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

Published: May 17, 2024

doi:

Gemma E. Martin, Ambrose Chan, Nicole M. Brossier

¹Department of Pediatrics,Washington University in St. Louis

Summary

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).

Abstract

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.

Introduction

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, 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 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 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.

Protocol

All animal procedures in this study followed NIH guidelines and were approved by the Institutional Animal Care and Use Committee of Washington University in St. Louis. Animals were housed with standard 12 h light:dark cycling and free access to food and water. 1. Maternal diet Place female mice on control chow (CD) or obesogenic diet (Ob) at 4 weeks of age. At 8-12 weeks of age, set up timed mating by monitoring the mucus plug of the dam in the early morn…

Representative Results

To illustrate the quality of brain tissue obtained via this technique, we show sample embryonic brains from Nestin-CFPnuc mice35, immunostained for GFAP per a previously reported technique32. Nestin+ cells are seen lining the lateral ventricle (Figure 2A), with GFAP+ filaments extending from the surface. We did not observe differences between Nestin or GFAP expression in the lateral ventricle of CD versus Ob-exposed mice i…

Discussion

Traditional methods for collecting blood from mice include retrobulbar, tail vein, saphenous vein, facial vein, and jugular vein bleeding⁴⁰^,⁴¹^,⁴². Unfortunately, these methods are not ideal for embryonic blood collection due to the size of the animal and small, delicate vasculature. Collection of blood via gravity-aided drainage after decapitation led to both low collection volumes and significant hemolysis in our samples. Previ…

Declarações

The authors have nothing to disclose.

Acknowledgements

N Brossier is supported by the Francis S. Collins Scholars Program in Neurofibromatosis Clinical and Translational Research funded by the Neurofibromatosis Therapeutic Acceleration Program (NTAP, Grant # 210112). This publication was supported in part by funding from the NTAP at the Johns Hopkins University School of Medicine. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of The Johns Hopkins University School of Medicine. Additional support by the St. Louis Children's Hospital (FDN-2022-1082 to NMB) and the Washington University in St. Louis Diabetes Research Core (NIH P30 DK020579). Microscopy was performed through the use of the Washington University Center for Cellular Imaging (WUCCI), supported by the Washington University School of Medicine, The Children's Discovery Institute of Washington University, and St. Louis Children's Hospital (CDI-CORE-2015-505 and CDI-CORE-2019-813) and the Foundation for Barnes-Jewish Hospital (3770 and 4642). Nestin-CFPnuc³⁵ mice were generously provided by Grigori Enikolopov (Renaissance School of Medicine, Stony Brook University, NY), and Nf1 mice heterozygous for either an R681X or C383X germline mutation³²^,³⁸^,³⁹ were generously provided by David Gutmann (Washington University School of Medicine, St. Louis, MO). Figure 1 was created with BioRender.com.

Materials

#5/45 Forceps	Dumont	11251-35	tip shape: angled 45°
4200 Tapestation	Agilent	G2991BA	Verify RNA integrity and quality, measurement of RIN values
Benchtop Liquid Nitrogen Container	Thermo Fisher	2122	Or other cryo-safe container
Control Chow	PicoLab	5053	Research diets D12328 (low-fat, low-sugar) may also be used.
Curved Forceps	Cole Parmer	UX-10818-25	Tip shape: curved 90°
Dissecting blade handle	Cole-Parmer Essentials	10822-20	SS Siegel-Type, #10 to #15 blades
EMS SuperCut Dissection Scissors	Electron microscope sciences	72996-01	5½" (139.7 mm), Straight
GFAP Antibody	Abcam	ab7260	Dilute 1:350. Block with 10% serum containing 0.3 M Glycine.
Glassvan Carbon Steel Surgical Blades, Size 11	MYCO medical	2001T-11	#11 blades allow straight, flat cut
Micro lab spoon	Az Scilab	A2Z-VL001	stainless steel, autoclavable
Micro scissors	Rubis	78180-1C3	model 1C300
Minivette POCT neutral	Sarstedt	17.2111.050	nominal volume: 50 µL, without preparation
Nanorop	Thermo Fisher	13-400-519	Measure RNA concentration, 260/280 ratios
Obesogenic diet	Researchdiets.com	D12331	High-fat, high-sucrose
Total Cholesterol Reagent	Thermo Fisher	TR13421	Colorimetric detection
β-actin antibody	Cell Signaling	8457	Dilute 1:1,000.

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