高压氧预适应对大鼠减压病的预防作用研究
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摘要
随着社会的发展,以海洋资源开发、考古探险或军事作业等目的的水下活动越来越普遍和频繁。人员进入水下,必须呼吸与周围水压相等的气体。呼吸气体中分压增高的惰性气体(指生理性惰性气体,最常见的是氮气),会随着潜水时间和深度的增加,而不断地溶解于机体组织中,这就要求随后的减压必须遵循一定的规则,否则惰性气体可能过快地溢出,在组织和血液中“就地”生成气泡,出现减压病(decompression sickness, DCS)。DCS可引起皮肤瘙痒、关节疼痛、呼吸循环障碍和神经系统损害的各类表现,严重者可以导致潜水员快速死亡。预防DCS成为水下作业安全保障的核心环节。
高压氧(hyperbaric oxygen, HBO)是治疗DCS的主要手段。近年研究发现,HBO预适应能够有效预防多种缺血缺氧性损伤。DCS也具有缺血缺氧的病理生理特点,HBO预适应对DCS是否也具有预防保护作用呢?
本课题旨在观察HBO预适应对于大鼠DCS的预防效应并初步探讨其机制。研究共分两部分进行。
第一部分,建立空气潜水减压病大鼠模型。以适当“压力-时程-减压速率”高气压暴露方案处理成年Sprague-Dawley (SD)大鼠,暴露结束后立即置于自行研制的电控转笼中,以3 m/min速度运动30 min。从整体行为学、大体及显微病理学、炎症反应等方面筛选建立评估指标体系。通过控制高气压暴露的“压力-时程-减压速率”以及转笼运动方法,可制备出具有稳定发病率的大鼠减压病模型,并能很好地观测大鼠行为学的变化；建立了包括行为学、肺及中枢神经系统大体病理和显微病理以及炎性指标在内的模型评估体系。
第二部分,探讨HBO预适应对于大鼠DCS的预防效应及其机制。研究又分两部进行。
①HBO预适应效应观察。成年SD大鼠以HBO(250 kPa-60min)处理(n=33),18 h后进行模拟空气潜水(700kPa-100min),快速减压(200 kPa/min)后动物在转笼中以3 m／min的速率运动30 min,观察DCS发病情况。对照动物分别以常压空气(n=30)或常氧高氮混合气(11=13)预处理。结果发现,HBO预处理组的发病率(30.3%)显著低于空气对照组(63.3%)(p<0.05)；常氧高氮预处理对DCS发病率无显著影响(61.5%)。
②HBO预适应机制研究,该部分又分为两步。第一,一氧化氮(nitric oxide, NO)在HBO预适应效应中的作用观察。检测了HBO暴露后即刻和18h大鼠大脑、脊髓和肺NO的含量；并观察了应用一氧化氮合酶(nitric oxide synthetase, NOS)抑制剂N-亚硝基-L-精氨酸甲酯(NG-Nitro-L-arginine Methyl Ester, L-NAME)对于HBO预防作用的影响。HBO暴露后即刻大鼠大脑、脊髓和肺NO含量显著升高(p<0.05),L-NAME可抑制此效应；暴露后18h各组织中NO含量无显著变化。L-NAME可显著对抗HBO对DCS的预防作用,发病率又回升至71.4%(p<0.05)。
第二,热休克蛋白(heat shock protein, HSP) 70在HBO通过NO预防DCS中的作用观察。检测了HBO暴露后18 h大鼠大脑、脊髓和肺HSP70的表达,观察了L-NAME和HSP抑制剂槲皮素(Quercetin, Q)对于HBO诱导HSP70表达的影响以及Q对HBO预防作用的影响。HBO暴露后18 h,大鼠大脑、脊髓和肺HSP70表达显著增高,L-NAME和Q均可抑制HSP70的增高。Q可有效对抗HBO对DCS的预防作用,动物DCS发病率显著回升至69.2%(p<0.05)。
上述结果可得出以下结论：
1.18 h前的HBO预适应能够有效预防大鼠DCS的发生；
2.HBO通过NO诱导HSP70表达,进而发挥对DCS损伤的保护作用。
Diving, which is an exposure to the underwater hyperbaric environment for recreational or occupational purposes, is growing in popularity every year. Personnel into the water, they must breathe with gas which has the same pressure with the surrounding water pressure. During a diving, inert gas (nitrogen is the most common one) becomes dissolved in tissues as a function of time and depth. This requires the following decompression must follow certain rules. As the partial pressure of inert gas in the blood and tissues exceeds ambient pressure, bubbles form in the tissues and blood vessels, which may result in the clincal syndrome of decompression sickness (DCS). It may present not only as minor symptoms such as skin itching and pain in joint (often termed "bends"), but also as serious neurological symptoms that can even lead to death. Prevention of DCS is becoming the core of the underwater security operations.
Hyperbaric oxygen (HBO) is a clinical therapy method for many diseases, such as carbon monoxide poisoning. HBO therapy is the most effective treatment for DCS. Furthermore, HBO preconditioning has been used to prevent various ischemia and anoxemic injuries. DCS also experiences the pathological process of ischemia and anoxemic. Does HBO preconditioning have the protective effect on DCS?
The objective of our study is to investigate the prophylactic effect of HBO preconditioning on DCS. The study is divided into two parts.
The first part is to establish the rat model of air diving DCS. Adult SD rats were exposed with appropriate "pressure-duration-decompression rate" profiles of hyperbaric air, and were subjected to run in the cylindrical cage rotating at 3 m/min for 30 min following exposure. Evaluating parameters were selected from the ethology, macropathology, micropathology and inflammatory indices. Our results suggested that controlling the pressure-duration of hyperbaric exposure and the rate of decompression, and making the animals exercises in a rotating cage at a constant speed, can produce a model of rat decompression sickness with a stable incidence, which could be observed efficiently. The evaluating parameters of ethology, macropathology, micropathology and inflammation were established.
The second part is to investigate the prophylactic effect of HBO preconditioning on DCS. The study is divided into two steps.
①HBO preconditioning effect observing. Adult SD rats were pretreated with HBO (250 kPa-60 min) (all the pressures described here are absolute pressure) 18 h before a simulated air dive (700 kPa-100 min) with fast decompression to the surface at the rate of 200 kPa/min (n=33). During the following 30 min, the rats walked in a 3 m/min rotating cage and were monitored for signs of DCS. The control rats were pretreated with normobaric air (n=30) or normoxic hyperbaric nitrox (250 kPa,8.4% O2) (n=13). The results showed that the incidence of DCS in rats pretreated with HBO was 30.3%, which was significantly lower than those treated with normobaric air (63.3%) (p<0.05); the hyperbaric nitrox preconditioning had no significantly effect on the incidence of DCS (61.5%).
②Study of mechanism is divided into two steps. First is the role of nitric oxide (NO) in the effect of HBO preconditioning on DCS. NO levels were recorded immediately and 18 h after HBO exposure in the brain, spinal cord and lung; the effect of the nitric oxygen synthetase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) on the prophylactic effect of HBO was observed. HBO increased NO level significantly in the rat brain, spinal cord and lung right after exposure; this effect was inhibited by L-NAME. L-NAME nullified the prophylactic effect of HBO on DCS, and dragged the morbidity back to 71.4%.
Second is the role of the heat shock protein (HSP) 70 in the prophylactic effect of HBO preconditioning on DCS via NO. HSP70 levels were detected 18 h after HBO exposure in the brain, spinal cord and lung; the effects of L-NAME and quercetin (Q) (the inhibitor of HSP) on the expression of HSP70 induced by HBO exposure were observed; the effect of Q on the prophylactic effect of HBO was also observed. It was showed that HBO increased HSP70 level in the rat brain, spinal cord and lung 18 h after HBO exposure, which could be inhibited by both L-NAME and Q. Moreover Q could nullify the prophylactic effect of HBO on DCS, and dragged the morbidity back to 69.2%.
The main conclusions are as follows:
1. HBO preconditioning 18 h ahead of diving could effectively prevent the DCS;
2. The prophylactic effect of the HBO preconditioning might be through the induced expression of HSP70, in which NO might be involved
高压氧(hyperbaric oxygen, HBO)是治疗DCS的主要手段。近年研究发现,HBO预适应能够有效预防多种缺血缺氧性损伤。DCS也具有缺血缺氧的病理生理特点,HBO预适应对DCS是否也具有预防保护作用呢?
本课题旨在观察HBO预适应对于大鼠DCS的预防效应并初步探讨其机制。研究共分两部分进行。
第一部分,建立空气潜水减压病大鼠模型。以适当“压力-时程-减压速率”高气压暴露方案处理成年Sprague-Dawley (SD)大鼠,暴露结束后立即置于自行研制的电控转笼中,以3 m/min速度运动30 min。从整体行为学、大体及显微病理学、炎症反应等方面筛选建立评估指标体系。通过控制高气压暴露的“压力-时程-减压速率”以及转笼运动方法,可制备出具有稳定发病率的大鼠减压病模型,并能很好地观测大鼠行为学的变化；建立了包括行为学、肺及中枢神经系统大体病理和显微病理以及炎性指标在内的模型评估体系。
第二部分,探讨HBO预适应对于大鼠DCS的预防效应及其机制。研究又分两部进行。
①HBO预适应效应观察。成年SD大鼠以HBO(250 kPa-60min)处理(n=33),18 h后进行模拟空气潜水(700kPa-100min),快速减压(200 kPa/min)后动物在转笼中以3 m／min的速率运动30 min,观察DCS发病情况。对照动物分别以常压空气(n=30)或常氧高氮混合气(11=13)预处理。结果发现,HBO预处理组的发病率(30.3%)显著低于空气对照组(63.3%)(p<0.05)；常氧高氮预处理对DCS发病率无显著影响(61.5%)。
②HBO预适应机制研究,该部分又分为两步。第一,一氧化氮(nitric oxide, NO)在HBO预适应效应中的作用观察。检测了HBO暴露后即刻和18h大鼠大脑、脊髓和肺NO的含量；并观察了应用一氧化氮合酶(nitric oxide synthetase, NOS)抑制剂N-亚硝基-L-精氨酸甲酯(NG-Nitro-L-arginine Methyl Ester, L-NAME)对于HBO预防作用的影响。HBO暴露后即刻大鼠大脑、脊髓和肺NO含量显著升高(p<0.05),L-NAME可抑制此效应；暴露后18h各组织中NO含量无显著变化。L-NAME可显著对抗HBO对DCS的预防作用,发病率又回升至71.4%(p<0.05)。
第二,热休克蛋白(heat shock protein, HSP) 70在HBO通过NO预防DCS中的作用观察。检测了HBO暴露后18 h大鼠大脑、脊髓和肺HSP70的表达,观察了L-NAME和HSP抑制剂槲皮素(Quercetin, Q)对于HBO诱导HSP70表达的影响以及Q对HBO预防作用的影响。HBO暴露后18 h,大鼠大脑、脊髓和肺HSP70表达显著增高,L-NAME和Q均可抑制HSP70的增高。Q可有效对抗HBO对DCS的预防作用,动物DCS发病率显著回升至69.2%(p<0.05)。
上述结果可得出以下结论：
1.18 h前的HBO预适应能够有效预防大鼠DCS的发生；
2.HBO通过NO诱导HSP70表达,进而发挥对DCS损伤的保护作用。
Diving, which is an exposure to the underwater hyperbaric environment for recreational or occupational purposes, is growing in popularity every year. Personnel into the water, they must breathe with gas which has the same pressure with the surrounding water pressure. During a diving, inert gas (nitrogen is the most common one) becomes dissolved in tissues as a function of time and depth. This requires the following decompression must follow certain rules. As the partial pressure of inert gas in the blood and tissues exceeds ambient pressure, bubbles form in the tissues and blood vessels, which may result in the clincal syndrome of decompression sickness (DCS). It may present not only as minor symptoms such as skin itching and pain in joint (often termed "bends"), but also as serious neurological symptoms that can even lead to death. Prevention of DCS is becoming the core of the underwater security operations.
Hyperbaric oxygen (HBO) is a clinical therapy method for many diseases, such as carbon monoxide poisoning. HBO therapy is the most effective treatment for DCS. Furthermore, HBO preconditioning has been used to prevent various ischemia and anoxemic injuries. DCS also experiences the pathological process of ischemia and anoxemic. Does HBO preconditioning have the protective effect on DCS?
The objective of our study is to investigate the prophylactic effect of HBO preconditioning on DCS. The study is divided into two parts.
The first part is to establish the rat model of air diving DCS. Adult SD rats were exposed with appropriate "pressure-duration-decompression rate" profiles of hyperbaric air, and were subjected to run in the cylindrical cage rotating at 3 m/min for 30 min following exposure. Evaluating parameters were selected from the ethology, macropathology, micropathology and inflammatory indices. Our results suggested that controlling the pressure-duration of hyperbaric exposure and the rate of decompression, and making the animals exercises in a rotating cage at a constant speed, can produce a model of rat decompression sickness with a stable incidence, which could be observed efficiently. The evaluating parameters of ethology, macropathology, micropathology and inflammation were established.
The second part is to investigate the prophylactic effect of HBO preconditioning on DCS. The study is divided into two steps.
①HBO preconditioning effect observing. Adult SD rats were pretreated with HBO (250 kPa-60 min) (all the pressures described here are absolute pressure) 18 h before a simulated air dive (700 kPa-100 min) with fast decompression to the surface at the rate of 200 kPa/min (n=33). During the following 30 min, the rats walked in a 3 m/min rotating cage and were monitored for signs of DCS. The control rats were pretreated with normobaric air (n=30) or normoxic hyperbaric nitrox (250 kPa,8.4% O2) (n=13). The results showed that the incidence of DCS in rats pretreated with HBO was 30.3%, which was significantly lower than those treated with normobaric air (63.3%) (p<0.05); the hyperbaric nitrox preconditioning had no significantly effect on the incidence of DCS (61.5%).
②Study of mechanism is divided into two steps. First is the role of nitric oxide (NO) in the effect of HBO preconditioning on DCS. NO levels were recorded immediately and 18 h after HBO exposure in the brain, spinal cord and lung; the effect of the nitric oxygen synthetase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) on the prophylactic effect of HBO was observed. HBO increased NO level significantly in the rat brain, spinal cord and lung right after exposure; this effect was inhibited by L-NAME. L-NAME nullified the prophylactic effect of HBO on DCS, and dragged the morbidity back to 71.4%.
Second is the role of the heat shock protein (HSP) 70 in the prophylactic effect of HBO preconditioning on DCS via NO. HSP70 levels were detected 18 h after HBO exposure in the brain, spinal cord and lung; the effects of L-NAME and quercetin (Q) (the inhibitor of HSP) on the expression of HSP70 induced by HBO exposure were observed; the effect of Q on the prophylactic effect of HBO was also observed. It was showed that HBO increased HSP70 level in the rat brain, spinal cord and lung 18 h after HBO exposure, which could be inhibited by both L-NAME and Q. Moreover Q could nullify the prophylactic effect of HBO on DCS, and dragged the morbidity back to 69.2%.
The main conclusions are as follows:
1. HBO preconditioning 18 h ahead of diving could effectively prevent the DCS;
2. The prophylactic effect of the HBO preconditioning might be through the induced expression of HSP70, in which NO might be involved
引文
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