MS-302三道测量分析系统的应用及HI-6在离体膈神经-膈肌上抗梭曼的作用机制

目的 呼吸肌麻痹是有机磷酸酯类化合物严重中毒致死的主要原因之一,由于膈肌是最主要的呼吸肌,因此,建立一种精确灵敏的实验方法评价药物对离体膈神经 膈肌的影响, 探讨相应的抗呼吸肌麻痹药物的实验显得尤为重要。本文在MS-302三道测量分析系统上探讨了抗神经毒有效化合物HI-6在离体膈神经-膈肌上抗梭曼的作用机制。方法 采用MS-302三道测量分析系统,观察大鼠离体膈神经-膈肌的收缩功能,此方法较既往测量方法实时、高效而灵敏。观察膈肌收缩功能的同时,待标本稳定30 min后取膈肌置-20℃冰箱,留待测定正常乙酰胆碱酯酶(AChE)活性,然后向麦氏槽中加入以台式液配置的药物,加入药液量一般不超过0.5 ml,使药物浓度为10 μmol·L^-1～1 mmol·L^-1, 对照组加入等量的生理盐水, 于加药后不同时间点留取膈肌样品测定AChE活性,观察药物对膈肌AChE活性的直接影响。结果 ① MS-302三道测量分析系统上,对膈神经-膈肌标本强直收缩曲线下的面积进行测量,能较为直接的反映膈神经-膈肌标本的生理功能,且结果精确。② HI-6 10和100 μmol·L^-1对正常膈神经-膈肌收缩功能影响不明显,当药物浓度为1 mmol·L^-1时, HI-6对正常膈神经-膈肌收缩功能有抑制作用;HI-6 10 μmol·L^-1对梭曼抑制的标本收缩功能既没有保护作用也没有拮抗作用; HI-6 100 μmol·L^-1有一定的保护和拮抗作用;当剂量增加到1 mmol·L^-1时,HI-6的保护作用和拮抗作用都明显增强,且保护作用在30 min内最佳。③ 预先给予HI-6不能减弱梭曼对大鼠离体膈肌AChE的抑制,提示HI-6对梭曼抑制的大鼠离体膈肌AChE无明显保护作用;梭曼中毒后给予HI-6对梭曼抑制的膈肌AChE活性无显著影响,提示HI-6对梭曼抑制的膈肌AchE无明显重活化作用。结论 HI-6对梭曼抑制的离体膈神经-膈肌收缩功能的恢复可能是直接生理对抗作用,在本实验条件下未见膈肌AChE活力有明显的恢复。     

HI-6对正常及梭曼抑制大鼠离体膈神经-膈肌标本收缩功能的影响

目的　探讨HI 6对抗梭曼中毒所致呼吸抑制重活化作用外的其他可能作用机制。方法　采用MS 30 2三道生理记录仪 ,建立大鼠离体膈神经 膈肌收缩标本 ,观察HI 6对梭曼中毒膈神经 膈肌标本收缩功能的影响。结果　①梭曼 (2mg·L- 1)中毒后 ,膈肌收缩功能明显下降 ,中毒 10min内强直收缩曲线下面积下降至最低点 ,且在 3h内无明显恢复。②梭曼中毒前给予不同浓度的HI 6 (0 .0 1,0 .1,1.0mmol·L- 1) ,发现HI 6 (0 .1,1.0mmol·L- 1)对梭曼中毒所致膈肌强直收缩具有明显保护作用 ,且保护作用随着浓度的增加逐渐增强。③梭曼中毒后再给予HI 6 (0 .0 1,0 .1,1.0mmol·L- 1) ,发现 0 .0 1mmol·L- 1HI 6对中毒大鼠离体膈肌收缩功能无任何拮抗作用 ;当浓度增至 0 .1mmol·L- 1时 ,对膈肌强直收缩有一定的拮抗作用 ,但无统计学意义 ;但 1.0mmol·L- 1HI 6可显著对抗梭曼中毒所致膈肌收缩功能下降。结论　HI 6对梭曼中毒所致大鼠离体膈神经 膈肌收缩功能抑制具有明显拮抗作用 ,提示HI 6对抗梭曼中毒所致呼吸抑制作用机制存在酶保护作用外的直接生理对抗作用     

MS-302三道生理信号测量分析系统应用及神经刺激装置的改进

目的 呼吸肌麻痹是有机磷酸酯类化合物严重中毒致死的主要原因之一，由于膈肌是最主要的呼吸肌，因此，建立在离体或在体膈神经-膈肌标本上寻找相应的抗呼吸肌麻痹药物的实验显得尤为重要。以往观察离体膈神经-膈肌标本的实验，多是在记纹鼓或平衡记录仪上进行的。它们均有一定的缺点。为此，我们率先引进了MS-302三道生理信号测量分析系统，该系统具有精确性高、可控性强及灵敏度好等优点，很好的满足了实验的要求。此外，实验中我们发现MS-302三道记录仪系统中的神经刺激器存在一定的局限性。如该刺激器只存在刺激膈神经的电极，只能刺激膈神经而不能刺激膈肌；只能用于研究整个膈神经-膈肌肌肉系统的功能状态，不能对神经-肌肉接头及膈肌本身的功能状态进行分别观察。针对上述缺点，我们对原有的单功能刺激电极进行了改进，改进后的刺激电极，可对肌肉和神经同时，分别或交替进行连续或不连续的刺激，并能将两者明确分开，可广泛应用于医学和生物学领域的各种实验，检测、生产和临床医疗。上述技术革新成果已成功申请专利(专利号为02247761．6)。     

HI-6对梭曼染毒大鼠膈肌胆碱酯酶的重活化作用

目的研究HI-6对膈肌局部梭曼染毒大鼠膈肌胆碱酯酶的重活化作用。方法将大鼠麻醉后固定,沿腹正中线切开腹腔,暴露膈肌。梭曼局部染毒膈肌1和10min后,局部或全身给予HI-6,于给药后24和72h处死大鼠,取出中毒部位膈肌。用硫胆碱酶法显示终板的AChE活性,观察HI-6对胆碱酯酶的重活化作用。结果膈肌局部梭曼中毒1和10min后．局部给予HI-624和72h对局部中毒膈肌胆碱酯酶均没有重活化作用．在光镜下观察不到棕褐色椭圆形的运动终板：而膈肌局部梭曼中毒1和10min后,全身给予HI-6对局部中毒膈肌胆碱酯酶有明显的重活化作用,在光镜下可见到肌纤维上又出现了棕褐色椭圆形的运动终板。结论HI-6全身给药对早期膈肌局部中毒的神经运动终板胆碱酯酶有重活化作用。     

HI-6对梭曼中毒活体膈肌胆碱酯酶的重活化作用

目的 研究HI-6对局部梭曼中毒活体膈肌胆碱酯酶(ChE)活性是否具有重活化作用。方法 大鼠戊巴比妥钠肌注40 mg·kg^-1麻醉后固定,沿腹正中线切开腹腔,暴露膈肌。以右膈神经穿过膈肌处为中心,在右侧膈肌上贴敷一片大小为0.5 cm×0.75 cm的棉纸。用微量进样器抽取10 μl 0.2%梭曼生理盐水溶液滴加其上。左侧膈肌为自体正常对照或全身系统给予HI-6的药物对照,中毒后暂闭腹腔,30 min后取出棉纸。实验分为两组：一组在大鼠膈肌局部梭曼中毒后1 min和10 min后肌注HI-6 144 mg·kg^-1;一组在大鼠膈肌局部梭曼中毒后1 min和10 min后局部给予180 ml（1 mmol·L^-1） HI-6, 分别于给药后24和72 h处死大鼠, 取出中毒部位膈肌,按Karnovsky等改良的硫胆碱法显示终板的AChE活性。将膈肌标本在4%甲醛2~4℃固定24 h后,冻切30 μm,37℃孵育30 min,在光镜下观察。结果 膈肌局部梭曼中毒后1 min和10 min后局部给予HI-6,24和72 h对局部中毒膈肌ChE均没有重活化作用,在光镜下均观察不到棕褐色椭圆形的运动终板;而膈肌局部梭曼中毒后1 min和10 min后,全身给予HI-6对局部中毒膈肌ChE有明显的重活化作用, 在光镜下可见到肌纤维上又出现了棕褐色椭圆形的运动终板。结论 HI-6对早期膈肌局部中毒的神经运动终板ChE有重活化作用。     

在不均匀牵拉的离体大鼠膈肌标本上梭曼的突触前和突触后作用

在离体大鼠膈肌上制备了一种不均匀牵拉肌肉以制动,便于用微电极记录终板电位(EPP)的标本(INSMP),避免了常规制动方法带来的制动药物、台氏液Ca~(2+),Mg~(2+)浓度改变或钳压肌肉导致膜电位(MP)下降的种种干扰。INSMP的MP和小终板电位(MEPP)正常,EPP幅度高达30mV以上,是研究药物对接头作用的较好标本。在INSMP上,梭曼(5.5μM)使MEPP频率加快,串刺激(50Hz)诱发的平均串EPPs幅度及其平均ACh 量子含量减少80％和77％。小剂量三碘季铵酚和六烃季铵可部分对抗梭曼的作用。梭曼引起强直收缩抑制主要原因为终板区蓄积的ACh作用于突触前N受体、负反馈地抑制ACh 量子释放;次要原因为作用突触后N受体、使其对ACh敏感性降低。     

离体大鼠膈肌标本上梭曼引起终板区持续性去极化的机理分析

在离休大鼠膈肌标本上，细胞内记录终板区微电泳给乙酰胆碱（ＡＣｈ）的电位，以ＡＣｈ电位幅度为突触后反应性指标，观察了梭曼中毒后间接串刺激产生终板区持续性去极化反应的性质并分析了其产生的机理。持续性去极化过程中不仅串终板电位消失，ＡＣｈ电位也消失，持续性去极化之后ＡＣｈ逐渐恢复。在正常大鼠和肉毒毒素Ａ中毒大鼠膈肌标本上，梭曼中毒后，串ＡＣｈ电位（５－１０Ｈｚ）也可诱发与持续性去极化性质相同的去极化反应，结果表明，持续性去极化主要是突触后Ｎ型ＡＣｈ受体对高浓度ＡＣｈ的一种反应，不支持突触前ＡＣｈ“再生性”释放的假说。     

罗哌卡因与左布比卡因角袋注射对锁骨上臂丛神经阻滞患者膈肌功能及术后疼痛介质表达的影响

目的 探讨罗哌卡因与左布比卡因角袋注射对锁骨上臂丛神经阻滞（SCBPB）患者膈肌功能及术后疼痛介质表达的影响。方法 选取2020年1月至2021年6月医院80例SCBPB患者作为研究对象,依据随机数字表法分为2组,各40例。对照组予以左布比卡因角袋注射,观察组予以罗哌卡因角袋注射。观察并比较2组神经阻滞情况、术后镇痛泵及舒芬太尼使用情况、膈肌麻痹情况、不良反应发生情况及心肌指标[血清肌钙蛋白（CTn I）和肌酸激酶同工酶（CK-MB）]水平。结果 观察组感觉阻滞及运动阻滞起效时间均较对照组长,感觉阻滞及运动阻滞持续时间均较对照组短（P <0.05）;2组首次臂丛神经阻滞成功率相比,差异无统计学意义（P> 0.05）。观察组术后首次按压镇痛泵时间短于对照组（P <0.05）,阻滞30 min平静及用力呼吸时膈肌总麻痹率低于对照组（P <0.05）,给药后24 h血清CTn I、CK-MB水平均低于对照组（P <0.05）。2组不良反应发生率经比较,差异无统计学意义（P> 0.05）。结论 罗哌卡因与左布比卡因用于锁骨上臂丛神经阻滞均可达到良好的镇痛与麻...     

A comparison of the oximes HS-6 and HI-6 in the therapy of soman intoxication in rodents

The bisquaternary mono-oximes HS-6 and HI-6 may both be considered as potential therapeutic agents for soman intoxication. It has been found that HI-6 was superior to an equal dose of /S-6 in the treatment of soman intoxication in mice and rats. Not only did HI-6 protect against higher levels of soman, but also fewer "delayed deaths" were seen following HI-6. In anesthetized atropinized rats, the administration of HI-6 resulted in a longer duration of spontaneous breathing and a higher degree of recovery of neuromuscular function than did the use of HS-6. HS-6 interacted with soman to alter the rate of excretion of the oxime. This problem was not encountered when HI-6 was used.     

HI-6 therapy of soman and tabun poisoning in primates and rodents

The bis-pyridinium oxime HI-6, in conjunction with atropine, was found to offer significant protection against multiple LD₅₀ challenges with the organophosphorus compounds soman and tabun. In adult rhesus macaques, the therapeutic administration of HI-6 with atropine and diazepam protected three of four animals from the lethal effects of 5 × LD₅₀ of soman and three of three animals from 5 × LD₅₀ of tabun. However, when toxogonin was substituted for HI-6 in the therapeutic mixture, all three animals poisoned with 5 × LD₅₀ of soman died. In rats, the 24 h protective ratios against tabun and soman with HI-6 were 2 and 3.5, respectively, whereas in guinea pigs these values were between 4 and 6 for both agents. No evidence was obtained for acetylcholinesterase (AChE) reactivation by HI-6 in tissue from tabun-poisoned rodents or following soman or tabun in primate plasma. The results underscore the significant therapeutic benefit of HI-6 in primates, a species specific efficacy against tabun, and argue for some mechanism of action of HI-6 at least partly unrelated to AChE reactivation.     

Cardiopulmonary effects of HI-6 treatment in soman intoxication

The cardiopulmonary effects of HI-6, together with atropine and soman, were studied in the rat. HI-6 is an effective antidote in acute poisoning with the nerve agent soman. The therapeutic efficiency of HI-6 is still unclear and cannot be explained entirely by the HI-6 reactivating ability of acetylcholinesterase (AChE). Other non-cholinergic factors must be involved. One possible detoxifying process might be an effect of HI-6 on the blood flow to sensitive organs. The purpose of the present study was to investigate 1) whether soman per se induces changes in regional blood flow and 2) whether the blood flow to different organs is affected when HI-6 (50 mg x kg(-1) i.m.) and atropine (10 mg x kg(-1) i.m.) are given either before or immediately after soman intoxication (90 microg x kg(-1) s.c.). For regional blood flow determinations the microsphere method was used with male Wistar rats weighing 300-400 g. The rats were anaesthetised and breathed spontaneously during the experiment. Three different blood flow measurements were made in the same animal and concomitant physiological parameters such as mean arterial blood pressure and respiratory rate were recorded. The blood AChE activity was followed throughout the experiment. Our results show that when HI-6 is given after intoxication with soman, dramatic changes in blood flow occur with a significant decrease in both respiratory rate and blood AChE activity. If HI-6 is given prior to the intoxication, however, all rats are unaffected and none of the parameters measured are changed.     

丁酮肟及酰胺磷定对大鼠血液中梭曼消除的影响

采用电鳐胆碱酯酶抑制法测定大鼠血液中梭曼残留浓度 ,考察了丁酮肟 (DAM)及酰胺磷定 (HI 6)对大鼠血液中梭曼消除的影响 .结果表明 ,4 15μg·kg- 1梭曼 ( 5LD50 )iv后 3min ,DAM预处理有加速梭曼在大鼠血液中消除的趋势 ,使血中梭曼浓度由( 86± 2 2 ) μg·L- 1下降到 ( 53± 9) μg·L- 1,这可能与DAM加速了大鼠体内主要解毒器官中膦酰化羧酸酯酶 (CaE)的脱膦酰化速率有关 ,使有限的酶分子结合了更多的梭曼 .相比之下 ,HI 6预处理则对大鼠血液中梭曼浓度没有明显影响 .提示在大鼠体内 ,CaE对梭曼的解毒作用比胆碱酯酶更为重要     

Mechanism of action of HI-6 on soman inhibition of acetylcholinesterase in preparations of rat and human skeletal muscle; comparison to SAD-128 and PAM-2

HI-6 is presently considered the most potent oxime antidote against soman poisoning in mice, rats, dogs and monkeys. However, it is still an open question whether efficiency of HI-6, observed in experimental animals, can be extrapolated to soman intoxicated humans. In this paper efficiency of HI-6 and possible mechanisms of action were compared in rat and human fresh muscle preparations. In rat muscle, about 50% of control AChE activity could be recovered by both therapeutic (5 min after soman) and prophylactic (5 min before soman) application of HI-6. On the other hand, in human muscle therapeutic treatment restored only 5%, while prophylactic application of HI-6 again resulted in about 50% recovery of control AChE activity. As revealed by comparison of the prophylactic effects of HI-6 and the non-oxime bispyridinium compound SAD-128, competitive inhibition of AChE plays a minor role as a protective mechanism. Immediate reactivation of rapidly aging human AChE must therefore be instituted for successful protective treatment by HI-6. Retardation of aging, a direct effect of SAD-128, was roughly estimated to improve reactivation by HI-6 for about 10% of control AChE activity of the human muscle. PAM-2 proved completely inefficient as a therapeutic and as a prophylactic agent on both rat and human muscle preparations.     

The pharmacokinetics of HI-6 in beagle dogs

The pharmacokinetics of HI-6 were studied following intravenous administration to beagle dogs (n = 7). The bioavailability of two different strength intramuscularly administered doses was also determined in the same animals. After a 20 mg kg-1 intravenous dose, the mean (+/- S.D.) initial HI-6 plasma concentration was 93.1 +/- 10.8 micrograms ml-1. The mean half-life was 48.2 +/- 17.7 min, the mean total body clearance was 5.16 +/- 0.81 ml min-1 kg-1, the mean apparent volume of distribution was 0.37 +/- 0.20 l kg-1 and 61.2 +/- 14.6 per cent of the dose was excreted as unchanged drug. The pharmacokinetic constants calculated following the 20 mg kg-1 intramuscular doses of 250 and 25 mg ml-1 solutions were not significantly different from those obtained following the intravenous dose. Also, the areas under the plasma concentration versus time curves were not significantly different indicating 100 per cent bioavailability from the intramuscular route of administration.     

Non-reactivating effects of HI-6 on hippocampal neurotransmission

 Effects of the oxime HI-6, unrelated to reactivation of acetylcholinesterase (AChE), on field potentials in the dentate gyrus of the rat hippocampus following AChE inhibition, were investigated both in vitro and in vivo. In hippocampal slices, AChE inhibition decreased the perforant path evoked population spike amplitude (PSA). This effect could be prevented by pre-incubation of the slices with atropine (0.1–1 μM) or with the M₁ muscarinic receptor antagonist pirenzepine (1 μM). A similar preventive effect was found after pre-incubation with the GABA_A antagonist picrotoxin (20 μM), suggesting that the effects of AChE inhibition in vitro may be due to an enhancement of GABAergic inhibitory activity via activation of M₁-muscarinic receptors. The effects of AChE inhibition in vivo were variable; both increases and decreases of the PSA were found. Following AChE inhibition, HI-6 increased the PSA dose-dependently, both in the in vivo and in the in vitro hippocampus. At higher oxime doses the perforant path stimulation elicited multiple population spikes. The effects of the oxime were presumably not mediated by an antagonism of cholinergic receptors, since they could not be mimicked with cholinergic antagonists like atropine, mecamylamine or gallamine. Further testing of the nature of the HI-6 effect in hippocampal slices in vitro, using a paired antidromic-orthodromic stimulation protocol, showed that HI-6 may interfere with GABAergic inhibition. Received: 21 March 1994 / Accepted: 13 June 1994     

Effect of poisoning by soman (pinacolyl methylphosphonofluoridate) on the serum half-life of the cholinesterase reactivator HI-6 in mice

The effect of fasting, atropine, and poisoning by an organophosphate anticholinesterase soman (pinacolyl methylphosphonofluoridate) on the pharmacokinetics of the acetylcholinesterase oxime reactivator HI-6 (CAS Reg. No. 34433-31-3; 1-[(4-(aminocarbonyl)pyridinio)methoxy)methyl)-2-(hydroxy imino)methyl) pyridinium dichloride) was investigated. Pharmacokinetic parameters (elimination half-life, volume of distribution, clearance rate) were determined for the following groups: (1) a 20 and 50 mg kg-1 dose of HI-6; (2) a 50 mg kg-1 dose of HI-6 after fasting for 18 h (water ad lib); (3) a 50 mg kg-1 dose of HI-6 at 0, 4, and 24 h after atropine (17.4 mg kg-1, i.p.) and soman (287 micrograms kg-1, s.c.); and (4) a 50 mg kg-1 dose of HI-6 at 0 and 4 h after soman (100 micrograms kg-1, s.c.). Fasting increased significantly (p less than 0.05) the elimination of half-life (t1/2) and tended to increase the volume of distribution (Vd) and decrease the clearance rate (CL). Following soman (287 micrograms kg-1) poisoning the t1/2 of HI-6 increased from 8.6 min to 21.6 min and the Vd increased to 0.731 kg-1. At the lower soman dose (100 micrograms kg-1) no significant effect on HI-6 pharmacokinetics was found. Atropine (17.4 mg kg-1: i.p.) pretreatment increased the t1/2 and CL while having no effect on the Vd. By 24 h the pharmacokinetic parameters of HI-6 in the various treatment groups were not significantly different from the control group. The changes in the pharmacokinetics of HI-6 following soman and atropine are probably the result of haemodynamic changes.     

梭曼对大鼠周围消化功能的影响

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