出生前尼古丁暴露對新生大鼠orexin A呼吸調(diào)節(jié)作用的影響
發(fā)布時間：2018-08-30 19:26
【摘要】： 目的 流行病學調(diào)查顯示,出生前暴露于煙霧環(huán)境是新生兒猝死綜合征發(fā)生的重要原因,尼古丁被認為是香煙煙霧中最主要的影響胎兒神經(jīng)發(fā)育的成份。近年的研究發(fā)現(xiàn),下丘腦的orexin神經(jīng)元分泌的orexin不僅與食欲、能量代謝、內(nèi)分泌、覺醒維持有關(guān),還參與對心血管與呼吸活動的調(diào)控。Orexin A是否可興奮新生大鼠呼吸活動,出生前尼古丁暴露是否會影響新生大鼠下丘腦的orexin A神經(jīng)元及其受體的表達,并影響新生大鼠orexin A對呼吸活動的調(diào)節(jié)等問題尚未可知。本工作觀察了orexin A對新生Sprague-Dawley(SD)大鼠離體延髓腦片舌下神經(jīng)根放電活動的調(diào)節(jié),以及orexin 1型受體是否參與此調(diào)節(jié)作用;觀察出生前尼古丁暴露模型的新生大鼠orexin A及其1型受體的表達,以及orexin A對呼吸調(diào)節(jié)作用的變化,并初步探討其調(diào)控機制。 方法 1.建立模型鼠:成年雌性大鼠,隨機分成尼古丁暴露模型組和正常對照組。從交配后第五天至分娩,在母鼠腹部皮下注射溶有酒石酸氫尼古丁的生理鹽溶液(3mg/kg,一日二次),其新生大鼠作為出生前尼古丁暴露模型組;正常對照組則在母鼠相同時間采用腹部皮下注射等量生理鹽水,取其新生大鼠作為對照組。 2.制備離體延髓腦片:選用新生大鼠(1～3天)制備離體延髓腦片,用振動切片機切取包含Ⅻ神經(jīng)根的700-800μm厚的延髓腦片,孵化后將腦片移入記錄槽內(nèi),氧飽和的人工腦脊液持續(xù)灌流,用玻璃微電極吸入舌下神經(jīng)根,記錄其節(jié)律性放電活動。 3.藥物灌流:在對照組和模型組新生鼠的延髓腦片,用不同濃度orexin A以及orexin A與orexin 1型受體阻斷劑SB408124的混合液分別灌流腦片達10分鐘,記錄并分析舌下神經(jīng)根放電活動的變化。 4.免疫組織化學的方法和圖像分析技術(shù):觀察和比較模型組下丘腦orexin A及延髓內(nèi)orexin 1型受體表達的變化。 5.數(shù)據(jù)統(tǒng)計分析:采用SPSS10.0統(tǒng)計軟件分析檢驗。單個濃度組內(nèi),用藥前后均數(shù)差別采用配對t檢驗法,三個濃度組間差別采用單因素方差分析(one-wayANOVA),生理鹽水對照組與尼古丁模型組間采用獨立樣本t檢驗法,當P＜0.05時,認為差別有統(tǒng)計學意義。 結(jié)果 1.Orexin A能使正常新生大鼠離體延髓腦片上舌下神經(jīng)根放電活動快速增加,沖洗后作用迅速消失;與灌流藥物前相比,放電頻率加快,積分面積增加,放電時間延長,并且呈劑量依賴性。 2.灌流液中同時給予100nM orexin A與orexin 1型受體阻斷劑(SB408124)100nM時,與僅灌流人工腦脊液相比,正常新生大鼠離體延髓腦片上舌下神經(jīng)根放電頻率、放電積分面積、放電時間的變化均沒有統(tǒng)計學意義。 3.出生前尼古丁暴露模型的新生大鼠(模型組)離體延髓腦片上灌流100nMorexin A,記錄舌下神經(jīng)根放電活動。觀察到orexin A沒有使其放電頻率增加,與對照組相比,差別具有統(tǒng)計學意義(P＜0.05)。而orexin A使模型組放電積分面積增加高于對照組(P＜0.05,)。灌流orexin A后,模型組放電持續(xù)時間與對照組相比,兩組間差別沒有統(tǒng)計學意義(P＞0.05)。 4.Orexin A免疫反應(yīng)陽性細胞在兩組新生大鼠下丘腦內(nèi)均有表達,主要分布在下丘腦背內(nèi)側(cè)區(qū)和穹窿周圍。模型組下丘腦orexin A的相對光密度值高于對照組,差別有統(tǒng)計學意義(P＜0.05)。orexin 1型受體免疫反應(yīng)陽性細胞在兩組新生大鼠延髓均有廣泛分布,尤其是在延髓腹外側(cè)區(qū)(VLM)和舌下神經(jīng)核(Ⅻ)。模型組orexin 1型受體免疫反應(yīng)陽性細胞的相對光密度值在VLM和Ⅻ均明顯高于對照組,差別有極顯著統(tǒng)計學意義(P＜0.001)。 結(jié)論 1.Orexin A是一種促進新生大鼠呼吸活動的神經(jīng)調(diào)節(jié)肽,其作用與orexin A濃度呈劑量依賴關(guān)系。 2.Orexin A的興奮呼吸作用主要是通過延髓orexin 1型受體介導的。 3.出生前尼古丁暴露可部分減弱orexin A興奮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腦orexin A和延髓orexin 1型受體表達增加。
[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.
【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R181.3;R714.7
本文編號：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.
【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R181.3;R714.7
【參考文獻】
相關(guān)期刊論文 前1條
1 寧穗;宋娜娜;劉自兵;李莉;曹銀祥;錢源;朱大年;沈霖霖;;orexin A和orexin 1型受體在新生大鼠與成年大鼠延髓分布的比較[J];神經(jīng)解剖學雜志;2008年04期
，本文編號：2214061
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