溴结构域蛋白4抑制剂JQ1在甲醛诱导小鼠炎性痛中的作用及机制

目的 探讨溴结构域蛋白4（BRD4）抑制剂JQ1在甲醛诱导炎性痛模型小鼠脊髓中的表达变化及作用。 方法 32只ICR小鼠随机分为生理盐水组、甲醛注射5 min、30 min和60 min组,每组8只,Western blotting（n=4/组）和Real-time PCR（n=4/组）检测各组小鼠脊髓BRD4的蛋白和mRNA的表达水平;66只小鼠随机分为甲醛注射组,溶媒（DMSO）加甲醛注射组,以及6.25、12.5、25 和50 mg/kg JQ1注射加甲醛溶液组,每组11只,观察BRD4抑制剂JQ1对每组小鼠自发性疼痛的影响（n=11/组）;免疫组织化学（n=3/组）、Real-time PCR（n=4/组）或Western blotting（n=4/组）的方法检测25 mg/kg JQ1对模型小鼠脊髓即早基因c-fos及谷氨酸受体2(GluR2)表达的影响。 结果 甲醛注射后30 min和60 min,小鼠脊髓BRD4的蛋白（P<0.01）和mRNA水平明显升高（P<0.05）;行为学结果显示,25 mg/kg和50 mg/kg JQ1处理组小鼠第Ⅱ相痛反应持续时间与溶媒（DMSO）组相比均明显缩短（P<0.05）; 免疫组织化学及Real-time PCR结果显示,25 mg/kg JQ1干预明显减少小鼠脊髓c-fos的阳性细胞数（P<0.05) 和mRNA水平（P<0.05）,Western blotting结果显示,25 mg/kg JQ1干预明显降低甲醛诱导的小鼠脊髓GluR2（P<0.001）的蛋白表达。 结论 BRD4在炎性痛中枢敏化中起重要作用,JQ1可能是通过抑制疼痛中枢敏化的形成从而缓解炎性疼痛。     

带气囊导管构建脊髓缺血模拟再灌注与缺血分离模型

背景：采用带有气囊的导管急性压迫脊髓缺血模拟人类损伤可以造成再灌注与缺血分离的动物模型。 目的：应用免疫组化和生物化学分析方法观察不同缺血时间窗处理对损伤脊髓的影响。 方法：SD大鼠36只,随机分为假手术组,带气囊导管造成大鼠脊髓缺血10,30,45,60,90 min组。 结果与结论：再灌注48 h后,随着缺血时间的延长,脊髓前角神经元坏死和凋亡逐渐加重,丙二醛水平逐渐增加,超氧化物歧化酶活性逐渐下降,大鼠的神经行为学病症加重。提示用带气囊的导管建立缺血再灌注大鼠模型成功,再灌注后大鼠脊髓的损伤随缺血时间的延长而加重。     

Analysis of FK506-mediated protection in an organotypic model of spinal cord damage: heat shock protein 70 levels are modulated in microglial cells

Functional loss after spinal cord injuries is originated by primary and secondary injury phases whose underlying mechanisms include massive release of excitatory amino acids to cytotoxic levels that contribute to neural death. Attenuation of this excitotoxicity is a key point for improving the functional outcome after injury. One of the drugs with potential neuroprotective actions is FK506, a molecule widely used as an immunosuppressant. FK506 may exert neuroprotection via inhibition of calcineurin by binding the FKBP12, or by binding other immunophilins such as FKBP52, leading to modulation of heat shock proteins (Hsp) 90 and 70. In the present study, we used an in vitro model of organotypic culture of rat spinal cord slices to assess whether FK506 is able to protect them against glutamate excitotoxicity. The results showed that FK506 promoted a significant protective effect on the spinal cord tissue at concentrations of 50 and 100 nM. Hsp70 induction was restricted to microglial cells in spinal cord slices treated with either glutamate or FK506. In contrast, the combination of both agents led to a transient reduction in Hsp70 levels in parallel to a marked reduction in IL-1beta precursor production by glial cells. The use of geldanamycin, which promotes persistent induction of Hsp70 in these cells as well as in motoneurons, did not produce tissue neuroprotection. These observations suggest that FK506 might protect spinal cord tissue by targeting on microglial cells and that transient downregulation of Hsp70 on these cells after excitotoxicity is a relevant mechanism of action of FK506.     

CNTF基因在大鼠脊髓中的表达及生后发育的变化

目的 观察CNTF基因在大鼠脊髓中的表达以及生后发育过程中的变化。方法 以地高辛标记（dig）－CNTF cDNA为探针，采用原位杂交法，观察CNTF mRNA在大鼠脊髓中的分布；采用TR－PCR法，半定量分析大鼠生后发育过程中，脊髓CNTF mRNA表达水平的变化。结果 CNTF mRNA原位杂交阳性信号存在于正常大鼠脊髓白质的腹索、外侧索周边的部分胶质细胞中；灰质中未能发现阳性杂交信号。RT－PCR结果显示，大鼠生后1d脊髓细胞中即可见CNTF mRNA表达，但表达量较低；出生15d表达量迅速增加；30d时最高；60d时的表达量有下降的趋势。结论 大鼠脊髓白质的部分胶质细胞可表达CNTF mRNA；大鼠出生后1d CNTF mRNA即有表达，之后随脊髓的发育而呈动态变化的趋势。     

Kinetics of collagenase and neutral protease release by neutrophils exposed to microcrystalline sodium urate

The rates of release of the various enzymes from PMN leukocytes exposed to MSU crystals were measured. Lysozyme and neutral protease appeared to be released simultaneously and release appeared to be essentially complete by 60 minutes. In contrast, collagenase was detected only after 30 minutes incubation, reached peak concentration at 90 minutes and dropped noticeably by 180 minutes. The presence of these enzymes was not due to cell lysis since only 10% of the total cellular LDH was present in the supernates. The levels of total and active collagenase in the supernatants were measured. In contrast to latent collagenase, active collagenase levels increased continually throughout the incubation period. The gradual increase in level of active collagenase may explain the corresponding drop of latent collagenase in the longer incubation (90 minutes or more) as the latter apparently is converted to active form. The effects of collagenase on Type I collagen were examined by SDS gel electrophoresis.     

谷氨酸诱导海马神经元凋亡涉及线粒体释放细胞色素C

目的：建立体外谷氨酸诱导神经元兴奋损伤模型，探索其凋亡发生是否通过线粒体信号转导途径介导的细胞色素C（Cyt C）释放而实现，为今后干预性使用神经保护剂提供依据。方法:分离及培养新生Wistar大鼠海马神经元，选用合适谷氨酸浓度建立神经元损伤模型；利用LDH测定及流式细胞仪Annexin V/PI双染色法检测谷氨酸暴露后不同时点神经元凋亡及坏死的动态改变；采用Western blotting法检测caspase-3活性及线粒体内和胞浆内Cyt C水平动态变化。结果:谷氨酸诱导神经元损伤呈明显浓度及时间依赖性，50 μmol/L浓度可使LDH释放量明显增加 (18.4%,P<0.05)，暴露后6 h凋亡率显著增加；凋亡发生前，神经元caspase-3活性已明显增高（3 h），6 h达高峰；线粒体Cyt C释放发生在caspase-3增高前，30 min时胞浆内Cyt C水平即明显增加（P<0.05），3 h胞浆内Cyt C水平超过线粒体内，而线粒体内Cyt C水平进行性减少。结论:50 μmol/L谷氨酸可诱导海马神经元凋亡，凋亡机制可能是通过损伤线粒体膜，使Cyt C易位释放入胞浆激活caspase级联反应而致。     

Voluntary exercise induces a BDNF-mediated mechanism that promotes neuroplasticity

We have investigated potential mechanisms by which exercise can promote changes in neuronal plasticity via modulation of neurotrophins. Rodents were exposed to voluntary wheel running for 3 or 7 days, and their lumbar spinal cord and soleus muscle were assessed for changes in brain-derived neurotrophic factor (BDNF), its signal transduction receptor (trkB), and downstream effectors for the action of BDNF on synaptic plasticity. Exercise increased the expression of BDNF and its receptor, synapsin I (mRNA and phosphorylated protein), growth-associated protein (GAP-43) mRNA, and cyclic AMP response element-binding (CREB) mRNA in the lumbar spinal cord. Synapsin I, a synaptic mediator for the action of BDNF on neurotransmitter release, increased in proportion to GAP-43 and trkB mRNA levels. CREB mRNA levels increased in proportion to BDNF mRNA levels. In separate experiments, the soleus muscle was paralyzed unilaterally via intramuscular botulinum toxin type A (BTX-A) injection to determine the effects of reducing the neuromechanical output of a single muscle on the neurotrophin response to motor activity. In sedentary BTX-A-treated rats, BDNF and synapsin I mRNAs were reduced below control levels in the spinal cord and soleus muscle. Exercise did not change the BDNF mRNA levels in the spinal cord of BTX-A-treated rats but further reduced the BDNF mRNA levels in the paralyzed soleus relative to the levels in sedentary BTX-A-treated rats. Exercise also restored synapsin I to near control levels in the spinal cord. These results indicate that basal levels of neuromuscular activity are required to maintain normal levels of BDNF in the neuromuscular system and the potential for neuroplasticity.     

CsA对树鼩海马微灌流谷氨酸和钙所致线粒体应激的影响

目的：观察环孢菌素A(CsA)对树鼩海马由谷氨酸（Glu）及钙（Ca²⁺）引起微环境改变所致线粒体应激的影响，并探讨其分子机制。方法:单泵等速微灌流系统行树鼩海马Glu及Ca²⁺微灌流，24 h后免疫组化法检测海马神经元细胞色素C（Cyt C）蛋白表达；低温差速离心分离海马神经元线粒体和胞质部分，免疫印迹（Western blotting）法检测Cyt C在胞内表达空间分布；实时荧光定量PCR技术检测海马caspase-3及caspase-9 mRNA的含量。微灌流Glu和Ca²⁺溶液后6 h于舌下iv CsA 40 mg/kg BW，24 h后观察上述指标的改变。结果:树鼩海马微灌流Glu和Ca²⁺溶液后24 h，海马神经元Cyt C表达增强，而线粒体Cyt C含量显著下降，同时胞质部分可见Cyt C；海马组织caspase-3、caspase-9mRNA明显升高；微灌流后6 h静脉注射CsA组, 海马神经元Cyt C表达显著减少，而线粒体Cyt C含量则显著增加，胞质部分未见Cyt C;海马组织caspase-3、caspase-9 mRNA降低。结论:海马微环境中Glu与Ca²⁺的大量堆积,可促进线粒体Cyt C释放，激活caspase级联反应而导致线粒体应激；CsA的神经保护效应可与其抑制线粒体通透性转导孔（MPT）开放，防止Cyt C释放及减少caspase-3和caspase-9的活化有关。     

Changes in transmitter release patterns in vitro induced by tremorgenic mycotoxins

The neurochemical effects of the tremorgenic mycotoxins Verruculogen and Penitrem A, which produce a neurotoxic syndrome characterized by sustained tremors, were studied using sheep and rat synaptosomes. The toxins were administered in vivo, either by chronic feeding (sheep) or ip injection 45 min prior to sacrifice (rat). Synaptosomes were subsequently prepared from cerebrocortical and spinal cord/medullary regions of rat, and corpus striatum of sheep. Penitrem A (400 mg mycelium/kg) increased the spontaneous release of endogenous glutamate, GABA, and aspartate by 213%, 455%, and 227%, respectively, from cerebrocortical synaptosomes. Verruculogen (400 mg mycelium/kg) increased the spontaneous release of glutamate and aspartate by 1,300% and 1,200% respectively, but not that of GABA, from cerebrocortical synaptosomes. The spontaneous release of the transmitter amino acids or other amino acids was not increased by the tremorgens in spinal cord/medullary synaptosomes. Penitrem A pretreatment reduced the Veratrine (75 microM) stimulated release of glutamate, aspartate and GABA from cerebrocortical synaptosomes by 33%, 46%, and 11% respectively, and the stimulated release of glycine and GABA from spinal cord/medulla synaptosomes by 67% and 32%, respectively. Verruculogen pretreatment did not alter the Veratrine-induced release of transmitter amino acids from cerebrocortex and spinal cord/medulla synaptosomes. Penitrem A pretreatment increased the spontaneous release of aspartate, glutamate and GABA by 68%, 62%, and 100%, respectively, from sheep corpus striatum synaptosomes but did not alter the synthesis and release of dopamine in this tissue. Verruculogen was shown to cause a substantial increase (300-400%) in the miniature-end-plate potential frequency at the locust neuromuscular junction. The response was detectable within 1 min, rose to a maximum within 5-7 min, and declined to the control rate over a similar period. No change in the amplitude of the m.e.p.p.s was observed. These effects of the tremorgens on transmitter release are interpreted in terms of their mode of action.     

葡萄糖调节蛋白75在缺糖损伤PC12细胞中的表达

目的观察葡萄糖调节蛋白75(GRP75)在缺糖条件下在PC12细胞中的表达。方法体外模拟能量代谢障碍建立细胞缺糖模型,MTT检测PC12细胞活率;LDH测定细胞膜损伤程度;流式细胞仪PI单染色法和An-nexinV/PI双染色法检测细胞的损伤形式;RT-PCR和W estern b lot分别检测GRP75基因在RNA和蛋白水平上的表达。结果随着缺糖时间的延长细胞活力逐渐降低,LDH释放率明显增加,缺糖死亡的细胞有部分凋亡细胞,并出现明显的凋亡峰,GRP75 mRNA在24 h内表达上调,同时蛋白表达上调。结论GRP75在缺糖损伤PC12细胞中有一定的保护作用。     

肌萎灵注射液对体外培养运动神经元的保护作用

目的：观察肌萎灵注射液(JWL)对体外培养运动神经元的保护作用。方法：应用密度离心法分离鼠胚脊髓运动神经元进行原代培养，加入合氨酸建立兴奋性氨基酸毒性损伤模型，评价不同浓度肌萎灵注射液对运动神经元的保护作用。MTT法检测细胞活力，NF-200免疫组化染色并进行图象分析，测定神经突起主干长度，生化分析仪检测培养上清中的乳酸脱氢酶(LDH)，TUNEL阳性神经元计数观察细胞凋亡。结果：(1)肌萎灵注射液能明显促进体外培养运动神经元的活力，促进神经突起的生长；(2)肌萎灵注射液可减少兴奋性氨基酸毒性损伤运动神经元LDH的漏出；(3)肌萎灵注射液能显著减少谷氨酸诱导的运动神经元凋亡。结论：肌萎灵注射液对正常运动神经元和兴奋性氨基酸毒性损伤运动神经元均具有保护作用．     

Preventive effects of intrathecal methylprednisolone administration on spinal cord ischemia in rats: the role of excitatory amino acid metabolizing systems

Spinal cord ischemic injury usually results in paraplegia, which is a major cause of morbidity after thoracic aorta operations. Ample evidence indicates that massive release of excitatory amino acids (EAAs; glutamate) plays an important role in the development of neuronal ischemic injuries. However, there is a lack of direct evidence to indicate the involvement of EAAs in the glutamate metabolizing system (including the glutamate transporter isoforms, i.e. the Glu-Asp transporter (GLAST), Glu transporter-1 (GLT-1), and excitatory amino acid carrier one (EAAC1); glutamine synthetase (GS); and glutamate dehydrogenase (GDH)) in spinal cord ischemia. In the present results, we found that methylprednisolone (MP; intrathecal (i.t.) injection, 200 mug twice daily administered for 3 days before ischemia), a synthetic glucocorticoid, is the therapeutic agent for the treatment of spinal injuries in humans, can significantly reduce the ischemia-induced motor function defect and down-regulate the glutamate metabolizing system (including GLAST, GLT-1, GS, and GDH) in male Wistar rats. The spinal cord ischemia-induced down-regulation of EAAC1 protein expression in the ventral portion of the lumbar spinal cord was partly inhibited by pretreatment with i.t. MP. However, MP did not affect the down-regulation of EAAC1 in the dorsal portion of the lumbar spinal cord after spinal cord ischemia. The i.t. injection of MP alone did not change the neurological functions and the expression of proteins of the glutamate metabolizing system in the spinal cord. Our results indicate that spinal cord ischemia-induced neurological deficits accompany the decrease in the expression of proteins of the glutamate metabolizing system in the lumbar portion of the spinal cord. The i.t. MP pretreatment significantly prevented these symptoms. These results support the observation that MP delivery through an i.t. injection, is beneficial for the treatment of spinal cord ischemic injuries.     

Suppressive effects of intrathecal granulocyte colony-stimulating factor on excessive release of excitatory amino acids in the spinal cerebrospinal fluid of rats with cord ischemia: role of glutamate transporters

Recently, the hematopoietic factor, granulocyte colony-stimulating factor (G-CSF), has been shown to exhibit neuroprotective effects in CNS injuries. Our previous study demonstrated that intrathecal (i.t.) G-CSF significantly improved neurological defects in spinal cord ischemic rats. Considerable evidence indicates that the release of excessive amounts of excitatory amino acids (EAAs) plays a critical role in neuron injury induced by ischemic insult. In the present study, we used a spinal cord ischemia-microdialysis model to examine whether i.t. G-CSF exerted antiexcitotoxicity effects in a rat model of spinal cord ischemia. I.t. catheters and a microdialysis probe were implanted in male Wistar rats. The results revealed that spinal cord ischemia-induced neurological defects were accompanied by a significant increase in the concentration of EAAs (aspartate and glutamate) in the spinal dialysates from 30 min to 2 days after reperfusion. I.t administration of G-CSF immediately after the performance of surgery designed to induce ischemia led to a significant reduction in ischemia-induced increases in the levels of spinal EAAs. Moreover, i.t. G-CSF also brought about a significant reduction in the elevation of spinal EAA concentrations induced by exogenous i.t. administration of glutamate (10 μl of 500 mM). I.t. G-CSF attenuated spinal cord ischemia-induced downregulation of expression of three glutamate transporters (GTs), glial transporter Glu–Asp transporter (GLAST), Glu transporter-1 (GLT-1), and excitatory amino acid carrier 1 (EAAC1) protein 48 h after spinal cord ischemic surgery. Immunohistofluorescent staining showed that i.t. G-CSF significantly upregulated expression of the three GTs in the gray matter of the lumbar spinal cord from 3 to 24 h after injection. We propose that i.t. G-CSF possesses an ability to reduce the extent of spinal cord ischemia-induced excitotoxicity by inducing the expression of glutamate transporters.     

非对称性二甲基精氨酸保护PC12细胞拮抗谷氨酸兴奋性毒性的损伤作用

 目的： 探讨一氧化氮合酶（NOS）抑制剂-非对称性二甲基精氨酸（ADMA）对谷氨酸（Glu）兴奋性毒性损伤PC12细胞的影响及其机制。方法: 用不同浓度的谷氨酸处理PC12 细胞,建立谷氨酸兴奋性神经毒性损伤细胞的实验模型;应用四甲基偶氮唑蓝（MTT）比色法检测细胞存活率;乳酸脱氢酶（LDH）释放试验评价细胞的损伤程度;双氢罗丹明123（DHR）染色后流式细胞仪（FCM）检测细胞内活性氧（ROS）水平;应用试剂盒及分光光度计测定NOS活性和NO水平。结果: 谷氨酸（1-6 mmol/L）处理PC12细胞24 h,可呈剂量依赖性地降低PC12细胞的存活率;在谷氨酸作用PC12 细胞前30min 给予ADMA,可明显地抑制谷氨酸引起的细胞存活率降低及LDH释放增加,减少谷氨酸引起的细胞内ROS堆积,抑制谷氨酸过度激活 NOS和增加NO的生成。结论: ADMA能显著地减弱谷氨酸对PC12细胞的兴奋性毒性损伤作用;其作用机制可能与抑制NOS活性,减少NO生成,进而减轻细胞内ROS的堆积有关。     

Cell viability in three ex vivo rat models of spinal cord injury

Spinal cord injury (SCI) is a devastating disorder that has a poor prognosis of recovery. Animal models of SCI are useful to understand the pathophysiology of SCI and the potential use of therapeutic strategies for human SCI. Ex vivo models of central nervous system (CNS) trauma, particularly mechanical trauma, have become important tools to complement in vivo models of injury in order to reproduce the sequelae of human CNS injury. Ex vivo organotypic slice cultures (OSCs) provide a reliable model platform for the study of cell dynamics and therapeutic intervention following SCI. In addition, these ex vivo models support the 3R concept of animal use in SCI research – replacement, reduction and refinement. Ex vivo models cannot be used to monitor functional recovery, nor do they have the intact blood supply of the in vivo model systems. However, the ex vivo models appear to reproduce many of the post traumatic events including acute and secondary injury mechanisms. Several well‐established OSC models have been developed over the past few years for experimental spinal injuries ex vivo in order to understand the biological response to injury. In this study, we investigated cell viability in three ex vivo OSC models of SCI: stab injury, transection injury and contusion injury. Injury was inflicted in postnatal day 4 rat spinal cord slices. Stab injury was performed using a needle on transverse slices of spinal cord. Transection injury was performed on longitudinal slices of spinal cord using a double blade technique. Contusion injury was performed on longitudinal slices of spinal cord using an Infinite Horizon impactor device. At days 3 and 10 post‐injury, viability was measured using dual staining for propidium iodide and fluorescein diacetate. In all ex vivo SCI models, the slices showed more live cells than dead cells over 10 days in culture, with higher cell viability in control slices compared with injured slices. Although no change in cell viability was observed between time‐points in stab‐ and contusion‐injured OSCs, a reduction in cell viability was observed over time in transection‐injured OSCs. Taken together, ex vivo SCI models are a useful and reliable research tool that reduces the cost and time involved in carrying out animal studies. The use of OSC models provides a simple way to study the cellular consequences following SCI, and they can also be used to investigate potential therapeutics regimes for the treatment of SCI.     

Omega-conotoxin MVIIC attenuates neuronal apoptosis in vitro and improves significant recovery after spinal cord injury in vivo in rats

Excessive accumulation of intracellular calcium is the most critical step after spinal cord injury (SCI). Reducing the calcium influx should result in a better recovery from SCI. Calcium channel blockers have been shown a great potential in reducing brain and spinal cord injury. In this study, we first tested the neuroprotective effect of MVIIC on slices of spinal cord subjected to ischemia evaluating cell death and caspase-3 activation. Thereafter, we evaluated the efficacy of MVIIC in ameliorating damage following SCI in rats, for the first time in vivo. The spinal cord slices subjected a pretreatment with MVIIC showed a cell protection with a reduction of dead cells in 24.34% and of caspase-3-specific protease activation. In the in vivo experiment, Wistar rats were subjected to extradural compression of the spinal cord at the T12 vertebral level using a weigh of 70 g/cm, following intralesional treatment with either placebo or MVIIC in different doses (15, 30 and 60 pmol) five minutes after injury. Behavioral testing of hindlimb function was done using the Basso Beattie Bresnahan locomotor rating scale, and revealed significant recovery with 15 pmol (G15) compared to other trauma groups. Also, histological bladder structural revealed significant outcome in G15, with no morphological alterations, and anti-NeuN and TUNEL staining showed that G15 provided neuron preservation and indicated that this group had fewer neuron cell death, similar to sham. These results showed the neuroprotective effects of MVIIC in in vitro and in vivo model of SCI with neuronal integrity, bladder and behavioral improvements.     

Murine strain differences in acute lung injury and activation of poly(ADP-ribose) polymerase by in vitro exposure of lung slices to bleomycin.

The DNA-cleaving, antitumor antibiotic bleomycin (BLM) causes pulmonary fibrosis, but the essential early events initiating the fibrotic state have not been well characterized. Thus, we have directly examined BLM-mediated pulmonary cell injury by monitoring lactate dehydrogenase (LDH) release and nuclear poly(ADP-ribose) polymerase (PAP) activity, which is stimulated by DNA breakage, using lung slices isolated from BLM-sensitive (C57B1/6) and BLM-resistant (BALB/c) mice. Lung slices were incubated continuously with or without the PAP inhibitor, 3-aminobenzamide (3-AB), and exposed to BLM for 45 min. LDH release from C57B1/6 lung slices increased 2-fold by 8.5 h after treatment with BLM. In contrast, BLM failed to enhance cumulative LDH release by BALB/c mouse lung slices. Co-incubation of C57B1/6 lung slices with 3-AB prevented BLM-induced LDH release. Nuclear PAP was activated 3- to 4-fold 1.25 h after exposure of C57B1/6 lung slices to BLM but returned to control levels by 3.75 h. Nuclear PAP was only marginally affected at these times in BALB/c lung slices. Co-incubation of C57B1/6 slices with 3-AB prevented the early increases in PAP activity. These results demonstrate that murine strain sensitivity to acute cell injury and early PAP activation by BLM in lung slices parallels the in vivo sensitivity of lungs. In addition, 3-AB suppresses PAP activation and acute cell injury in lung slices. Differential activation of PAP appears to govern murine strain variation in response to BLM and is consistent with the hypothesis that activation of PAP participates in acute pneumocyte injury, initiating the process of BLM-induced fibrosis.     

Glutamate neurotoxicity in spinal cord cell culture.

The neurotoxicity of glutamate was investigated quantitatively in mixed neuronal and glial spinal cord cell cultures from fetal mice at 12-13 days of gestation. Five-minute exposure to 10-1000 microM glutamate produced widespread acute neuronal swelling, followed by neuronal degeneration over the next 24 h (EC50 for death about 100-200 microM); glia were not injured. Glutamate was neurotoxic in cultures as young as four days in vitro, although greater death was produced in older cultures. By 14-20 days in vitro, 80-90% of the neuronal population was destroyed by a 5-min exposure to 500 microM glutamate. Acute neuronal swelling following glutamate exposure was prevented by replacement of extracellular sodium with equimolar choline, with minimal reduction in late cell death. Removal of extracellular calcium enhanced acute neuronal swelling but attenuated late neuronal death. Both acute neuronal swelling and late degeneration were effectively blocked by the noncompetitive N-methyl-D-aspartate receptor antagonist dextrorphan and by the novel competitive antagonist CGP 37849. Ten micromolar 7-chlorokynurenate also inhibited glutamate neurotoxicity; protection was reversed by the addition of 1 mM glycine to the bathing medium. These observations suggest that glutamate is a potent and rapidly acting neurotoxin on cultured spinal cord neurons, and support involvement of excitotoxicity in acute spinal cord injury. Similar to telencephalic neurons, spinal neurons exposed briefly to glutamate degenerate in a manner dependent on extracellular Ca2+ and the activation of N-methyl-D-aspartate receptors.     

LPS对大鼠脊髓背角星形胶质细胞CXCL1和CCL2表达的影响

目的:观察脂多糖(LPS)诱发的大鼠脊髓背角星形胶质细胞趋化因子CXCL1和CCL2的表达及释放,分析Toll样受体2(TLR2)以及Toll样受体4(TLR4)的作用。方法:培养新生SD大鼠(<3d)脊髓背角星形胶质细胞,免疫荧光鉴定纯度达95%之后分为空白对照组、LPS处理组、TAK-242+LPS处理组、LPS-RS+LPS处理组。在LPS(1μg/ml)作用下,采用real time RT-PCR法检测SD大鼠(<3d)脊髓背角星形胶质细胞CXCL1和CCL2 mRNA表达;Western Blot用于检测胶质纤维酸性蛋白(GFAP)、TLR2、TLR4、CXCL1以及CCL2表达;ELISA检测CXCL1和CCL2释放。结果:与正常对照组相比,LPS作用6 h,背角星形胶质细胞CXCL1和CCL2在mRNA和蛋白水平的表达均上调(P<0.05),CXCL1和CCL2释放增加(P<0.05)。LPS刺激CXCL1和CCL2表达和释放增加的效应可以被TLR4受体拮抗剂TAK-242(50 nmol)以及TLR2/TLR4受体拮抗剂LPS-RS(500 ng/ml)明显阻断(P<0.05)。与正常组相比,LPS刺激星形胶质细胞GFAP、TLR2、TLR4和核转录因子-κBp65(NF-κBp65)表达上调(P<0.05),该效应亦可被TAK-242和LPS-RS所阻断(P<0.05)。此外,与LPS处理组相比,TAK-242预处理背角星形胶质细胞可明显抑制LPS诱发的TLR2表达上调。结论:TLR4/NF-κB信号可能参与了LPS诱导的培养的大鼠脊髓星形胶质细胞趋化因子CXCL1和CCL2表达上调和释放增加。     

The effects of chemical or surgical deafferentation on [3H]-acetylcholine release from rat spinal cord

Cholinergic modulation of nociceptive transmission through both nicotinic and muscarinic receptors in the spinal cord represents an important mechanism in pain signaling. However, what neuronal elements release acetylcholine and how release might change in response to deafferentation are unclear. The present studies demonstrated Ca++- and K+-dependent release of [3H]-acetylcholine from slices of regional areas of rat spinal cord. That [3H]-acetylcholine was synthesized from [3H]-choline was demonstrated by the lack of [3H]-acetylcholine release following incubation with either the choline uptake inhibitor hemicholinium or the choline acetyltransferase inhibitor bromoacetylcholine. Rats treated neonatally with capsaicin or with spinal nerve ligation as adults showed a significantly decreased K+-stimulated release of [3H]-acetylcholine from dorsal horn but not ventral horn lumbar spinal cord slices. In rats subjected to dorsal rhizotomy, while basal release from lumbar dorsal spinal cord slices was reduced, K+-stimulated [3H]-acetylcholine release, while decreased, was not significantly different compared with controls. The data presented here show that there are regional differences in the release of acetylcholine from spinal cord and that this release can be modulated by chemical or surgical deafferentation. These results also indicate that the source of acetylcholine in the dorsal cord originates mainly from resident somata and their collaterals, interneurons and/or descending terminals, with only very minor contributions coming from primary afferents. The present data help to further elucidate the role of acetylcholine in spinal signaling, particularly with respect to the effects of nerve injury and nociceptive neurotransmission.