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

<meta charset="utf8"></head><body>Gleichzeitige Entnahme des Gehirns und des Serums aus dem Mausfötus

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 im Rampenlicht: Sammeln des Gehirns und des Serums desselben Mäusefötus zur Untersuchung der Entwicklung von Hirntumoren

Gleichzeitige Entnahme von fötalem murinem Gehirn und Serum zur Beurteilung der Auswirkungen der mütterlichen Ernährung auf die Ernährung und Neuroentwicklung bei Neurofibromatose Typ 1

Entnahme des Fötus aus dem Muttertier für die Entnahme des fötalen Gehirns und des Serums

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

53 Aufrufe. Um den Fötus aus dem ordnungsgemäß eingeschläferten Muttertier zu entfernen, legen Sie das Tier mit der Bauchseite nach oben in eine 100-Millimeter-Schale. Kneifen Sie die Haut zusammen, die den Bauch bedeckt, und machen Sie mit einer Präparierschere einen Schnitt in der Mittellinie. Machen Sie zusätzliche Schnitte senkrecht zum Mittellinienschnitt, um Hautlappen zu erzeugen, die die Gebärmutter mit den Föten freilegen.Ziehe die Gebärmutter au...

Serie: Entnahme des Fötus aus dem Muttertier für die Entnahme des fötalen Gehirns und des Serums

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, ...