APA微囊化BCCs移植逆转神经性疼痛大鼠背根神经节Navl.8 mRNA表达下调

目的观察APA微囊化牛肾上腺嗜铬细胞(BCCs)移植对坐骨神经慢性挤压伤(CCI)大鼠背根神经节(DRG)Nav1.8 mRNA表达的影响.方法 SD大鼠分为4组,每组5只,即对照组(C组),正常大鼠;CCI组,右侧坐骨神经结扎;APA组,CCI模型后7 d,蛛网膜下腔移植500～600个APA空囊;APA-BCCs组,CCI模型后7 d蛛网膜下腔移植5×106个APA微囊化BCCs.测定各组移植前和移植后7d的触诱发痛阈值(g)和CO2激光刺激痛阈值(ms).移植后7 d行为学测定后取DRG冰冻切片,按照原位杂交法检测各组DRG中Nav1.8 mRNA表达的变化.结果 CCI组和APA组大鼠L4和L5 DRG Nav1.8杂交信号较C组明显降低(P＜0.01),两组间差异无显著性(P＞0.05).APA-BCCs组扎侧触诱发痛阈值和CO2痛阈值高于CCI组和APA组结扎侧(P＜0.01),同时DRG中Nav1.8杂交信号较CCI组和APA组明显升高(P＜0.01),于C组差异无显著性(P＞0.05).结论 APA微囊化BCCs蛛网膜下腔移植可使CCI大鼠DRG中表达量下降的Nav1.8 mRNA恢复正常表达,APA微囊化BCCs蛛网膜下腔移植的镇痛作用和促进DRG中Nav1.8 mRNA表达恢复有关.     

微囊化牛嗜铬细胞移植对大鼠镇痛作用的研究

目的探讨海藻酸钠-多聚赖氨酸-海藻酸钠(APA)微囊化牛嗜铬细胞(BCC)蛛网膜下腔移植镇痛的作用机制和APA微囊的免疫隔离效果。方法取SD大鼠96只，随机分为空囊组(APA组)、微囊化细胞组(APA—BCC组)和裸细胞组(BCC组)，每组又分为4个亚组(2周组、4周组、6周组和8周组)．在三组大鼠的脊髓蛛网膜下腔分别植入含空囊的DMEM、微囊化BCC和单纯BCC。测定移植术后各组大鼠脑脊液灌流液中儿茶酚胺含量的变化、8周组大鼠对福尔马林刺激的行为学变化及脊髓后角c—fos基因表达的变化。结果微囊化细胞组和裸细胞组大鼠脑脊液中儿茶酚胺含量高于空囊组；微囊化细胞组大鼠的缩腿舔爪时间短于空囊组和裸细胞组，脊髓后角c—fos表达阳性神经元数目少于空囊组和裸细胞组。结论微囊化牛嗜铬细胞移植对福尔马林致痛大鼠有镇痛作用，可以提高脑脊液中儿茶酚胺的含量，可以抑制福尔马林致痛大鼠脊髓后角c—fos基因的表达。     

阿霉素减轻坐骨神经慢性缩窄性损伤大鼠的神经病理性疼痛及其机制

目的观察阿霉素(DOX)对坐骨神经慢性缩窄性损伤(CCI)模型大鼠的镇痛作用,并从形态学及组织凋亡蛋白的角度对其机制进行分析。方法将SD大鼠随机分为4组:假手术组(Sham)、CCI模型组(Model)、假手术+阿霉素5 mg·kg^-1组(Sham+DOX)、CCI模型+阿霉素5 mg·kg^-1组(Model+DOX)。造模成功后,各组采用尾静脉注射的方式给药,Sham组和Model组给予等量生理盐水,检测各组大鼠机械痛阈值和热痛阈值。在行为学检测结束后,即手术后d 15取大鼠右侧L4-5DRG,观察DRG细胞形态、超微结构及DOX的分布情况,采用Western blot法测定DRG组织中Bax、Bcl-2、PKCɑ、PKCδ及PKCε的蛋白表达。结果静脉注射DOX可在DRG组织检测到其自发荧光表达。与Sham组相比,Sham+DOX组痛阈值在整个观察期未见差别,而Model组在术后d 7痛阈值明显降低。与Model组相比,Model+DOX组的痛阈值在给药后明显回升,并表现出DRG细胞明显损伤,Bax/Bcl-2升高以及PKCδ、PKCε的蛋白表达量降低等现象。结论 DOX静脉注射可以到达并蓄积于DRG组织,明显减轻CCI大鼠的疼痛反应,这一作用与其降低PKCδ和PKCε的蛋白表达,诱导DRG的凋亡有关。     

神经病理性疼痛大鼠背根神经节TRPM8表达变化的研究

目的: 观察慢性神经病理性疼痛大鼠背根神经节TRPM8表达的变化.方法: 健康成年雄性SD大鼠72只,体质量250~280 g,随机分为慢性坐骨神经束缚性损伤(CCI)组和假手术组(n = 36), CCI或假手术前1 d,术后1、4、7、10、14 d每组各随机取6只大鼠,测定冷痛阈值、热痛阈值和机械痛阈值后立即处死大鼠,取L5背根神经节行免疫组织化学染色,观察瞬时受体电位melastatin 8(TRPM8)在慢性神经病理性疼痛下的表达变化.结果: CCI组于术后4 d术侧冷痛阈值、机械痛阈值和热痛阈值开始下降,至术后14 d仍处于较低水平,冷痛阈值和热痛阈值于术后10 d降至最低,机械痛阈值术后14 d降至最低(P ＜ 0.05或P ＜ 0.01);CCI组术侧L5背根神经节TRPM8的表达于术后4 d开始增加,术后10 d达到高峰,至术后14 d仍维持在高水平(P ＜ 0.05或P ＜ 0.01).结论: 背根神经节TRPM8受体表达的上调参与神经病理性疼痛的发生和维持.     

脊髓背角神经元上调Nav1.8通道参与缺血再灌注损伤后痛觉过敏的机制

目的观察鞘内注射钠通道抑制剂619C89对脊髓缺血再灌注损伤引起的痛觉过敏大鼠的痛阈、脊髓背角神经元中Nav1.8通道表达的影响。方法 SD大鼠随机分为3组:假手术组(S组)、痛觉过敏组(H组,缺血再灌注前3 d鞘内注射30μL生理盐水)、钠通道抑制剂组(I组,缺血再灌注前3 d鞘内注射619C895μg/30μL)。S组仅暴露主动脉弓而不结扎,其他各组开胸后无创动脉夹夹闭主动脉弓14 min后再开放,建立SCIRI引起的痛觉过敏模型。各组手术前均于L_(5-6)鞘内置管并连续注射3 d。术后1、3、5、7、14 d分别测定各组大鼠的热痛阈及机械性痛阈;取第4~6节腰段脊髓,采用免疫双荧光法观察背角神经元状态及其Nav1.8的表达,Real time-PCR法检测各组大鼠脊髓组织Nav1.8表达。结果与S组相比,术后各观察点(尤以第7天为著)H组大鼠热痛阈和机械性痛阈降低,损伤后7 d脊髓组织中Nav1.8 mRNA的表达增加(P<0.05);I组大鼠热痛阈和机械性痛阈值明显提高(P<0.05),脊髓组织中Nav1.8 mRNA的表达降低(P<0.05)。免疫双荧光染色显示,损伤后7 d,H组大鼠脊髓背角Nav1.8的荧光强度明显增加,且主要表达在NeuN表达阳性的神经元的胞浆中;且与S组相比,H组中NeuN/Nav1.8双阳性的细胞数量明显增多,而I组双阳性的细胞数量减少(P<0.05)。结论脊髓背角神经元通过上调Nav1.8通道参与缺血再灌注损伤后痛觉过敏的形成。     

坐骨神经慢性挤压伤大鼠背根神经节GAT-1的表达

目的 观察背根神经节(DRG)γ-氨基丁酸转运体-1(GAT-1)在大鼠坐骨神经慢性挤压伤(CCI)后的表达,探讨慢性神经痛的发生机制.方法 60只雄性SD大鼠随机分成空白对照组(Naive组)、假手术组(Sham组)和坐骨神经结扎组(CCI组,又分为结扎后3d组、7d组、14d组和28d组).分别在各自的时间点灌注取材L4～5背根神经节,用免疫组织化学方法观察GAT-1表达.结果 CCI组(3、7和14d)背根神经节GAT-1的免疫阳性神经元数和染色深度均比假手术组和空白对照组显著增加(P＜0.05和P＜0.01).结论 坐骨神经慢性挤压伤后背根神经节GAT-1表达增加,这可能与神经性疼痛的病理过程相关.     

鞘内注射美洛昔康对神经损伤大鼠背根神经节内GAP-43和NGF表达影响

目的研究鞘内注射美洛昔康(Mel)对CCI模型疼痛治疗效果和背根神经节(DRG)内生长相关蛋白-43(GAP-43)、神经生长因子(NGF)表达的影响。方法成年SD雄性大鼠随机分成四组:正常对照(C)组不做处理,CCI组不予注药,生理盐水(NS)治疗组予注射生理盐水,美洛昔康治疗(Mel)组予注射不同剂量美洛昔康(Mel组100μg,qd、Mel2组200μg,qd、Mel3组100μg,bid、Mel4组200μg,bid);每(亚)组均为5只大鼠。35只大鼠分别于术前及术后3d、7d测试机械性缩足反射阈值(MWT)和热缩腿潜伏期(TWL)。术后7d取材大鼠DRG,检测其GAP-43、NGF表达水平。结果MWT、TWL方面CCI组、NS组和Mel组较正常组显著下降(P0.05);美洛昔康组比CCI、NS组下降幅度小(P0.05),且剂量越大MWT、TWL下降幅度越小(P0.05)。GAP-43、NGF表达方面CCI组、NS组Mel组C组(P0.05),且美洛昔康剂量越大,表达越少。结论鞘内注射美洛昔康能明显减轻大鼠CCI模型的机械痛敏和热痛敏,并降低DRG内GAP-43、NGF的表达水平。     

坐骨神经慢性压迫损伤性疼痛与脊髓背角P物质关系研究

目的观察大鼠坐骨神经慢性压迫性损伤（CCI）后脊髓背角P物质表达的变化,探讨P物质在疼痛发生机制中的作用。方法SD雄性大鼠60只,随机分为：A组：CCI组（30只）;B组：对照组（30只）。术前及术后3、7、14、28 d分别测定大鼠热痛阈值、机械痛阈值和行为学评分。术后3、7、14、28d每组取4只,麻醉后用4%多聚甲醛灌注固定,取L4-6段脊髓,以备免疫组化,测定SP的变化。结果所有CCI动物从术后第3天起,出现明显的疼痛行为学改变和热痛阈值、机械痛阈值的降低,与对照组比较差异有统计学意义（P〈0.05或P〈0.01）。免疫组织化学结果表明,A组术后术侧明显高于B组（P〈0.05或P〈0.01）;A组术侧明显高于健侧（P〈0.05或P〈0.01）;而B组仅在第4天术侧高于健侧（P〈0.05）。结论慢性坐骨神经损伤后,脊髓背角SP的表达增加,而且表达增加与CCI大鼠的痛觉过敏、行为变化在时相上基本一致,说明CCI大鼠痛觉过敏与脊髓背角SP的表达增加有关。     

CD55蛋白的表达在大鼠神经病理性疼痛发病机制中的作用

目的 观察CD55蛋白在2种神经病理性疼痛模型（NPP）大鼠脊髓背角的表达情况,旨在探索它在NPP发病机制中的作用.方法 60只健康雄性SD大鼠随机分为假手术组、慢性坐骨神经缩窄损伤模型（CCI）组、保留性脊神经损伤模型（SNI）组,每组20只.分别于术前、术后1、3、7 d,用热刺激和机械刺激法测定大鼠的痛阈值变化,评估其可靠性,术后第7天处死大鼠,用Western-blot和免疫组化两种技术测定大鼠脊髓中的CD55蛋白表达情况.结果 CCI组和SNI组大鼠术后1、3、7 d痛阈值与本组术前比较显著降低,术后3、7 d较假手术组同时点有显著差异（P＜0.05）,模型制作成功.免疫组化染色显示：CCI组、SNI组大鼠脊髓背角CD55蛋白表达阳性细胞较假手术组显著减少（P＜0.05）.Western-blot检测显示,3组模型大鼠脊髓背角CD55蛋白表达发生不同程度降低,CCI组、SNI组与假手术组相比有统计学差异（P＜0.05）.结论 NPP大鼠脊髓背角CD55蛋白表达下降,可能是此处发生补体级联反应的重要原因,CD55在NPP的形成中发挥作用.     

川芎嗪对P2X_3受体介导的神经病理痛的作用研究

目的探讨川芎嗪(tetramethylpyrazine,TMP)对P2X3受体介导的神经病理痛的作用。方法建立大鼠坐骨神经慢性压迫性损伤(CCI)神经病理痛模型,应用vonFrey细丝法和热辐射法测定机械缩足反射阈值(mechanical withdrawal threshold,MWT)和热缩足反射潜伏期(thermal withdrawal latency,TWL),观察川芎嗪对CCI大鼠MWT和TWL的影响。结合免疫组织化学方法观察大鼠L4/L5段脊髓P2X3受体的表达变化。结果术后14d,Ⅴ组(CCI模型组)和Ⅰ组(生理盐水对照组)、Ⅱ组(TMP对照组)、Ⅲ组(假手术组)、Ⅳ组(CCI模型+TMP治疗组)相比较大鼠后爪的机械和热痛敏阈值明显降低(P<0.05),大鼠脊髓P2X3受体表达明显升高(P<0.05);Ⅰ、Ⅱ、Ⅲ、Ⅳ组之间相比大鼠后爪的机械和热痛敏阈值差异没有显著性(P>0.05),大鼠脊髓P2X3受体表达差异没有显著性(P>0.05);Ⅳ组脊髓P2X3受体的表达较Ⅴ组低(P<0.05)。结论川芎嗪对P2X3受体介导的神经病理痛有抑制作用。     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Aquatic Insects and Trace Metals: Bioavailability,Bioaccumulation, and Toxicity

Abstract

The uptake of metals from food and water sources by insects is thought to be additive. For a given metal, the proportions taken up from water and food will depend both on the bioavailable concentration of the metal associated with each source and the mechanism and rate by which the metal enters the insect. Attempts to correlate insect trace metal concentrations with the trophic level of insects should be made with a knowledge of the feeding relationships of the individual taxa concerned. Pathways for the uptake of essential metals, such as copper and zinc, exist at the cellular level, and other nonessential metals, such as cadmium, also appear to enter via these routes. Within cells, trace metals can be bound to proteins or stored in granules. The internal distribution of metals among body tissues is very heterogeneous, and distribution patterns tend to be both metal and taxon specific. Trace metals associated with insects can be both bound on the surface of their chitinous exoskeleton and incorporated into body tissues. The quantities of trace meals accumulated by an individual reflect the net balance between the rate of metal influx from both dissolved and particulate sources and the rate of metal efflux from the organism. The toxicity of metals has been demonstrated at all levels of biological organization: cell, tissue, individual, population, and community. Much of the literature pertaining to the toxic effects of metals on aquatic insects is based on laboratory observations and, as such, it is difficult to extrapolate the data to insects in nature. The few experimental studies in nature suggest that trace metal contaminants can affect both the distribution and the abundance of aquatic insects. Insects have a largely unexploited potential as biomonitors of metal contamination in nature. A better understanding of the physico-chemical and biological mechanisms mediating trace metal bioavailability and exchange will facilitate the development of general predictive models relating trace metal concentrations in insects to those in their environment. Such models will facilitate the use of insects as contaminant biomonitors.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.     

Alzheimer’s disease – current trends in basic and clinical research. Highlights from the 16th ECNP

Advances in the molecular biological knowledge of neuronal nicotinic acetylcholine receptors (nAChRs) have led to a growing interest by the pharmaceutical industry in the development of novel compounds that selectively modulate nAChR function. The ability of (-)-nicotine, an activator of nAChRs, to enhance attentional aspects of cognition in animals and humans, to exert neuroprotective and anxiolytic-like effects, and presumably to mediate the negative correlation between smoking and Alzheimer's (and Parkinson's) Disease, has focused interest on the potential therapeutic utility of modulators of nAChR function for treatment of some of the deficits associated with these progressive, neurodegenerative conditions. Numerous compounds are known which activate nAChRs and which might serve as lead compounds toward the development of such agents. The pharmacologic diversity of neuronal nAChR subtypes suggests the possibility of developing selective compounds which would have more favourable side-effect profiles than existing agents. This broader class of agents, collectively called cholinergic channel modulators (ChCMs), is anticipated to encompass compounds which would have more favourable side-effect profiles than existing agents, which generally exhibit low selectivity. This selectivity may be achieved by preferentially activating some subtypes of nAChRs (i.e., Cholinergic Channel Activators, ChCAs) or inhibiting the function of other subtypes (Cholinergic Channel Inhibitors, ChCIs). An overview of the biology of nAChRs and the rationale for the use of ChCMs for the treatment of dementia related to neurodegenerative diseases are presented, followed by a discussion of lead compounds and compounds under consideration for clinical evaluation.