摘要
有机磷中毒引起的迟发性神经病(0PIDN)发生在急性中毒后l~2
周,表现为感觉异常和运动障碍,是严重的中毒性神经病。以往许多文
献报告0PIDN选用大剂量B族维生素、ATP、地巴唑、硝酸一叶秋碱治疗
以及一些中药制剂,但疗效不确切。目前普遍认为糖皮质激素可以加速
0PIDN的恢复,但副作用大,禁忌症较多,因此寻找一种安全有效的治疗
药物迫在眉睫。三磷酸胞苷二钠(CTP)是核苷酸衍生物,为辅酶类药,
在体内参与磷脂的合成代谢,能提高神经细胞生物膜结构的稳定性和重
建能力,对心肌和脑组织有营养再生和修复作用,该药对0PIDN的肌肉
病变是否有保护作用,目前尚未见报道。本研究用甲胺磷使leghorn鸡
染毒,采用肌肉病理组化分析和超微结构观察,了解甲胺磷中毒后神经
肌病的肌肉病理变化特点,明确地塞米松、CTP对0PIDN是否有保护作用。
Leghorn鸡l05只,按体重随机分为5组,正常组、地塞米松组、大
剂量CTP组、小剂量CTP组及盐水对照组。用甲胺磷使动物染毒后制成
迟发性神经肌病的模型,方法如下:断食24小时后用植物油溶解的甲胺
磷(10mg/kg)按2m1/kg体积通过套管直接灌入鸡素囊中,染毒的鸡用
阿托品保持度过急性期,染毒前20分钟8 mg/kg肌注,染毒后3、6、l0
小时4 mg/kg肌注。从染毒后第二天开始各组均连续给药一周,所有药
物均按照1ml/kg肌注,地塞米松组每日1 mg/kg体重,CTP剂量以体表
面积成人剂量计算,CTP用生理盐水稀释,小剂量cTP组每日1.5 mg/kg
体重,大剂量CTP组每日3.0 mg/kg体重,盐水对照组用0.9%生理盐水
每日按lml/kg体重肌注。
染毒后观察鸡流涎、流泪、呼吸、瞳孔、肌束颤动、步态等症状变
化,每周称重,并每日观察迟发神经毒性的临床体征,临床0PIDN的分
级标准按照l987年R0bertson的分级标准:0级一没有体征;1级一步
态异常;2级一动物用后臀休息但还能站立;3级一完全瘫痪。染毒后l、
2、3、4、8周末在水合氯醛腹腔深麻醉后,将Leghorn鸡固定于实验台
上,进行肌肉活检,取鸡小腿腓肠肌准备制成病理标本,肌肉标本在手
术台上即分为两部分,大组织块用液氮冷冻到一160℃,再放入一20℃恒
冷切片机切片,切片的厚度一般为8um,连续切片,在常温下自然干燥
10—20分钟后,进行常规苏木素一伊红染色(HE)、改良Gomorri三色染
色、还原型辅酶工一四唑氮还原酶染色(NADH—TR)、ATP酶染色。小组织
块在手术台上取出l×l×lmm,约米粒大小,直接放入2.5%戊二醛中固
定,修块后包埋,超薄切片进行电镜观察。
本实验结果显示鸡经甲胺磷染毒后用地塞米松治疗组第l、2周与对
照组的肌肉病理改变未见明显差异,第3、4、8周肌肉坏死部分面积比
例明显小于盐水对照组,而且第3周I、II型纤维萎缩程度也小于盐水
对照组,说明激素可以减轻症状,加速恢复。大剂量CTP组肌肉坏死部
分面积比例在第3周也明显小于盐水对照组,此时I型纤维萎缩程度轻
于对照组,说明CTP对中毒后肌肉损害有一定的保护修复作用,但在第4、
8周与对照组比较病理改变无明显差异(P>0.05)。
人们对0PTDN的研究已经超过70年,动物实验及人体肌肉活检均证
实0PIDN的病理改变主要为周围神经脱髓鞘,轴索肿胀,肌肉变性坏死,
中毒性肌病和中毒性神经病同时存在。甲胺磷近年广泛使用的有机磷杀
虫剂,又名多灭磷或克螨隆(Methamidophos/Tamaron),化学名0,S一
二甲基一硫代磷酰胺(0,S—dimethyl phosphoramidothiolate),高毒,
属硫代磷酸脂类化合物。甲胺磷中毒性肌病的发病机理尚不清楚,推测
其发病过程可能既有甲胺磷对横纹肌细胞的直接细胞毒作用,亦有甲胺
磷酰化,形成老化磷酰化酯酶(半抗原--载体蛋白)成为完全性抗原,
产生细胞免疫反应,横纹肌为靶细胞,致发生变性、坏死等超微结构的
改变,特别是使线粒体变性、萎缩或消失、肌浆网扩大等。线粒体是细
胞内能量转运的重要细胞器。线粒体变性、空泡化、脊凝改变及肌浆网
扩张,导致横纹肌细胞代谢障碍。
激素起作用的原理可能是通过抑制中毒患者体内毒性物质转化、毒性
物质对神经肌肉组织的直接损伤或者通过增强线粒体膜,加速糖原颗粒形
成而起到减轻中毒后神经肌肉损害和促进病损组织恢复,使得迟发神经肌
病提前治愈。但激素组动物同样出现0PIDN,说明激素虽然可以减轻症状,
促进恢复,却并不能防止0PIDN的出现。目前国内外尚无CTP用于有机磷
中毒后保护迟发性神经肌肉损害的报道,起作用的原理可能有两方面,一
是由于CTP为辅酶类药,在体内参与磷脂的合成代谢,可以提供能量,提
高肌肉细胞抗损伤的能力,对肌肉组织有营养再生和修复作用。二是由于
CTP是核苷酸衍生物,是核酸代谢的中间产物和能量来源,能提高神经细胞
生物膜结构的稳定性和重建能力,提高神经细胞抗损伤能力,促进神经突
起的再生长,从而能营养肌肉细胞,提高抗损伤的能力。
本实验1周后出现周围神经病步态改变的发病率为20%左右,第4
周时所有存活的动物
OPlON ,induced by the organophosphorus compounds,is a severe toxic neuropathy,which behaves the symptom of paresthesia and akinesis after acute poisoning 1 or 2 weeks.Many literature reported that big dosage vitamine B family ,ATP(adenosine triphosphate),bendazol,securinine and some Chinese traditional medicine preparations can cure OPIDN .however, the curative effect is not reliable.lt is urgent to find one safe and effective medcine to cure OPIDN.though it is believed that the glucocorticoid can quicken up the recovery of OPIDN.it has many side-effects and tabus.CTP (Cytidine disodium Triphosphate) .which is nucleotide ramification and the coenzyme medicine, participates in the lecithoid anabolism in the body , at the same time, can boosts the stability and the rebuiding capacity of the cell membrane.CTP also can regenerate and reedify the myocardium and encephalon tissue.This research ,poisoned the white leghorn hens by the methamidophos and made use of the muscle histochemical analysis and the ultrastructure evaluation, is to comprehend the characteristics of the neuropathy's muscle pathology poisoned by the methamidophos and make it certain that the dexamethasone and CTP has curative effect to the OPIDN whether or not.
There are 105 white Leghorn chicken ,and we divided them into 5 groups randomly according to the body weight .that is the normal group.dexamethasone group.big dosage CTP group.little dosage group and the brine contrast group.After poisoned the hens by the methamidophos.we made them into the OPIDN.The method is just as follows: methamidophos (10mg/kg) were dissolved in com oil and administered in volume of 2ml/kg by oral cannula directly into the crops of hens fasted for 24 hous.and
the poisoned hens received atropine sulfate, 8mg/kg im,20 minutes before poisoning and 4mg/kg 3,6,10hr afterwards.From the second day of the poisoning .muscle injection had been given to every groups for 1 weeks in volume of 1 ml/kg. Dexamethasone group received 1mg/kg everyday , the CTP was dissolved in physiological
saline.little dosage CTP group received 1.5mg/kg everyday.big dosage CTP group received 3.0mg/kg,the brine contrast group received physiological saline alone.
Live hens in each groups were observed daily for clinical signs such as salivation, lacrimation, respiration, pupil, fascicle tremors,gait,and hens were weighed in every week,meanwhile,we scored signs of the OPIDN every day following the designations of Robertson in 1987:0 level —no signs,1 level—gait abnormalities, 2level—animal observed resting on haunches but the abilty to stand still remains,3level—complete prostration. At the weekend of 1,2,3,4,8 week after poisoning.the hens were made celiac deep anaesthesia by chloral hydrate.and then we fixed the leghorn hens to the labortaory desk to make the muscle biopsy.after that ,we choosed the gastrocnemius muscle of the calf to make the pathology specimen.which would be divided into 2 parts on the laboratory desk.the big tissue was freezed to — 160°C in the liquid nitrogen ,then sliced up under — 20°C in cryomat ,the thickness of each slice is 8 um.after desiccated for about 10~20minutes under normal temperature.we take the Eaenatoxylin-Hosin stain, improved Gomorri tripcolour stain, NADH-Tetrazolium Reductase stain.Adenosine Triphosphatase stain.The small tissue was taken out 1x1x1 mm from the laboratory desk just a grain size.and then fixed it in the 2.5% glutaraldehyde .after that embedded .sliced and observed under the electron -microscope.
The result of the research shows that the muscle's pathology change of hens .poisoned by methamidophos, cured with dexamethasone manifests no more
evidence than the brine contrast group at the first and the second week.meantime, the percentage of muscle necrosis area is much smaller than the brine compared group at the third.fouth and eighth week.and the I and II type fibre atrophy extent is much smaller than the brine compared group.AII these illuminate that glucocorticoid can alleviate symptom.quicken up recovery.At the third w
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