Modifying Levels of Maternal Dietary Folic Acid or Choline to Study the Impact of Deficiencies on Offspring Health Outcomes

Summary

This protocol describes the method for reducing dietary intake of folic acid or choline in female mice prior to pregnancy with the objective of investigating the impact of maternal diet on offspring health outcomes.

Abstract

Maternal nutrition during pregnancy and lactation plays an important role in the neurodevelopment of offspring. One-carbon (1C) metabolism, which centers around folic acid and choline, as well as other B vitamins, plays a key role during the closure of the neural tube of the developing fetus. However, the impact of these maternal nutritional deficiencies during pregnancy on offspring health outcomes after birth remains relatively undefined. Furthermore, maternal dietary deficiencies in folic acid or choline may impact other health outcomes in offspring - making this a valuable model. This protocol aims to outline the procedure for inducing a deficiency in 1C metabolism in female mice through dietary modifications. Females are placed on diets at weaning, up to 2 months of age, for 4-6 weeks prior to mating and remain on diet throughout pregnancy and lactation. Offspring from these females can be evaluated for health outcomes. Females can be used multiple times to generate offspring, and tissues from females can be collected to measure for 1C metabolite measurements. This protocol provides an overview of how to induce maternal dietary deficiencies in folic acid or choline to study offspring health outcomes.

Introduction

Maternal nutrition during pregnancy and lactation plays a vital role in offspring development. One-carbon (1C) metabolism is a key metabolic network that integrates nutritional signals with biosynthesis, redox homeostasis, and epigenetics. Folic acid and choline are central components of 1C that play an important role in the closure of the neural tube. It is widely recommended for women to supplement with folic acid and choline during their childbearing years¹. While the critical role of folic acid in fetal development is clearly established, the underlying mechanisms involved remain unclear. Women of childbearing are recommended to take folic acid prior to becoming pregnant since the closure of the neural tube occurs within the first trimester². Pregnant mothers who are deficient in folic acid or other components of 1C, like choline, have a higher incidence of birthing offspring with neural tube defects due to the high demand for nucleotide synthesis and other processes required by the early gestational period³.

The long-term impact of maternal diets deficient in 1C metabolism intake on offspring health outcomes is poorly understood. Research from our laboratories, as well as others, has demonstrated that maternal diets deficient in either folic acid or choline do indeed impact offspring health outcomes after ischemic stroke using preclinical models^4,5,6. The impact of maternal diet has also been shown in clinical studies where smaller brain volumes and altered methylation levels have been described⁷. The Developmental Origins of Health and Disease (DOHaD) theory suggests that prospective chronic diseases are programmed in utero^8,9, and maternal diet plays an important role.

This protocol is designed to outline the method for altering 1C dietary consumption in female mice before pregnancy, with the purpose of examining the effects of maternal nutrition on the health outcomes of the offspring.

Protocol

This protocol was approved, and all the animal subjects were used in accordance with the guidelines of the Institutional Animal Care and Use Committee of Midwestern University (Protocol 2983). Female C57BL/6J mice, 2 months of age (body weight, ~20 g), were used in this study. In general, female mice of reproductive age can be used¹⁰. To ensure litter effects are avoided, multiple females need to be used for each diet¹¹. The details of the reagents and the equipment used in the study are listed in the Table of Materials.

1. Experimental preparation

1. Obtain folic acid or choline-deficient diet from commercial rodent food manufacturers. Refer to Table 1 for the detailed list of micro- and macro-nutrients, including the exact amount of folic acid or choline needed in deficient diets.
   NOTE: A corresponding control diet needs to also be ordered; details are listed in Table 1. These diets were based on AIN93G and contained the recommended nutrients for rodents¹⁰.
2. In collaboration with the manufacturer, determine the coloring of the diets, as this can help with the distribution of correct diets during experimental studies. This study used green for folic acid deficient diets, yellow for choline, and white for control diet. They are all easily distinguishable.
   NOTE: There are no restrictions on water consumption. The water does not contain any folic acid, choline, or other 1C metabolites.

2. Inducing folic acid or choline dietary deficiency

1. Before putting the females on diets, weigh all the animals. This will be referred to as the baseline weight.
2. Depending on the individual laboratory cage setup (ventilated or unventilated, see Table of Materials), replace or top up the special diet pellets for each cage a few times a week. Do not overfill the cage food hoopers with special diets, as this may lead to the deterioration of pellets once they interact with saliva from animals and spoilage over time.
   NOTE: Ensure that diet stock is stored as per manufacturer guidelines and used prior to the expiration date.
3. Maintain the female mice on the diets for 4-6 weeks prior to mating.
4. Introduce the male mice into the female cages for mating. Ensure that males are introduced into female cages after females have been on special diets for a minimum of 4-6 weeks. Introduce one male into one female cage for one to several days.
   NOTE: In the present study, males were kept with the females for the entire duration of the experiment (~4 months) to increase the number of pups produced since mating in mice can occur after birth¹². Females after 6 months of age were not used for any studies.
5. Check the female mice daily for a copulatory plug, which indicates breeding has occurred.
6. Collect the weight measurements of the female mice every week to track their health and pregnancy status.
   NOTE: It is safe for females to be maintained on the diet for pregnancy (~21 days) and lactation (~21 days).
7. When offspring are weaned from the mother (~21 days after birth), place them on a folic acid and choline-deficient diet, as well as a control diet. For details, see the results section.
   NOTE: When studying the impact of maternal diet on offspring health outcomes, it is recommended that the offspring are placed on a control diet after being weaned. The control diet must contain adequate amounts of vitamins and nutrients for survival.
8. At the end of the study (determining the weekly weights to monitor the overall health of females maintained on folic acid or choline-deficient diets), euthanize the mothers using an overdose of CO₂ (dosage, 10%-30% volume displacement per minute, following institutionally approved protocols) and collect the tissues (step 3) to confirm dietary deficiencies.

3. Tissue collection for the measurements of 1C and the related metabolites

NOTE: When euthanizing animals, it is important to make sure the animals are not responsive to a toe pinch. Once the animals are not responsive, the dissection of tissues can begin. The tissue that will be dissected needs to be determined beforehand^13,14, and materials need to be prepared. These materials include tubes for storage, appropriate dissection tools, and dry ice or liquid nitrogen.

1. Collect blood and liver tissues following previously published protocols^15,16.
   1. Collect ~500-1000 µL of blood using a cardiac puncture following the steps below. For details, refer to Parasuraman et al.¹⁷:
      1. Make a V-shaped cut through the skin and the abdominal wall. Move the abdominal organs aside and insert a needle through the diaphragm and into the heart to collect the blood sample.
      2. Place the blood samples into EDTA-coated tubes. The tube can be left at room temperature until all samples have been collected.
   2. Spin down the blood samples in a centrifuge at 1000-2000 x g for 7 min at 4 °C to isolate the blood plasma. Collect the plasma into a 1.5 mL microfuge tube and then store it at -80 °C until analysis.
   3. To remove the liver, open the abdomen and dissect the entire liver following previously published protocol¹⁵.
      1. After removing the liver from the body cavity, cut the tissue into smaller pieces with a small scissor and snap freeze them using dry ice or liquid nitrogen.
         NOTE: The frozen pieces of tissue can be placed in a 1.5 mL cryogenic microcentrifuge tube and kept at -80 °C until analysis.

Results

The focus of this study was to investigate the impact of maternal dietary deficiencies on folic acid or choline on offspring health outcomes. Weekly weights were determined to monitor the overall health of females maintained on folic acid or choline-deficient diets. In our previous work, no changes in body weight were observed between maternal dietary groups⁴. Figure 1 depicts the baseline and weekly weights for females maintained on control, choline, or folic acid de...

Discussion

During the implementation of this protocol in research laboratories, there are several important notes that researchers need to consider prior to starting experiments. Firstly, in diet manufacturing, enough lead time needs to be given to manufacture the diet before the start of the experimental study. Lead times can vary from company to company, and shipment of diet also needs to be factored in. Female mice that are at reproductive age (~2-2.5 months in age) can be placed on the diet 4-6 weeks prior to mating

Materials

Name	Company	Catalog Number	Comments
1.5 mL tubes	Eppendorf
C57BL/6 Female Mice	Charles River	27	Mice can be bred in house as well.
Choline Deficient Diet	Envigo	TD.06119
Control Diet	Envigo	TD.190790
EDTA tubes
Folic Acid Deficient Diet	Envigo	TD.01546
GraphPad prism			https://www.graphpad.com/features
Housing for rodents	Lab Products, LLC	One CageVentilated Racks & Cages
Liquid chromatography-tandem mass spectrometry (LC-MS/MS)	SCIEX, Framingham, MA	5500 QTrap
Ultra-performance liquid chromatography (UPLC)	Shimadzu, Columbia, MD, USA	Nexera