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

<meta charset="utf8"></head><body>Colhendo simultaneamente o cérebro e o soro do feto murino

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>Autor em destaque: Coletando o cérebro e o soro do mesmo feto de camundongos para estudar o desenvolvimento do tumor cerebral

Coleta Simultânea de Cérebro Murino Fetal e Soro para Avaliar os Efeitos da Dieta Materna na Nutrição e Neurodesenvolvimento na Neurofibromatose Tipo 1

Our group is interested in the neurodevelopmental as well as the environmental factors that influence whether or not brain tumors will form in a child. We are addressing how to obtain both brains and serum from the same embryonic mice, so we can reduce the total number of animals needed for a given study. The brains allow us to study neurodevelopment, and then the serum allows us to link those observations to systemic factors.This protocol allows us to collect both brains and serum reproducibly from embryonic mice. We are able to obtain higher volumes of serum, and the serum is non-hemolyzed. We have recently identified that maternal obesogenic diet exposure affects neurodevelopmental phenotypes.It increases the risk of optic pathway glioma formation in neurofibromatosis type one related mirroring models. We developed this protocol in order to determine whether these findings are related to changes in the macronutrient profiles. We will focus on the mechanism by which obesogenic diet affects neurodevelopment and increases the risk of NF1 optic pathway gliomas.

Removendo o feto da barragem para colher o cérebro fetal e o soro

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

44 visualizações. Para remover o feto da mãe devidamente sacrificada, coloque o lado ventral do animal para cima em um prato de 100 milímetros. Aperte a pele que cobre o abdômen e, com uma tesoura de dissecção, faça uma incisão na linha média. Faça incisões adicionais perpendiculares à incisão da linha média para criar retalhos de pele, expondo o útero que contém os fetos.Puxe o útero para fora da cavidade abdominal. Usando uma tesoura, desconecte o úte...