Analyses, using toxin fragment, of disturbances in autonomic nervous system in tetanus intoxication, a possible major cause of tetanus death

采用高效液相色谱-凝胶渗透和吸附柱层析技术，从木瓜蛋白酶消化的毒素中纯化出破伤风毒素的A-B和C片段。片段A-B制剂（200 <mu> g），以抗片段C抗体为配体进一步亲和层析纯化，无破伤风毒素特有的痉挛性毒性，也不阻断大鼠神经肌肉传递，但肌内或静脉注射小鼠时表现出明确的非典型毒性（“弛缓性麻痹”或“虚弱”），2-4天内死亡。与整个毒素不同，纯化片段A-B注射到猫脊髓后，几乎同时阻断了抑制性和兴奋性突触。在小鼠死亡前1 ~半天肌肉或静脉注射毒素均未引起高血压，但有明显的低血压。纯化片段A-B在小鼠中显示出类似的活性。毒素（0.64-2.6 ng）静脉注射到大鼠体内，与肌肉注射的毒素不同，引起异常高血压和随后的死亡。大鼠脑室内注射毒素比静脉注射具有更好的重复性。间接荧光免疫细胞化学研究显示，毒素定位于延髓后脑区和孤核交界。目前正在利用小鼠单克隆抗体对毒素片段进行进一步的精确研究。静脉注射毒素（第一次0.3 <mu> g，第7天注射到30 <mu> g）后，兔血压出现明显波动，出现反复的低血压期，与重症破伤风患者相似。在全脊髓麻醉下，通过静脉注射肾上腺素来控制血压。该方法已成功应用于破伤风患者的治疗。

Fragments(A-B and C) of tetanus toxin were highly purified from mildly papain-digested toxin by HPLC-gel permeation and adsorption column chromatograpies. Fragment A-B preparation(200 <mu> g), further purified by affinity chromatography using anti-fragment C antibody as ligand, had no spastic toxicity which is typical for tetanus toxin, nor blocked neuromuscular transmission in rat, but showed definite, atypical toxicity("flaccid paralysis" or "weakness") when injected intramuscularly or intravenously into mice and killed them in 2-4 days. Unlike whole toxin, purified fragment A-B, blocked both inhibitory and excitatory synapses almost at the same time after injection into cat spinal cord. Toxin injected intramuscularly or intravenously into mice did not elicit hypertension but remarkable hypotension one to half day before death. Purified fragment A-B showed similar activity in mice. Toxin(0.64-2.6 ng) injected intravenously into rat, unlike toxin injected intramuscularly, elicited abnormal hypertension and subsequent death. Intracerebroventicular injection of toxin into rat gave more reproducible effects than intravenous injection. Immunocytochemical studies by indirect fluorescent technique showed localizatin of toxin in border between area postrema and nucleus solitarius in medulla oblongata. Further exact studies are being carried out using mouse monoclonal anti-bodies against toxin fragments. Toxin injected intravenously into rabbits (first 0.3 <mu> g and then 30 <mu> g 7 days later) showed remarkable fluctuations in blood pressure having repeated hypotensive phases resembling those in severe cases of tetanus patients. Control of blood pressure in these rabbits was attempted by intravenous administration of epinephine under total spinal anaesthesia. The method was successfully applied to treatment of tetanus patients.