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).
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.
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.
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
2. Embryo removal from the dam
3. Fetal serum collection
4. Embryonic brain collection
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; Nf1mut/+ 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.
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.
The authors have nothing to disclose.
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.
#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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