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

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 glioma^1,2, high-grade glioma^3,4) and peripheral (plexiform neurofibroma^5,6, malignant peripheral nerve sheath tumor^7,8) nervous system as well as bony dysplasias⁹ and skin pigmentary abnormalities¹⁰ (axillary freckling, café-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 disorder^11,12,13. However, there is significant heterogeneity in the development of these phenotypes between patients^{13,14,15,16,17}, 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 population^{18,19,20,21,22,23,24,25,26,27,28}, 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 hyperactivity^{18,19,20,23,25,26}, autism^19,24,27, executive function deficits^21,23, and have lower full-scale IQ scores^22,28. However, patients with NF1 have altered metabolic phenotypes compared to the general population, including decreased incidence of obesity and diabetes^29,30,31, 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 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.