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

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

doi:10.3791/66226

Genetics

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: 10.3791/66226

Gemma E. Martin¹, Ambrose Chan¹, Nicole M. Brossier¹

¹Department of Pediatrics, Washington University in St. Louis

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.

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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 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 embryonic time point was chosen due to neurodevelopmental changes previously observed at this time in obesogenic diet-exposed animals (data not shown).

2. Embryo removal from the dam

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.
Euthanize the dam by placing it into a chamber with a paper towel containing 3 mL of isoflurane. 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.
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.
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 embryos on ice.
NOTE: As each litter typically contains 6-10 pups that will be harvested for both brains and serum, embryos are kept on ice to avoid tissue degradation. If only serum collection is desired, room temperature collection may be preferred.

3. Fetal serum collection

Remove the embryo 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.
Gently blot the animal on a paper towel to remove residual amniotic fluid and PBS from the specimen.
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.
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.
Place the head in a fresh 60 mm dish containing cold, sterile 1x PBS for further tissue collection (section 4).
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.
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.
Coagulate the blood at room temperature for 30 min.
Centrifuge the blood samples at 4 °C, 16,000 × g for 20 min.
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 embryo should yield 15-30 µL of serum.
Store the collected serum at -80 °C.

4. Embryonic brain collection

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.
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.
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.

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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 in either WT or Nf1-heterozygous strains (Figure 2A,B). We next assessed the quality of microdissected tissue. We first performed western blotting³⁶ for the housekeeping protein β-actin on the microdissected tissue isolated from the lateral ventricles of CD versus Ob-exposed mice. All samples displayed strong expression of β-actin (Figure 2C), as expected. We then isolated RNA from the microdissected tissue per the previously reported methodology³². Isolated RNA displayed both high purity (260/280 ≥ 2) and integrity (RIN values ≥ 9), indicative of high-purity, non-degraded RNA (Figure 2D).

We then compared the different methods of serum isolation. Using our minivette method, we routinely collected larger volumes of non-hemolyzed serum, while gravity drainage and thoracic puncture resulted in highly variable volume collection and hemolysis in many samples (Figure 3A,B). To illustrate the quality of serum obtained via this technique, we measured total serum cholesterol levels using the total cholesterol reagent per the manufacturer's instructions. Fetal cholesterol levels were within the expected range for late gestation pups based on prior studies in human fetuses³⁷. Levels were increased in Ob-exposed mice compared to CD-exposed for both wild-type (Figure 3C, left) and Nf1-heterozygous pups (Figure 3C, right). Note that the right panel depicts combined data from two different Nf1-heterozygous strains with patient-identified germline mutations (R681X, C383X)³²^,³⁸^,³⁹ as similar profiles were seen in both strains.

Figure 1: Graphical abstract of workflow. Pups are first isolated from pregnant dams. Serum is then harvested from the pups. Lastly, whole brains are isolated from pups, and, if desired, microdissected for regions of interest. Graphic created with BioRender.com. Please click here to view a larger version of this figure.

Figure 2: Illustrative results, brain tissue. (A) Cryosectioned brains from Nestin-CFPnuc mice, which express a nuclearly-localized cyan fluorescent protein under the control of the Nestin promoter, were harvested with this method and immunostained for GFAP (red) per previously reported methodology³². Nestin-positive cells are visualized via endogenous Nestin-CFPnuc expression (green). Nuclei were counterstained with bis-benzimide (blue). There were no significant differences between CD and Ob-exposed WT brains. Scale bar = 100 µm. (B) Nestin-CFPnuc; Nf1^mut/+ pups were similarly assessed. GFAP and Nestin-CFP distribution appeared similar to WT and did not vary with dietary conditions. Scale bar = 100 µm. (C) Protein was isolated from microdissected tissue from the LV. Western blotting was performed per previously reported methodology³⁶ with 20 µg of protein loaded per lane. Strong β-actin expression was detectable in all samples. (D) RNA was isolated from microdissected tissue per previously reported methodology³². RNA quality was assessed via 260/280 ratios and RIN values. All samples displayed excellent 260/280 ratios (≥2) and RIN values (≥9), indicating good isolation of non-degraded RNA. Abbreviations: CFP = cyan fluorescent protein; GFAP = glial fibrillary acidic protein; WT = wild-type; LV = lateral ventricle; RIN = RNA integrity number. Please click here to view a larger version of this figure.

Figure 3: Illustrative results and serum. (A) Serum was collected from each of the described methods. Collection with the minivette enabled routine collection of unhemolyzed serum compared to gravity-aided drainage and thoracic puncture. (B) Typical collection volumes of serum are shown. Minivette collection allowed for a more reproducible collection of larger volumes of serum. Statistical significance determined by one-way ANOVA with multiple comparisons. Error bars: SEM. (C) Total serum cholesterol was measured from CD vs Ob-exposed WT (left) and Nf1-heterozygous (right) pooled fetal serum; Ob-exposed animals displayed higher serum cholesterol in both genotypes. Statistical significance determined by t-test. Error bars: SEM. Abbreviations: WT = wild type; CD = control diet; Ob = obesogenic diet. Please click here to view a larger version of this figure.

Supplemental Table S1: Dietary Compositions. The composition of the control and obesogenic diets utilized in this manuscript are shown. NOTE: For a low-fat, no-sucrose diet that better controls for the individual dietary components in D12331, the D12328 diet (researchdiets.com) may be preferred. Please click here to download this File.

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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. Previous protocols reported improved volumes by using a modified 20 G needle to drain blood from thoracic blood vessels³³; however, we found this technique technically challenging and observed high rates of hemolysis, making it unsuitable for many downstream applications. Our method has the advantage of collection via passive blood flow, lessening the risk of hemolysis from aspiration-induced sheer stress. The plunger on the capillary tube also provides a quick and efficient way to deliver blood into the collection tube, minimizing exposure to air and limiting fragmentation of blood clots. Importantly, whole or regionally microdissected brain tissue can subsequently be collected from the same animal, reducing total experimental animal needs.

This protocol is not without adjustments to optimize collection quality and efficiency. For serum collection, it is crucial to gently remove embryonic fluid and extra PBS from the animal before blood collection as excess fluid may make the animal slippery and increase the likelihood of poor or unstable positioning negatively affecting the quality of blood collection. Additionally, finger positioning while holding the embryo is critical, as blood will stick to a gloved finger during collection if the finger is adjacent to the capillary tube. It is also important to hold the opening of the capillary tube adjacent to, rather than submerged in, the flowing blood; the capillary collection will be disrupted if the tube is submerged. Finally, one must consider the downstream application carefully to optimize serum collection. For instance, glucose degrades over time in untreated tubes at room temperature, so collection into sodium fluoride or EDTA-containing tubes may be necessary to accurately quantify glucose levels⁴³^,⁴⁴.

For brain tissue collection, the careful removal of the skin and transparent cranium on both sides of the head is critical for a clean extraction. If only one side is dissected away, the brain may stick to the skull with attempted removal, resulting in tearing and distortion of the tissue. For sectioning the brain, it is important to make single, clean slices to separate the anterior from the mid-cortex and the mid-cortex from the hindbrain. Having multiple cuts can disrupt the structural integrity of the tissue or obscure your reference point for subsequent microdissection. Finally, keeping samples on ice until fixation or snap freezing is crucial to prevent sample degradation.

While we prefer this method due to the routine collection of higher-volume, non-hemolyzed serum, it is not without drawbacks. Although hemolysis is reduced compared to other techniques, this method does not eliminate the risk. The capillary tube can also become clogged with a blood clot once blood stops flowing, so it is important to work quickly after decapitation to mitigate the risk of failed collection due to coagulation. Keeping samples on ice, which is done to preserve sample integrity in this protocol, may also adversely affect some phenotypes and may not be appropriate for all applications.

In conclusion, this methodology can be used to simultaneously isolate serum as well as whole or regionally microdissected brains from the same embryo. This technique can be utilized to address how systemic factors, such as a maternal diet-induced change in macronutrient profile, might affect neurodevelopment in NF1 or other disorders.

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Disclosures

The authors do not have any conflicts of interest.

Acknowledgments

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

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.

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Genetics