Concurrent Collection of Fetal Murine Brain and Serum to Assess Effects of Maternal Diet on Nutrition and Neurodevelopment in Neurofibromatosis Type 1
May 17th, 2024
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).
Our group is interested in the neurodevelopmental as well as the environmental factors that influence whether or not brain tumors will form in a child. We are addressing how to obtain both brains and serum from the same embryonic mice, so we can reduce the total number of animals needed for a given study. The brains allow us to study neurodevelopment, and then the serum allows us to link those observations to systemic factors.
This protocol allows us to collect both brains and serum reproducibly from embryonic mice. We are able to obtain higher volumes of serum, and the serum is non-hemolyzed. We have recently identified that maternal obesogenic diet exposure affects neurodevelopmental phenotypes, it increases the risk of optic pathway glioma formation in neurofibromatosis Type 1-related mirroring models.
We developed this protocol in order to determine whether these findings are related to changes in the macronutrient profiles. We will focus on the mechanism by which obesogenic diet affects neurodevelopment, and increases the risk of NF1 optic pathway gliomas. To remove the fetus from the properly euthanized dam, place the animal ventral side up in a 100 millimeter dish.
Pinch the skin covering the abdomen, and with dissection scissors, make a midline incision. Make additional incisions perpendicular to the midline incision to create skin flaps, exposing the uterus containing the fetuses. Pull the uterus out of the abdominal cavity.
Using scissors, disconnect the uterus from the dam. Transfer the intact fetal sack containing the fetuses, resembling pearls on a string, into cold, sterile PBS in a dish. Place the dish of fetuses on ice, to eventually harvest fetal serum and brain.
Proceed to harvest the fetal serum from the fetal sac previously isolated from the dam. Using a number five forceps, carefully dissect the fetal sac to remove the fetus for serum collection. With curved forceps, transfer the fetus to a dish contain fresh, cold, sterile PBS.
Gently blot the fetus on a paper towel to remove residual amniotic fluid and PBS from the specimen. Hold the fetus by the abdomen, partially decapitate the fetus with micro scissors by making an incision at the ventral neck. Ensure the blood vessels are cut, but the skin keeping the head is still attached.
Hold the body at a 45-degree angle with the incision facing down. Hold a 50-microliter minovet or capillary tube parallel to the incision site where the blood forms a droplet. Carefully allow the capillary tube to fill with blood.
Place the head in a fresh 60-millimeter dish containing cold, sterile PBS for further tissue collection. After the blood is collected within the capillary tube, press the plunger quickly into a 1.5-milliliter tube Flick, tap, or quickly spin the tube to ensure the blood is in the bottom. Centrifuge the blood samples at four degrees Celsius and 16, 000 G.Using a 200-microliter pipette, transfer the serum to fresh 500-microliter tubes without disturbing the blood cell palate at the bottom of the tube.
Proceed to collect the brain from the previously isolated head of the fetus under a dissecting microscope. Using curved forceps, hold the snout and use a number five forceps to carefully peel off the skin from the skull cap, keeping it attached to the snout for ease of holding. Holding at about 75 degrees relative to the brain, run the forceps along the midline until the clear membrane of the developing skull parts to each side.
Shift the forceps parallel to the cranium, insert it underneath the skull cap, and pinch, and peel to one side. After one side is removed, grasp the remaining side with the forceps and peel to the other side. Using a micro spoon, gently scoop the brain out of the skull.
Transfer the brain to a fresh 60-millimeter dish containing cold PBS. 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, then, press down firmly at the mid-cortical region.
Still holding the hindbrain, make a second incision in front of the hindbrain to separate it from the forebrain. In the middle section, look for a small linear space on the ventral surface, which is the third ventricle. Using number five forceps, carefully pinch off the lining on either side of the ventricular surface, then, transfer it to a fresh, clean, 1.5-milliliter micro centrifuge tube.
Place the tube quickly into a CryoSafe container of liquid nitrogen.
Overview
This protocol describes a method for the simultaneous collection of fetal brain tissue and high-quality, non-hemolyzed serum from the same mouse embryo. This technique is utilized to study the impact of maternal dietary exposure on fetal neurodevelopment in mice with Neurofibromatosis Type 1.
Key Study Components
Area of Science
- Neurodevelopment
- Maternal dietary exposure
- Fetal brain and serum collection
Background
- The study focuses on neurodevelopmental factors influencing brain tumor formation.
- It aims to reduce the number of animals used in research by collecting both brains and serum from embryonic mice.
- Higher volumes of non-hemolyzed serum can be obtained using this method.
- Maternal obesogenic diet exposure has been linked to neurodevelopmental phenotypes and increased risk of glioma formation.
Purpose of Study
- To investigate how maternal diet affects fetal neurodevelopment.
- To link neurodevelopmental observations with systemic factors through serum analysis.
- To provide a reproducible method for collecting fetal brain and serum samples.
Methods Used
- Simultaneous collection of fetal brain and serum from mouse embryos.
- Utilization of a reproducible protocol to ensure high-quality samples.
- Analysis of neurodevelopmental outcomes in relation to maternal diet.
- Assessment of serum quality to ensure non-hemolyzed samples.
Main Results
- Successful collection of both brain and serum samples from embryonic mice.
- Identification of maternal dietary impacts on fetal neurodevelopment.
- Increased risk of optic pathway glioma formation linked to maternal diet.
- Establishment of a method that reduces animal usage in research.
Conclusions
- The protocol allows for efficient collection of critical samples for neurodevelopmental research.
- Maternal dietary factors significantly influence fetal brain development.
- This method can aid in understanding the environmental influences on brain tumor formation.
Frequently Asked Questions
Executive Industry Relevance
Simultaneous collection of fetal murine brain and non-hemolyzed serum enables direct linkage of neurodevelopmental phenotypes to systemic metabolic factors in preclinical models of Neurofibromatosis Type 1 (NF1). This approach supports mechanistic de-risking of maternal diet effects on neurodevelopment and tumor predisposition, informing early-stage target validation and translational biomarker strategies. The protocol reduces animal usage while enhancing predictive confidence in environmental modulation of disease-relevant pathways.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of maternal diet as a modifier of neurodevelopmental risk in NF1 models.
- Supports mechanistic de-risking by linking systemic metabolic changes to brain phenotypes.
- Facilitates functional target validation for environmental and genetic interactions.
Screening & Assay Development
- Provides high-quality, non-hemolyzed serum and intact brain tissue for downstream molecular assays.
- Standardizes sample collection for reproducibility across experimental cohorts.
- Enables quantitative assessment of macronutrient profiles and neurodevelopmental markers.
Translational & Preclinical Research
- Aligns preclinical models with human-relevant environmental exposures for translational continuity.
- Supports identification of serum biomarkers linked to neurodevelopmental outcomes.
- Informs risk-adjusted advancement of candidate interventions targeting metabolic or neurodevelopmental pathways.
Pipeline & Workflow Integration
This method integrates into the early discovery and preclinical continuum, bridging environmental exposure studies with molecular and phenotypic analyses in disease-relevant systems.
- Discovery Biology: Enables hypothesis testing on maternal diet impact and mechanistic pathway clarification in NF1 models.
- Screening: Delivers reproducible, quantitative outputs for serum and brain-based assays.
- Analytics: Supports comparative analysis of metabolic and neurodevelopmental readouts across experimental groups.
- Translational Research: Facilitates alignment of preclinical findings with potential human biomarkers and exposures.
- Enterprise Reuse: Establishes a standardized protocol adaptable to other genetic or environmental neurodevelopmental studies.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in environmental risk factors and target validation for neurodevelopmental disorders.
- Operational Value: Enhances reproducibility and standardization of sample collection across studies.
- Strategic Value: Improves go/no-go decision-making by linking systemic and neurodevelopmental data in a single workflow.
- Portfolio Impact: Supports risk-adjusted prioritization of early-stage interventions targeting metabolic or neurodevelopmental pathways.
Implementation Considerations
- Requires expertise in murine fetal dissection and sample handling to ensure tissue and serum integrity.
- Needs access to specialized dissection tools, centrifugation, and cryopreservation infrastructure.
- Demands cross-team standardization for reproducibility in multi-site studies.
- Adaptable to various genetic backgrounds and dietary interventions with protocol optimization.
- Sample quality and yield may vary with fetal stage and operator proficiency.
Biopharma FAQs
Chapters in this video
0:00
Introduction
1:19
Removing the Fetus from the Dam for Harvesting the Fetal Brain and Serum
2:09
Harvesting Serum from the Murine Fetus to Study Neurodevelopment in Neurofibromatosis Type 1
3:54
Harvesting the Brain from the Murine Fetus to Study Neurodevelopment in Neurofibromatosis Type 1
