生。结论:椎弓根钉棒系统加植骨融合能有效治疗胸腰椎爆裂性骨折并脊髓神经损伤,术后患者神经功能恢复较好,并发症较少,值得临床推广使用。     

Developmental change and sexual difference in synaptic modulation produced by oxytocin in rat substantia gelatinosa neurons

We have previously reported that oxytocin produces an inward current at a holding potential of ?70 mV without a change in glutamatergic excitatory transmission in adult male rat spinal lamina II (substantia gelatinosa; SG) neurons that play a pivotal role in regulating nociceptive transmission. Oxytocin also enhanced GABAergic and glycinergic spontaneous inhibitory transmissions in a manner sensitive to a voltage-gated Na⁺-channel blocker tetrodotoxin. These actions were mediated by oxytocin-receptor activation. Such a result was different from that obtained by other investigators in young male rat superficial dorsal horn neurons in which an oxytocin-receptor agonist enhanced glutamatergic and GABAergic but not glycinergic spontaneous transmissions. In order to know a developmental change and also sexual difference in the actions of oxytocin, we examined its effect on spontaneous synaptic transmission in adult female and young male rat SG neurons by using the whole-cell patch-clamp technique in spinal cord slices. In adult female rats, oxytocin produced an inward current at ?70 mV without a change in excitatory transmission. GABAergic and glycinergic transmissions were enhanced by oxytocin, the duration of which enhancement was much shorter than in adult male rats. In young (11–21 postnatal days) male rats, oxytocin produced not only an inward but also outward current at ?70 mV, and presynaptically inhibited or facilitated excitatory transmission, depending on the neurons tested; both GABAergic and glycinergic transmissions were enhanced by oxytocin. The inhibitory transmission enhancements in adult female and young male rats were sensitive to tetrodotoxin. Although the data may not be enough to be estimated, it is suggested that synaptic modulation by oxytocin in SG neurons, i.e., cellular mechanism for its antinociceptive action, exhibits a developmental change and sexual difference.