removing the fetus from the dam for harvesting the fetal brain and serum

<meta charset="utf8"></head><body>同时从小鼠胎儿中采集大脑和血清

Neurofibromatosis Type 1 (NF1) is considered a RASopathy, a group of disorders characterized by germline genetic mutations resulting in activation of the RAS/MAPK (RAt Sarcoma virus/Mitogen-activated Protein Kinase) signaling pathway. Patients with the NF1 RASopathy are at risk for developing many different manifestations, including both benign and malignant tumors of the central (optic pathway glioma1,2, high-grade glioma3,4) and peripheral (plexiform neurofibroma5,6, malignant peripheral nerve sheath tumor7,8) nervous system as well as bony dysplasias9 and skin pigmentary abnormalities10 (axillary freckling, caf&#233;-au-lait macules). The effect of this disorder on cognition and neurodevelopment is increasingly being recognized, with NF1 patients displaying an increased incidence of learning deficits, hyperactivity, and autism spectrum disorder11,12,13. However, there is significant heterogeneity in the development of these phenotypes between patients13,14,15,16,17, and it is unclear why some patients display significant cognitive impairments while others are unaffected. Maternal diet-induced obesity has been shown to similarly affect learning and behavior in the general population18,19,20,21,22,23,24,25,26,27,28, suggesting that differential maternal dietary exposures in NF1 could be one source of this clinical heterogeneity. In particular, children of obese mothers display an increased risk of developing hyperactivity18,19,20,23,25,26, autism19,24,27, executive function deficits21,23, and have lower full-scale IQ scores22,28. However, patients with NF1 have altered metabolic phenotypes compared to the general population, including decreased incidence of obesity and diabetes29,30,31, making it unclear whether they would respond similarly to dietary stimuli.
To address these questions, we wished to determine whether obesogenic-diet-induced changes to the macronutrient profile in fetal offspring with Nf1 contributed to neurodevelopmental changes. We have previously collected high-quality whole and regionally micro-dissected tissue appropriate for neurodevelopmental applications from the fetal brain32. However, fetal blood collection is challenging due to the small body size and low blood volume33. The collection of blood via gravity-aided drainage after decapitation led to low collection volumes and significant hemolysis in our samples, which can affect downstream application interpretation. Collection via aspiration from the fetal heart or thoracic vessels, as has been previously reported33, was technically challenging and also resulted in frequent hemolysis. We thus developed a method for fetal serum collection, which utilizes specialized capillary tubes to allow for higher volume collection without significant shear stress.
Here, we present this method to simultaneously collect embryonic brains and fetal serum from Nf1-heterozygous pups exposed to a high-fat, high-sugar diet versus a control diet in utero (Figure&#160;1 and Supplemental Table S1). Brains were cryo-embedded for subsequent analysis by immunofluorescence or regionally micro-dissected and flash-frozen for subsequent use in molecular biology applications. High-quality serum was obtained, suitable for downstream applications such as macronutrient profiling. Utilizing this method, we identified that maternal high-fat, high-sucrose dietary exposure leads to the elevation of serum cholesterol levels in both WT and Nf1-heterozygous pups.

<meta charset="utf8"></head><body>作者聚焦：收集同一小鼠胎儿的大脑和血清以研究脑肿瘤的发展

同时收集胎鼠大脑和血清以评估母体饮食对 1 型神经纤维瘤病营养和神经发育的影响

从母带中取出胎儿以收获胎儿大脑和血清

removing-fetus-from-dam-for-harvesting-fetal-brain

Research

JoVE Journal

Genetics

Removing the Fetus from the Dam for Harvesting the Fetal Brain and Serum

51 次观看. 要从适当安乐死的母带中取出胎儿，请将动物腹侧朝上放入 100 毫米的培养皿中。捏住覆盖腹部的皮肤，然后用解剖剪刀做一个中线切口。在垂直于中线切口的地方做额外的切口，形成皮瓣，露出含有胎儿的子宫。将子宫从腹腔中拉出。使用剪刀断开子宫与母膜的连接。将含有胎儿的完整胎囊（类似于绳子上的珍珠）转移到培养皿中冷无菌的 PBS...

视频: 从母带中取出胎儿以收获胎儿大脑和血清

Concurrent Collection of Fetal Murine Brain and Serum to Assess Effects of Maternal Diet on Nutrition and Neurodevelopment in Neurofibromatosis Type 1

Maternal diet-induced obesity has been demonstrated to alter neurodevelopment in offspring, which may lead to reduced cognitive capacity, hyperactivity, and impairments in social behavior. Patients with the clinically heterogeneous genetic disorder Neurofibromatosis Type 1 (NF1) may present with similar deficits, but it is currently unclear whether environmental factors such as maternal diet influence the development of these phenotypes, and if so, the mechanism by which such an effect would occur. To enable evaluation of how maternal obesogenic diet exposure affects systemic factors relevant to neurodevelopment in NF1, we have developed a method to simultaneously collect non-hemolyzed serum and whole or regionally micro-dissected brains from fetal offspring of murine dams fed a control diet versus a high-fat, high-sucrose diet. Brains were processed for cryosectioning or flash frozen to use for subsequent RNA or protein isolation; the quality of the collected tissue was verified by immunostaining. The quality of the serum was verified by analyzing macronutrient profiles. Using this technique, we have identified that maternal obesogenic diet increases fetal serum cholesterol similarly between WT and Nf1-heterozygous pups.

In this protocol, we describe a method for simultaneous collection of fetal brain tissue as well as high-quality, non-hemolyzed serum from the same mouse embryo. We have utilized this technique to interrogate how maternal dietary exposure affects macronutrient profiles and fetal neurodevelopment in mice heterozygous for Nf1 (Neurofibromatosis Type 1).

Maternal diet-induced obesity has been demonstrated to alter neurodevelopment in offspring, which may lead to reduced cognitive capacity, hyperactivity, ...