Pathogenesis of neuromuscular synaptic dysfunction and transmission failure in organophosphate toxicity

每年，在世界各地，成千上万的人死于有机磷农药中毒。其他人群面临恐怖分子使用OP神经毒剂的风险（如最近在中东使用的）。这些化合物会干扰大脑和肌肉中的信号，导致呼吸衰竭时瘫痪和死亡。然而，如果病人及时到达医院，他们可以接受治疗：首先使用解毒剂，然后在重症监护病房通过机械通气辅助呼吸。不幸的是，由于未知的原因，一些患者无法自己呼吸数周，使他们面临致命并发症的危险，如肺炎。我们正试图揭示OP中毒导致瘫痪和呼吸停止的机制。我们首先建立动物中毒模型，重点关注一种特殊的农业有机磷农药，乐果EC40。这种杀虫剂含有两种基本成分：乐果本身和一种有机溶剂环己酮。这些物质在体内通过代谢被分解：乐果在肝脏转化为乐果；环己酮在胃和肠中转化为环己醇。到目前为止，我们的调查让我们有了一个惊人的发现：两种代谢物，即乐果和环己醇，对运动神经元和肌肉细胞之间的神经-肌肉连接处的毒性比杀虫剂本身的任何一种主要成分都要大得多。乙酰胆碱是一种神经递质分子，它将信号从运动神经末梢传递到这些神经肌肉连接处的肌肉。我们的数据强烈表明，氧乐果和环己醇共同作用，直接影响位于神经肌肉连接处肌肉侧的乙酰胆碱蛋白受体。在这个项目中，我们将以三种协调一致的方式关注和研究这一理论。首先，我们将把乙酰胆碱受体蛋白的人类DNA编码注射到组织培养的细胞中。这些细胞会将DNA转化为可工作的受体，使我们能够使用一种称为“贴片夹”的精密电子技术进行录音。通过这种方式，我们可以直接测试和测量氧乐果、环己醇或两者一起如何影响受体分子对乙酰胆碱的反应。其次，我们将使用一种新型的组织培养试验，测量在24小时内，氧乐果和环己醇对神经肌肉连接处传递的进行性毒性，这段时间通常是患者病情恶化的时间。为了进行这些测量，我们将利用一种小鼠（称为wld）的神经和肌肉，这种小鼠的神经肌肉连接通常对神经损伤具有相当的抵抗力。这个特征给了我们足够的时间来找出为什么农药及其代谢物会导致肌肉逐渐瘫痪。这个实验也给我们提供了有节奏地刺激神经（就像我们用来呼吸的肌肉自然发生的那样）的选择，以测试这种活动是否真的会使农药中毒更严重。最后，我们将结合电记录和一项令人兴奋的新技术，称为共聚焦内窥镜，并将其应用于活体神经肌肉连接的直接观察。我们将使用这种方法来评估保护乙酰胆碱受体的不同种类药物的益处，这些药物应该可以防止农药及其分解产物破坏受体。我们将在dna注射细胞中测试这些药物，在我们的小鼠组织培养中测试，最后在麻醉的猪身上测试，因为我们发现猪对OP农药的反应与人类非常相似。有效的治疗方法将在临床试验中进行测试，在我们目前在斯里兰卡医院治疗的OP农药中毒的患者中进行测试。一种有效的治疗方法每年将挽救数万人的生命。

Every year, across the world, hundreds of thousands of people die following poisoning with organophosphorus (OP) pesticides. Other populations are at risk from terrorist use of OP nerve agents (as recently used in the Middle East). These compounds interrupt signalling in the brain and muscles, causing paralysis and death when breathing fails. However, if patients reach hospital in time, they can be treated: first with antidotes and then with assisted breathing, by mechanical ventilation, in an intensive care unit. Unfortunately and for unknown reasons, some patients are unable to breathe for themselves for several weeks, leaving them dangerously at risk of lethal complications, like pneumonia. We are trying to uncover the mechanism linking OP poisoning to paralysis and cessation of breathing. We started by setting up animal models of poisoning, focusing on a particular agricultural OP pesticide, dimethoate EC40. This pesticide contains two essential ingredients: dimethoate itself and an organic solvent, cyclohexanone. These substances are broken down in the body by metabolism: dimethoate is converted into omethoate in the liver; and cyclohexanone is converted to cyclohexanol in the stomach and intestines. Our investigations have so far led us to a surprising discovery: the two metabolites, omethoate and cyclohexanol, are much more toxic to the connections between motor neurones and muscle cells, at their nerve-muscle ("neuromuscular") junctions, than either of the main ingredients in the pesticide itself. Acetylcholine is the neurotransmitter molecule that carries signals from the motor nerve endings to the muscle at these neuromuscular junctions. Our data strongly suggest that omethoate and cyclohexanol act aggressively together, directly affecting the protein receptors for acetylcholine, located on the muscle side of neuromuscular junctions. In this project, we will home in on and investigate this theory in three, concerted ways. First of all, we will inject the human DNA code for the acetylcholine receptor proteins into cells in tissue culture. The cells will turn the DNA into working receptors, allowing us to make recordings using an exquisite electrical technique called patch-clamping. In this way we can test and measure directly how omethoate, cyclohexanol or both together affect the way the receptor molecules respond to acetylcholine. Secondly, we will use a new type of tissue culture assay, to measure the progressive toxicity of omethoate and cyclohexanol on transmission at neuromuscular junctions over a period of 24 hours, the same period over which patients typically deteriorate. For these measurements, we will take advantage of nerves and muscles in a breed of mouse (called WldS) whose neuromuscular junctions are normally quite resistant to nerve damage. This feature gives us the time we need to find out why pesticide and its metabolites cause muscles to become progressively paralysed. This assay also gives us the option of rhythmically stimulating the nerve (as occurs naturally in muscles we use for breathing) to test whether this activity actually makes the pesticide poisoning worse. Finally, we will combine electrical recording with an exciting new technology, called confocal endomicroscopy, and apply these to direct observation of living neuromuscular junctions. We will use this approach to assess the benefits of different kinds of drugs that protect acetylcholine receptors, which should prevent pesticide and their breakdown products from damaging the receptors. We will test these drugs in our DNA-injected cells, in our WldS mouse tissue cultures, and lastly in anaesthetised pigs, because we have found that pigs react to OP pesticides in a very similar way to humans. Treatments that work will then be tested in clinical trials, in patients poisoned by OP pesticides that we currently look after in hospitals in Sri Lanka. An effective treatment will save tens of thousands of lives every year.