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

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 glioma^1,2, high-grade glioma^3,4) and peripheral (plexiform neurofibroma^5,6, malignant peripheral nerv....

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

1. Place female mice on control chow (CD) or obesogenic diet (Ob) at 4 weeks of age.
2. At 8-12 weeks of age, set up timed mating by monitoring the mucus plug of the dam in the early morn.......

Discussion

Traditional methods for collecting blood from mice include retrobulbar, tail vein, saphenous vein, facial vein, and jugular vein bleeding^40,41,42. 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.......

Materials

Name	Company	Catalog Number	Comments
#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.