Development of artificial environments to regulate fetal growth through controlled light exposure

我们研究的目的是阐明光照条件对环境传感器基因表达和未成熟器官发育的影响。通过羊胎和新生大鼠模型，我们研究了光对周围组织或器官（如单核血细胞和肾上腺）的影响。绵羊胎儿模型：妊娠102 d时，定时妊娠的Suffolk母羊在鞘内麻醉盐酸丁卡因和静脉注射盐酸氯胺酮的情况下进行手术。剖腹、剖宫产后，将3个电极固定在胎儿胸壁上，将聚乙烯导管置入胎儿上腔静脉、下腔静脉、腹主动脉远端、羊膜腔。所有导管和电极通过每只母羊侧面的一个小切口外置。母羊不受约束，被安置在单独的笼子里，在整个研究期间，在12小时的光照（荧光灯，200勒克斯）：12小时的黑暗（LD…更多）循环下自由获取水和食物。实验前给予7 d恢复期，在此期间给予母体、胎儿和羊膜腔适当的抗生素治疗。在妊娠109 d时，通过导管从上腔静脉采集胎儿血液。用Ficoll-Hypaque溶液从5ml全血中分离出单核细胞（MNCs）。从MNCs中提取绵羊mrna，通过实时荧光定量PCR检测绵羊Perl、Per2、Bmall和Cryl的表达。本研究成功建立了绵羊妊娠模型，分析光照条件对绵羊胎儿环境传感器基因表达和未成熟器官发育的影响。大鼠新生模型：新生大鼠从出生（出生后第0天）至出生后第4天，与母鼠保持12 h光照（荧光，200勒克斯）：12 h黑暗（LD）循环。从出生后第4天的黑暗期开始，将新生大鼠和鼠坝转入持续黑暗2天。在出生后第6天的昼夜节律时间16，即黑暗开始后4小时，将新生大鼠暴露在3000勒克斯的光刺激下30分钟。原位杂交结果显示，与光刺激开始时相比，光刺激后120 min新生大鼠肾上腺皮质Perl mRNA表达显著增加（p < 0.05，单因素方差分析）。然而，新生大鼠肾上腺皮质未检测到Per2 mRNA表达的增加。本研究表明：1)光刺激对新生大鼠肾上腺皮质在昼夜节律时间（主观夜间）的影响；2)在出生后早期发育过程中，光环境可以影响肾上腺时钟等外周器官。少

The purpose of our study was to clarify the effects that lighting conditions have on gene expressions of environmental sensors and on development of immature organs. By using the models of sheep fetus and rat neonates, we examined the effects of light on peripheral tissues or organs such as mononuclear blood cells and adrenals.1. Sheep fetus model: At 102 d gestation, Suffolk ewes with timed pregnancies underwent surgery while anesthetized with intrathecal tetracaine hydrochloride and intravenous ketamine hydrochloride. After laparotomy and hysterotomy, three electrodes were fixed to the fetal chest wall and polyvinyl catheters were placed in the fetal superior vena cava, inferior vena cava, distal abdominal aorta, and amniotic cavity. All catheters and electrodes were exteriorized through a small incision in the flank of each ewe. The ewes were unrestrained and housed in individual cages, with free access to water and food under a 12 h light (fluorescent light, 200 lux): 12 h dark (LD … More ) cycle throughout the study period. A recovery period of 7 d was allowed before the experiments, and during that time appropriate antibiotics were administered to the mother, fetus, and amniotic cavity. At 109 d gestation the fetal blood was sampled from the superior vena cava through the catheters. The mononuclear cells (MNCs) were isolated from 5 mL of the whole blood by single-step separation over a Ficoll-Hypaque solution. From the MNCs ovine mRNAs were extracted and ovine Perl, Per2, Bmall and Cryl expression were confirmed by real-time PCR. In this study we successfully established a pregnant sheep model to analyze the effects of lighting conditions on gene expressions of environmental sensors and on development of the immature organs of sheep fetuses.2. Rat neonate model: Neonatal rats were maintained under a 12 h light (fluorescent light, 200 lux): 12 h dark (LD) cycle with their dams which had free access to food and water from birth (postnatal day 0) to postnatal day 4. From the dark period of postnatal day 4, both neonatal rats and a dam were transferred to constant dark for 2 days. At circadian time 16 of postnatal day 6, which is the equivalent time of 4 hours after dark onset, neonatal rats were exposed to light stimulus of 3000 lux for 30 min. In situ hybridization revealed that Perl mRNA expression in the adrenal cortex of neonatal rats significantly increased in the 120 min after light exposure compared to that in the beginning of the light stimuli (p>0.05, one-way ANOVA). However, increase in Per2 mRNA expression was not detected in the adrenal cortex of the neonatal rats. The study demonstrated 1) that the adrenal cortex of rat neonates was affected by light stimulus at circadian time 16 (during subjective night) and 2) peripheral organs such as the adrenal clock can be entrained by the light environment in early postnatal development. Less