摘要
一氧化碳(carbon monoxide, CO)中毒是急性中毒最常见的原因之一，也是急性中毒死亡的主要原因之一。据报道，美国每年约有3800例患者死于CO中毒，大约占一半的致死性中毒是由于CO中毒引起的[Blumenthal I, 2001]业国内尚无每年CO中毒发病人数、发病率、死亡率等方面的直接统计资料。但根据各相关资料来看，在我国CO中毒也很常见。1992～1994年，在国内报导的急性职业性中毒2842例中，急性CO中毒占1384例，居首位[高春锦，1997]。
自上个世纪60年代首次发现高压氧(hyperbaric oxygen, HBO)对急性CO中毒具有很好的疗效以来，高压氧成为治疗急性CO中毒的首选方法。人们随即对高压氧治疗急性CO中毒的机理进行了大量的研究。研究发现高压氧能加速碳氧血红蛋白解离和CO排出，增加机体的血氧含量，提高氧分压，迅速纠正组织缺氧[Myers RA,1998]；改善微循环和血液流变性，增加损伤组织血液灌注；改善细胞内能量代谢，降低细胞内钙浓度，减轻钙超载[Rogatsky GG,1999]；调节脂质过氧化过程和氧自由基损伤[Raub JA, 2000]。以上发现和结论很好的解释了高压氧治疗急性CO中毒
第口军医大学硕士学位论文
一
的作用，但要完全弄清高压氧治疗CO中毒的机理，尤其是治疗CO中毒
迟发性脑病的机理，则还需要做更深一步的研究。其中亟待解抉的几个问
题是：回．急性CO中毒迟发性脑病的发病机制为何？2高压氧对急性CO中
毒迟发性脑病是否具有治疗作用，其作用机理是什么？
为了探讨以上问题，我们参照Ischiropoulos的CO染毒方法制备急
性CO中毒动物模型。采用组织病理学、免疫组织化学。行为心理学等方
法检测大鼠染毒后 6h、id、3d、sd、7d、14d和 Zd等各个时问点脑组织
病理改变和学习、记忆功能变化的特点并与高压氧治疗组相比较，依此评
价高压氧的治疗作用。通过细胞超微结构的观察、BC 12蛋白免疫组化染
色和原位末端转移酶标记（TUNEL）技术等三种方法进行细胞凋亡的检测，
观察高压氧对急性CO中毒导致神经元凋亡的作用。其主要结果如下：
（一）建立了可靠的急性CO中毒动物模型 制定了不同浓度CO中
毒大鼠血HbCO浓度曲线，确定了适宜的中毒浓度和中毒时问。暴露在
2500Ppm（parts per million）CO中60min，可以使大鼠血中HbCO浓度
达到 65～70％，大鼠无一例外发生昏迷，且死亡率不高（2％）。
（二）急性CO中毒大鼠脑内发生急性水肿和广泛的病理损伤CO暴
露后前3 天，大鼠出现脑水肿，脑组织含水量明显高于正常对照组
（P＜001）。与co 中毒组相比，高压氧治疗组脑组织含水量明显减轻
（P＜005）。CO中毒后大鼠大脑皮质、海马、纹状体和小脑等部位神经元
出现变性坏死，其中大脑皮质、海马等部位损伤较重。此现象与大脑不同
区域对缺氧的敏感程度有关。高压氧治疗可以减少大鼠脑内神经元变性坏
死，实验中各时间点大鼠海马区损伤均轻于CO中毒组。
（三）急性CO 中毒大鼠海马区迟发性神经元损伤和迟发性健忘症
HE、TUN’EL染色和电镜观察表明CO中毒大鼠海马神经元发生凋亡，凋亡
神经元从染毒后第 3d开始显著增加，第 7d达到高峰（P＜001。。ontrol
group），以后逐渐减少，直到 CO暴露后 ZId实验期结束时还可观察到凋
＊2．
第四军医大学硕士学位论文
一
亡神经元。通过卜迷宫实验和跳台实验发现CO中毒大鼠的正确反应率和
跳台潜伏期＊t印刁OW川at＊Cy SDL）均明显低于正常对照组，尤其是 CO
暴露后第3d开始，大鼠＊L出现显著下降，至第7d达到最低（P＜ 0刀】s
control groupL 以上说明 CO中毒导致大鼠海马区迟发性神经元损伤
（delayed neuronal damage，DND）和迟发性学习记忆能力下降，亦称为
迟发性健忘症（delay。d amn。sia）。迟发性健忘症与海马区迟发性神经元
损伤在发生时间卜是一致的。因此认为，海马区迟发性神经元损伤是迟发
性健忘症的发生原因之一。凋亡是迟发性神经元损伤的形式之一，它参与
了迟发性健忘症的发生。
（四）高压氧对CO中毒大鼠脑神经元的保护作用
1）高压氧抑制CO 中毒大鼠海马区神经元凋亡 根据HE染色和
TUNEL染色进行凋亡细胞记数发现与CO中毒组相比，HBO治疗组大鼠凋亡
神经元数目减少，尤以＊中毒后sd和N 明显（P＜001），说明mO可
以抑制CO中毒后神经元凋亡，减轻DND。
2）高压氧促进 CO中毒大鼠海马区h 12蛋白表达 正常对照组和
CO中毒组大鼠海马区不表达或很少表达BC12蛋白，两组之间比较无差
别。HBO治疗组与 C0rl，毒组相比 BC］－2蛋白阳性细胞增多，尤以 CO暴露
后3、sd明显（P＜001），说明nBO暴露可以促进仇一2蛋白表达。BCI－2
蛋白可以抑制神经元凋亡［Walton MI，1993」，HBO可能通过促进 Bcl－2蛋
白表达从而起到减少神经元凋亡的作用。
3）高压氧改善CO中毒大鼠学习记忆功能Y－迷宫实验中，HBO治
疗组大鼠的正确反应率在CO暴露后第id到第7d都明显高?
Acute carbon monoxide (CO) poisoning is one of the common reasons for acute poisoning, and it is one of the main reasons of the death caused by acute poisoning. It's reported that there are 3800 patients died because of acute carbon monoxide poisoning. The number is about one half of that died of acute poisoning. At present, there are not detailed data of morbidity and mortality about acute CO poisoning in out country. But according as all kinds of data, the morbidity of acute CO poisoning is comparative high in our country. From 1992 to 1994, 2842 accidental deaths
due to acute vocationally poisoning are reported in China. Thereinto, there are 1384 accidental deaths due to acute carbon monoxide poisoning. The proportion of carbon monoxide poisoning to acute vocationally poisoning is highest.
Since the effect of hyperbaric oxygen (HBO) on acute carbon monoxide was known in 1960s, hyperbaric oxygen has became the mainstay of treatment for acute carbon monoxide poisoning. People have made a lot of research on the mechanism of hyperbaric oxygen. All these research found that Hyperbaric oxygen has many benefits. The half-life of carboxyhaemoglobin at 3 ATA (absolute atmospheres) of oxygen is only 23 minutes. Other benefits are improved mitochondrial function, impairment of platelet adhesion in the capillaries and inhibition of lipid peroxidation. But contrary to expectation, clinical trials of hyperbaric oxygen have given conflicting results. A recent Cochrane review of three major randomized controlled trials concluded that there is as yet no evidence of neurological benefit at one month. Ongoing trials will soon provide further information. In the absence of firm evidence most centres continue using hyperbaric oxygen if the carboxyhaemoglobin is above 25-30%. Myocardial ischaemia and neurological signs, especially coma, are treated with hyperbaric oxygen irrespective of the concentration. All these found and conclusions have explained the effect of hyperbaric oxygen on acute carbon monoxide poisoning. However this research is only in the beginning and there are several questions to be considered firstly: 1) what is the mechanism of delayed neurological syndrome (DNS) induced by acute carbon monoxide poisoning? 2) Whether hyperbaric oxygen treatment has the effect on delayed neurological syndrome induced by acute carbon monoxide poisoning and what is the mechanism of hyperbaric oxygen treatment.
-6-
Based on these questions, we produced acute carbon monoxide poisoning animal model designed by Ischiropoulos. General hematoxylin-eosin(H&E) stain and immunohistochemistry stain were used to observe the neurological damage in the brain of rat at different time after CO exposure (include 6h> ld> 3d, 5d> 7d, 14d and 21d). Y-maze and Step-Down type passive avoidance task were used to assess the effects of the CO exposure on learning and memory. We compared HBO-treated group with CO-exposed group to assess the effects of hyperbaric oxygen on carbon monoxide poisoning. At the same time to detect neural apoptosis, electron microscopy, immunohistochemistry stain of Bcl-2 and terminal deoxy-nucleotidyl transferase mediated UTP end labeling (TUNEL) were used to assess the effect of HBO on neural apoptosis induced by CO poisoning. The main results of present work are as follows:
I) A stable method for the acute CO poisoning animal model: we
described the curve of arterial HbCO in the rats which were exposed to CO with different concentration and confirmed feasible exposing concentration and time. Exposed in 2500 parts per million (ppm) carbon monoxide will make the arterial HbCO of rats increased to 65%-70%. All rats loss of conscious (LOG) and the mortality is low (2%).
II) Acute oedema and diffuse pathology injury in the brain of rats following acute CO poisoning: Oedema was observed in the brain of rats in the first 3 days after CO exposed. Compared with control group, the brain water content increased significantly (P<0.01). Neural necrosis was observed in cortex, hippocampus, striatum, and cerebellum
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