出生前尼古丁暴露对新生大鼠orexin A呼吸调节作用的影响
发布时间：2018-08-30 19:26
【摘要】： 目的 流行病学调查显示,出生前暴露于烟雾环境是新生儿猝死综合征发生的重要原因,尼古丁被认为是香烟烟雾中最主要的影响胎儿神经发育的成份。近年的研究发现,下丘脑的orexin神经元分泌的orexin不仅与食欲、能量代谢、内分泌、觉醒维持有关,还参与对心血管与呼吸活动的调控。Orexin A是否可兴奋新生大鼠呼吸活动,出生前尼古丁暴露是否会影响新生大鼠下丘脑的orexin A神经元及其受体的表达,并影响新生大鼠orexin A对呼吸活动的调节等问题尚未可知。本工作观察了orexin A对新生Sprague-Dawley(SD)大鼠离体延髓脑片舌下神经根放电活动的调节,以及orexin 1型受体是否参与此调节作用;观察出生前尼古丁暴露模型的新生大鼠orexin A及其1型受体的表达,以及orexin A对呼吸调节作用的变化,并初步探讨其调控机制。 方法 1.建立模型鼠:成年雌性大鼠,随机分成尼古丁暴露模型组和正常对照组。从交配后第五天至分娩,在母鼠腹部皮下注射溶有酒石酸氢尼古丁的生理盐溶液(3mg/kg,一日二次),其新生大鼠作为出生前尼古丁暴露模型组;正常对照组则在母鼠相同时间采用腹部皮下注射等量生理盐水,取其新生大鼠作为对照组。 2.制备离体延髓脑片:选用新生大鼠(1～3天)制备离体延髓脑片,用振动切片机切取包含Ⅻ神经根的700-800μm厚的延髓脑片,孵化后将脑片移入记录槽内,氧饱和的人工脑脊液持续灌流,用玻璃微电极吸入舌下神经根,记录其节律性放电活动。 3.药物灌流:在对照组和模型组新生鼠的延髓脑片,用不同浓度orexin A以及orexin A与orexin 1型受体阻断剂SB408124的混合液分别灌流脑片达10分钟,记录并分析舌下神经根放电活动的变化。 4.免疫组织化学的方法和图像分析技术:观察和比较模型组下丘脑orexin A及延髓内orexin 1型受体表达的变化。 5.数据统计分析:采用SPSS10.0统计软件分析检验。单个浓度组内,用药前后均数差别采用配对t检验法,三个浓度组间差别采用单因素方差分析(one-wayANOVA),生理盐水对照组与尼古丁模型组间采用独立样本t检验法,当P＜0.05时,认为差别有统计学意义。 结果 1.Orexin A能使正常新生大鼠离体延髓脑片上舌下神经根放电活动快速增加,冲洗后作用迅速消失;与灌流药物前相比,放电频率加快,积分面积增加,放电时间延长,并且呈剂量依赖性。 2.灌流液中同时给予100nM orexin A与orexin 1型受体阻断剂(SB408124)100nM时,与仅灌流人工脑脊液相比,正常新生大鼠离体延髓脑片上舌下神经根放电频率、放电积分面积、放电时间的变化均没有统计学意义。 3.出生前尼古丁暴露模型的新生大鼠(模型组)离体延髓脑片上灌流100nMorexin A,记录舌下神经根放电活动。观察到orexin A没有使其放电频率增加,与对照组相比,差别具有统计学意义(P＜0.05)。而orexin A使模型组放电积分面积增加高于对照组(P＜0.05,)。灌流orexin A后,模型组放电持续时间与对照组相比,两组间差别没有统计学意义(P＞0.05)。 4.Orexin A免疫反应阳性细胞在两组新生大鼠下丘脑内均有表达,主要分布在下丘脑背内侧区和穹窿周围。模型组下丘脑orexin A的相对光密度值高于对照组,差别有统计学意义(P＜0.05)。orexin 1型受体免疫反应阳性细胞在两组新生大鼠延髓均有广泛分布,尤其是在延髓腹外侧区(VLM)和舌下神经核(Ⅻ)。模型组orexin 1型受体免疫反应阳性细胞的相对光密度值在VLM和Ⅻ均明显高于对照组,差别有极显著统计学意义(P＜0.001)。 结论 1.Orexin A是一种促进新生大鼠呼吸活动的神经调节肽,其作用与orexin A浓度呈剂量依赖关系。 2.Orexin A的兴奋呼吸作用主要是通过延髓orexin 1型受体介导的。 3.出生前尼古丁暴露可部分减弱orexin A兴奋新生大鼠呼吸的作用。 4.出生前尼古丁暴露可使新生大鼠下丘脑orexin A和延髓orexin 1型受体表达增加。
[Abstract]:objective
Prenatal exposure to smoke is an important cause of sudden neonatal death syndrome. Nicotine is considered to be the most important component of cigarette smoke affecting fetal neural development. Recent studies have shown that orexin secreted by orexin neurons in the hypothalamus is not only associated with appetite, energy metabolism, endocrine and awakening. It is unknown whether Orexin A can stimulate respiratory activity in neonatal rats, whether nicotine exposure before birth affects the expression of orexin A neurons and their receptors in hypothalamus of neonatal rats, and whether orexin A can regulate respiratory activity in neonatal rats. The regulation of orexin A on the discharge of hypoglossal nerve roots in neonatal Sprague-Dawley (SD) rat medulla oblongata slices in vitro and whether orexin type 1 receptor is involved in this regulation were studied. Control mechanism.
Method
1. Establishment of model rats: adult female rats were randomly divided into nicotine exposure model group and normal control group. From the fifth day after mating to delivery, rats were subcutaneously injected with physiological saline solution containing nicotine tartrate (3mg/kg, twice a day), and neonatal rats were used as prenatal nicotine exposure model group. At the same time, the newborn rats were taken as the control group by abdominal subcutaneous injection of the same physiological saline.
2. Preparation of in vitro medulla oblongata slices: neonatal rats (1-3 days) were used to prepare in vitro medulla oblongata slices. 700-800 micron thick medulla oblongata slices containing_nerve roots were harvested by a vibratory section machine. After hatching, the slices were moved into a recording slot. Oxygen-saturated artificial cerebrospinal fluid (ACF) was perfused continuously. The rhythmic discharge activities of the slices were recorded by inhaling the hypoglossal nerve roots Move.
3. Drug perfusion: In the medulla oblongata slices of control group and model group, the slices were perfused with different concentrations of orexin A and the mixture of orexin A and orexin 1 receptor blocker SB408124 for 10 minutes, respectively. The discharges of hypoglossal nerve roots were recorded and analyzed.
4. Immunohistochemical method and image analysis technique: To observe and compare the expression of orexin A in hypothalamus and orexin 1 receptor in medulla oblongata in model group.
5. Statistical analysis of data: SPSS10.0 statistical software was used for analysis and test. In a single concentration group, paired t test was used for the mean difference before and after treatment, one-way ANOVA was used for the difference among the three concentration groups, and independent sample t test was used for the normal saline control group and the nicotine model group. Academic significance.
Result
1. Orexin A could rapidly increase the discharge activity of hypoglossal nerve roots on normal neonatal rat medulla oblongata slices in vitro, and the effect disappeared after irrigation.
2. When 100 nM orexin A and orexin type 1 receptor blocker (SB408124) were administered in perfusate at the same time, the discharging frequency, integral area and discharging time of hypoglossal nerve roots on the isolated medulla oblongata slices of normal neonatal rats had no statistical significance compared with that of artificial cerebrospinal fluid only.
3. Prenatal nicotine exposure model of neonatal rats (model group) in vitro medulla oblongata slices perfusion 100N Morexin A, recording the activity of hypoglossal nerve root discharge. It was observed that orexin A did not increase its discharge frequency, compared with the control group, the difference was statistically significant (P After perfusion of orexin A, the duration of discharge in the model group was not significantly different from that in the control group (P > 0.05).
4. Orexin A immunoreactive cells were expressed in the hypothalamus of both groups, mainly in the dorsomedial and perifornical areas of the hypothalamus. The relative optical density of orexin A in the hypothalamus of the model group was higher than that of the control group, and the difference was statistically significant (P < 0.05). The relative optical density of orexin type 1 receptor immunoreactive cells in the model group was significantly higher than that in the control group (P < 0.001).
conclusion
1. Orexin A is a neuromodulatory peptide that promotes respiratory activity in neonatal rats. Its effect is dose-dependent with orexin A concentration.
The excitatory respiration of 2.Orexin A is mediated by orexin 1 receptor of medulla oblongata.
3. prenatal nicotine exposure partially attenuated the respiratory function of orexin A in neonatal rats.
4. prenatal nicotine exposure can increase the expression of orexin A and medullary orexin 1 receptor in the neonatal rat hypothalamus.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R181.3;R714.7
本文编号：2214061
[Abstract]:objective
Prenatal exposure to smoke is an important cause of sudden neonatal death syndrome. Nicotine is considered to be the most important component of cigarette smoke affecting fetal neural development. Recent studies have shown that orexin secreted by orexin neurons in the hypothalamus is not only associated with appetite, energy metabolism, endocrine and awakening. It is unknown whether Orexin A can stimulate respiratory activity in neonatal rats, whether nicotine exposure before birth affects the expression of orexin A neurons and their receptors in hypothalamus of neonatal rats, and whether orexin A can regulate respiratory activity in neonatal rats. The regulation of orexin A on the discharge of hypoglossal nerve roots in neonatal Sprague-Dawley (SD) rat medulla oblongata slices in vitro and whether orexin type 1 receptor is involved in this regulation were studied. Control mechanism.
Method
1. Establishment of model rats: adult female rats were randomly divided into nicotine exposure model group and normal control group. From the fifth day after mating to delivery, rats were subcutaneously injected with physiological saline solution containing nicotine tartrate (3mg/kg, twice a day), and neonatal rats were used as prenatal nicotine exposure model group. At the same time, the newborn rats were taken as the control group by abdominal subcutaneous injection of the same physiological saline.
2. Preparation of in vitro medulla oblongata slices: neonatal rats (1-3 days) were used to prepare in vitro medulla oblongata slices. 700-800 micron thick medulla oblongata slices containing_nerve roots were harvested by a vibratory section machine. After hatching, the slices were moved into a recording slot. Oxygen-saturated artificial cerebrospinal fluid (ACF) was perfused continuously. The rhythmic discharge activities of the slices were recorded by inhaling the hypoglossal nerve roots Move.
3. Drug perfusion: In the medulla oblongata slices of control group and model group, the slices were perfused with different concentrations of orexin A and the mixture of orexin A and orexin 1 receptor blocker SB408124 for 10 minutes, respectively. The discharges of hypoglossal nerve roots were recorded and analyzed.
4. Immunohistochemical method and image analysis technique: To observe and compare the expression of orexin A in hypothalamus and orexin 1 receptor in medulla oblongata in model group.
5. Statistical analysis of data: SPSS10.0 statistical software was used for analysis and test. In a single concentration group, paired t test was used for the mean difference before and after treatment, one-way ANOVA was used for the difference among the three concentration groups, and independent sample t test was used for the normal saline control group and the nicotine model group. Academic significance.
Result
1. Orexin A could rapidly increase the discharge activity of hypoglossal nerve roots on normal neonatal rat medulla oblongata slices in vitro, and the effect disappeared after irrigation.
2. When 100 nM orexin A and orexin type 1 receptor blocker (SB408124) were administered in perfusate at the same time, the discharging frequency, integral area and discharging time of hypoglossal nerve roots on the isolated medulla oblongata slices of normal neonatal rats had no statistical significance compared with that of artificial cerebrospinal fluid only.
3. Prenatal nicotine exposure model of neonatal rats (model group) in vitro medulla oblongata slices perfusion 100N Morexin A, recording the activity of hypoglossal nerve root discharge. It was observed that orexin A did not increase its discharge frequency, compared with the control group, the difference was statistically significant (P After perfusion of orexin A, the duration of discharge in the model group was not significantly different from that in the control group (P > 0.05).
4. Orexin A immunoreactive cells were expressed in the hypothalamus of both groups, mainly in the dorsomedial and perifornical areas of the hypothalamus. The relative optical density of orexin A in the hypothalamus of the model group was higher than that of the control group, and the difference was statistically significant (P < 0.05). The relative optical density of orexin type 1 receptor immunoreactive cells in the model group was significantly higher than that in the control group (P < 0.001).
conclusion
1. Orexin A is a neuromodulatory peptide that promotes respiratory activity in neonatal rats. Its effect is dose-dependent with orexin A concentration.
The excitatory respiration of 2.Orexin A is mediated by orexin 1 receptor of medulla oblongata.
3. prenatal nicotine exposure partially attenuated the respiratory function of orexin A in neonatal rats.
4. prenatal nicotine exposure can increase the expression of orexin A and medullary orexin 1 receptor in the neonatal rat hypothalamus.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R181.3;R714.7
【参考文献】
相关期刊论文 前1条
1 宁穗;宋娜娜;刘自兵;李莉;曹银祥;钱源;朱大年;沈霖霖;;orexin A和orexin 1型受体在新生大鼠与成年大鼠延髓分布的比较[J];神经解剖学杂志;2008年04期
，本文编号：2214061
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