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 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.

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 morning to determine the correct collection age. To verify diet-induced obesity, weigh females at mating.
   NOTE: A range of 8-12 weeks was used for the initial mating setup to ensure that the obesogenic diet-fed females were overweight compared to their counterparts, which did not always occur by 8 weeks of age. The day of the plug is considered embryonic day 1 (E1), and all the animals used in this study were collected at E19. The late fetal time point was chosen due to neurodevelopmental changes previously observed at this time in obesogenic diet-exposed animals (data not shown).

2. Fetus removal from the dam

1. First, ensure that the dam appears gravid (abdominal shape and fullness, abdominal palpation, or fetal movement). If in doubt, weigh the female again to measure weight gain.
   NOTE: Late-term pregnant mice should display at least 3-4 g of weight gain. To increase the likelihood of conception in the desired timeframe, dirty (urine- and pheromone-containing) sawdust from a male's cage can be added to the female's cage daily for 3 days prior to mating to induce estrus³⁴.
2. Euthanize the dam by placing it into a chamber with a paper towel containing 3 mL of isoflurane, being careful to avoid direct contact between dam and the anesthetic-soaked paper towel. After the animal is sedated, place it on a paper towel, hold it by the tail, place dissection scissors at the neck, and then force the scissors upward towards the head to perform cervical dislocation. Alternatively, perform decapitation after isoflurane sedation.
   NOTE: Serum can be collected from the dam at this step, using a similar methodology as described below but with larger capillary tubes. If this is desired, decapitation rather than cervical dislocation should be performed. Care should be taken to ensure that the phenotype of interest is not altered by the administration of isoflurane immediately before euthanasia.
3. Place the dam ventral side up in a 100 mm dish. Pinch the skin covering the abdomen and make a midline incision with dissection scissors. Make additional incisions perpendicular to the midline incision to create skin flaps to expose the uterus containing the embryos.
4. Pull the uterus out of the abdominal cavity. Using scissors, disconnect the uterus from the dam. Place the intact embryonic sac ("pearls on a string") containing the embryos into a dish with cold sterile 1x phosphate-buffered saline (PBS). Place the dish of fetuses on ice.
   NOTE: As each litter typically contains 6-10 pups that will be harvested for both brains and serum, fetuses are kept on ice to avoid tissue degradation. Placing pups on ice additionally ensures adequate euthanasia of pups, as maternal euthanasia alone may not always be sufficient. If only serum collection is desired, room temperature collection may be preferred.

3. Fetal serum collection

1. Remove the fetus from the embryonic sac by carefully dissecting with #5 forceps. Transfer the embryo to a dish using curved forceps with fresh cold sterile 1x PBS.
2. Gently blot the animal on a paper towel to remove residual amniotic fluid and PBS from the specimen.
3. Hold the animal by the abdomen. Partially decapitate the animal with micro scissors by making an incision at the ventral neck. Make sure the blood vessels are cut, but the skin keeping the head is still attached.
   NOTE: The head must remain attached so that blood can be collected from the body and head simultaneously.
4. Hold the body at a 45° angle with the incision facing down. Hold the 50 µL minivette (capillary tube) parallel to the incision site of the dam where the blood forms a droplet. Carefully allow the 50 µL capillary tube to fill with blood.
   NOTE: Capillary tube x2 or 100 µL capillary tube can be used to collect up to ~75 µL of blood.
5. Place the head in a fresh 60 mm dish containing cold, sterile 1x PBS for further tissue collection (section 4).
6. After the blood is within the capillary tube, press the plunger quickly into a 1.5 mL tube. Flick the tube or quickly spin it to make sure the blood is in the bottom of the tube.
7. Repeat for the remainder of the litter.
   NOTE: Keeping the pups on ice and moving quickly between is essential to reduce the risk of postmortem coagulation that will limit collection volume.
8. Coagulate the blood at room temperature for 30 min.
9. Centrifuge the blood samples at 4 °C, 16,000 × g for 20 min.
10. Transfer the serum to fresh 500 µL tubes with a 200 µL pipette, without disturbing the blood cell pellet at the bottom of the tube.
    NOTE: Serum should be straw-colored with no red debris. Each fetus should yield 15-30 µL of serum.
11. Store the collected serum at -80 °C.

4. Fetal brain collection

1. Looking through the dissecting microscope, hold the snout using curved forceps and use #5 forceps to carefully peel off the skin from the skull cap, keeping it attached at the snout for ease of holding. Angle #5 forceps to 45° relative to the cranium, carefully puncture the clear membrane that is the developing skull, shift the forceps parallel to the cranium, insert it underneath the skull cap, and then pinch and peel to one side. After one side is removed, grasp the remaining side with #5 forceps and peel to the other side.
2. Gently scoop the brain out of the skull using a micro spoon and transfer the brain to a fresh 60 mm dish containing cold 1x PBS.
   NOTE: At this point, whole brains can be fixed and embedded for immunostaining. See previously published methodology³².
3. If tissue must be snap-frozen for other molecular applications, sub-dissect the area of interest. Isolation of the periventricular tissue around the lateral, third, and fourth ventricles is described below.
   1. Using the curved forceps, hold the brain at the junction of the hindbrain and forebrain with the non-dominant hand. With the dominant hand, hold the scalpel with the blade parallel to the surface of the brain. Press down firmly at the mid-cortical region (Figure 1).
   2. Still holding the hindbrain, make a second incision in front of the hindbrain to separate it from the forebrain (Figure 1). In the anterior-most section, look for two small semicircular spaces located within the horizontal plane, which are the lateral ventricles. Use the curved forceps placed in the center of each ventricle to hold the brain in place, carefully dissect the lining using #5 forceps, and remove to a fresh clean 1.5 mL microcentrifuge tube.
   3. In the middle section, look for a small linear space on the ventral surface, which is the third ventricle. Using #5 forceps, carefully pinch off the lining on either side of the ventricular surface, then remove to a fresh clean 1.5 mL microcentrifuge tube.
   4. In the posterior-most section, look for a small linear space central within the hindbrain, which is the fourth ventricle. Dissect the lining on both sides using #5 forceps, then remove to a fresh clean 1.5 mL microcentrifuge tube.
      NOTE: If slicing the hindbrain is not a clean cut, the fourth ventricle tissue may be obscured and/or destroyed.
   5. For steps 4.3.2-4.3.4, immediately after the tissue has been placed into microcentrifuge tubes, place the tubes quickly into a cryo-safe container containing liquid nitrogen.
      NOTE: Tubes can be kept in liquid nitrogen until the remaining samples have been dissected. CAUTION: Liquid nitrogen can cause severe frostbite. Do not stick hands directly in liquid nitrogen and avoid splashes.
   6. Place frozen tube(s) on dry ice and transfer frozen tissue into a -80 °C freezer until use.

Results

To illustrate the quality of brain tissue obtained via this technique, we show sample fetal 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 in ei...

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.