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
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Inducing folic acid or choline dietary deficiency
<ol>
	<li>Before putting the females on diets, weigh all the animals. This will be referred to as the baseline weight.</li>
	<li>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.</li>
	<li>Maintain the female mice on the diets for 4-6 weeks prior to mating.</li>
	<li>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.</li>
	<li>Check the female mice daily for a copulatory plug, which indicates breeding has occurred.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
</ol>
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 beforehand13,14, and materials need to be prepared. These materials include tubes for storage, appropriate dissection tools, and dry ice or liquid nitrogen.
<ol>
	<li>Collect blood and liver tissues following previously published protocols15,16.
	<ol>
		<li>Collect ~500-1000 &#181;L of blood using a cardiac puncture&#160;following the steps below. For details, refer to Parasuraman et al.17:
		<ol>
			<li>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.</li>
			<li>Place the blood samples into EDTA-coated tubes. The tube can be left at room temperature until all samples have been collected.</li>
		</ol>
		</li>
		<li>Spin down the blood samples in a centrifuge at 1000-2000 x g for 7 min at 4 &#176;C to isolate the blood plasma. Collect the plasma into a 1.5 mL microfuge tube and then store it at -80 &#176;C until analysis.</li>
		<li>To remove the liver, open the abdomen and dissect the entire liver following previously published protocol15.
		<ol>
			<li>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 &#176;C until analysis.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

Effect of Maternal Folic Acid and Choline Deficiencies on Offspring Health

<ol>
	<li>Van Gool, J. D., Hirche, H., Lax, H., De Schaepdrijver, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folic+acid+and+primary+prevention+of+neural+tube+defects:+A+review.">Folic acid and primary prevention of neural tube defects: A review.</a> Reprod Toxicol. 80, 73-84 (2018).</li><li>Ali, S. A., Economides, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folic+acid+supplementation.">Folic acid supplementation.</a> Curr Opin Obstet Gynecol. 12 (6), 507-512 (2000).</li><li>Osterhues, A., Ali, N. S., Michels, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+folic+acid+fortification+in+neural+tube+defects:+a+review.">The role of folic acid fortification in neural tube defects: a review.</a> Crit Rev Food Sci Nutr. 53 (11), 575966 (2013).</li><li>Hurley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+dietary+deficiencies+in+folic+acid+and+choline+result+in+larger+damage+volume,+reduced+neuro-degeneration+and+-inflammation+and+changes+in+choline+metabolites+after+ischemic+stroke+in+middle-aged+offspring.">Maternal dietary deficiencies in folic acid and choline result in larger damage volume, reduced neuro-degeneration and -inflammation and changes in choline metabolites after ischemic stroke in middle-aged offspring.</a> Nutrients. 15 (7), 1556 (2023).</li><li>Clementson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+dietary+deficiencies+in+folic+acid+or+choline+worsen+stroke+outcomes+in+adult+male+and+female+mouse+offspring.">Maternal dietary deficiencies in folic acid or choline worsen stroke outcomes in adult male and female mouse offspring.</a> Neural Regen Res. 18 (11), 2443-2448 (2023).</li><li>Pull, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+maternal+dietary+folic+acid+or+choline+dietary+deficiencies+on+vascular+function+in+young+and+middle-aged+female+mouse+offspring+after+ischemic+stroke.">Impact of maternal dietary folic acid or choline dietary deficiencies on vascular function in young and middle-aged female mouse offspring after ischemic stroke.</a> Am J Physiol Heart Circ Physiol. 325 (6), H1354-H1359 (2023).</li><li>Virdi, S., Jadavji, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+maternal+folates+on+brain+development+and+function+after+birth.">The impact of maternal folates on brain development and function after birth.</a> Metabolites. 12 (9), 8766 (2022).</li><li>Barker, D. J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+nutrition+and+cardiovascular+disease+in+later+life.">Fetal nutrition and cardiovascular disease in later life.</a> Br Med Bull. 53 (1), 96-108 (1997).</li><li>Barker, D. J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+nutrition,+fetal+nutrition,+and+disease+in+later+life.">Maternal nutrition, fetal nutrition, and disease in later life.</a> Nutrition. 13 (9), 807-813 (1997).</li><li>Jackson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+age+matter?+The+impact+of+rodent+age+on+study+outcomes.">Does age matter? The impact of rodent age on study outcomes.</a> Lab Anim. 51 (2), 160-169 (2017).</li><li>Jim&#233;nez, J. A., Zylka, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+litter+effects+to+enhance+rigor+and+reproducibility+with+rodent+models+of+neurodevelopmental+disorders.">Controlling litter effects to enhance rigor and reproducibility with rodent models of neurodevelopmental disorders.</a> J Neurodev Disords. 13 (1), 2 (2021).</li><li>Breeding and Reproduction of Mice - All Other Pets. Merck Veterinary Manual Available from: <a target="_blank" href="https://www.merckvetmanual.com/all-other-pets/mice/breeding-and-reproduction-of-mice">https://www.merckvetmanual.com/all-other-pets/mice/breeding-and-reproduction-of-mice</a> (2024)</li><li>Jena, S., Chawla, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anatomy+of+the+laboratory+mouse.">The anatomy of the laboratory mouse.</a> Essentials of Laboratory Animal Science: Principles and Practices. , 159-185 (2021).</li><li>. The anatomy of the laboratory mouse Available from: <a target="_blank" href="https://www.informatics.jax.org/cookbook/imageindex.shtml">https://www.informatics.jax.org/cookbook/imageindex.shtml</a> (2024)</li><li>Mortimer, T., Welz, P. S., Benitah, S. A., Sassone-Corsi, P., Koronowski, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collecting+mouse+livers+for+transcriptome+analysis+of+daily+rhythms.">Collecting mouse livers for transcriptome analysis of daily rhythms.</a> STAR Protoc. 2 (2), 100539 (2021).</li><li>Aboghazleh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+brain+extraction+and+dissection:+A+comprehensive+approach.">Rodent brain extraction and dissection: A comprehensive approach.</a> MethodsX. 12, 102516 (2024).</li><li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sample+collection+in+small+laboratory+animals.">Blood sample collection in small laboratory animals.</a> J Parmacol Pharmacother. 1 (2), 87-93 (2010).</li><li>Jadavji, N. M., Deng, L., Malysheva, O. V., Caudill, M. A., Rozen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MTHFR+deficiency+or+reduced+intake+of+folate+or+choline+in+pregnant+mice+results+in+impaired+short-term+memory+and+increased+apoptosis+in+the+hippocampus+of+wild-type+offspring.">MTHFR deficiency or reduced intake of folate or choline in pregnant mice results in impaired short-term memory and increased apoptosis in the hippocampus of wild-type offspring.</a> Neuroscience. 300, 1-9 (2015).</li><li>Pickell, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+intake+of+folic+acid+disrupts+embryonic+development+in+mice.+Birth+defects+research.">High intake of folic acid disrupts embryonic development in mice. Birth defects research.</a> Birth Defects Res A Clin Mol Teratol. 91 (1), 8-19 (2011).</li><li>Mikael, L. G., Deng, L., Paul, L., Selhub, J., Rozen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moderately+high+intake+of+folic+acid+has+a+negative+impact+on+mouse+embryonic+development.">Moderately high intake of folic acid has a negative impact on mouse embryonic development.</a> Birth Defects Res A Clin Mol Teratol. 97 (1), 47-52 (2013).</li><li>Mikael, L. G., Pancer, J., Wu, Q., Rozen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disturbed+one-carbon+metabolism+causing+adverse+reproductive+outcomes+in+mice+is+associated+with+altered+expression+of+apolipoprotein+ai+and+inflammatory+mediators.">Disturbed one-carbon metabolism causing adverse reproductive outcomes in mice is associated with altered expression of apolipoprotein ai and inflammatory mediators.</a> J Nutr. 142 (3), 411-418 (2012).</li></ol>

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 mating4,6,14. Previous studies have only used C57Bl/6J mice4,5,6,18, but other groups have used BALB/c mice in similar studies19,20,21.
The manufactured diet used in this study contains 1% succinyl sulfathiazole to help reduce folate synthesis by intestinal flora; this is another reason that a control diet needs to be used, as it contains the same antibiotics that are present in the deficient diets. Once the diet has been manufactured and shipped, its storage is vital. It is important to follow the manufacturer&#39;s instructions so that the diet can last for the duration of the experimental study. It is important to note that standard mouse chow is formulated with elevated levels of folic acid and choline to support pregnancy and the overall health of mouse colonies. Therefore, the use of a control diet in studies primarily aimed at exploring the effects of folic acid and/or choline deficiency needs to be used. For such experimental studies, it is recommended to obtain control diets from the manufacturers with the minimum recommended amount of folic acid (2.0 mg/kg) or choline bitrate (1150 mg/kg)10 (Table 1).
Furthermore, combining both dietary folic acid and choline deficiency in females has not been tried yet. Both folic acid and choline are important methyl donors, and there is a high demand for them during pregnancy for developing offspring. Previous work has demonstrated that folate and choline metabolism are tightly linked to normal brain function16,17. This has also been demonstrated by others in other tissues18,19. It is unknown whether a diet deficient in folic acid and choline would yield viable offspring.
When considering experimental design, it is important to note that dietary deficiencies in 1C in males have been reported to negatively impact offspring health outcomes11. This is an important point to consider when placing males into cages with females that are fed either a folic acid or choline-deficient diet. At this point, the impact of short-term dietary deficiency in males remains unknown.
Successful mouse breeding can introduce challenges in experimental progress. It is recommended to consult animal care staff at the institution or the literature, including vendors that successfully breed mice. Weighing females on a weekly basis helped monitor pregnancy progression.
The long-term impact of maternal deficiencies in 1C during pregnancy on offspring health outcomes requires more investigation4,14. This protocol has described how to induce maternal folic acid or choline dietary deficiencies in a mouse model, as well as how to confirm it. To determine other potential markers of dietary deficiencies in folic acid or choline, ensure that Figure 2 is consulted18. This experimental model facilitates the study of maternal dietary deficiencies in folic acid or choline during pregnancy and lactation on long-term offspring health outcomes.

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 models4,5,6. 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 utero8,9, 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.

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

modifying levels of maternal dietary folic acid or choline to study the impact of deficiencies on offspring health outcomes

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.

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 deficient diets during the four weeks prior to mating (F (1.144,4.577) = 11.96, p = 0.196). A general observation made is that the number of offspring on these special diets is reduced in the number of offspring compared to standard mouse chow. This has not been determined to be statistically significant.
When the female mice are fed diets deficient in 1C, either folic acid or choline, there are changes in metabolites within body tissues that process 1C. Measurements of 1C and related metabolites can be taken using a variety of techniques. In the present study, liquid chromatography-tandem mass spectrometry with ultra-performance liquid chromatography (UPLC) was used, as previously described12. Both liver (Table 2) and plasma (Table 3) 1C measurements for total homocysteine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine, cystathionine, betaine, and choline are presented. There are different ways to measure these metabolites; LC-MS is just one way.
The data presented in Table 2 and Table 3 show some minor changes in metabolite levels. The current measurements were taken when females were mating on diets for 6 months. It might be better to measure the metabolites after 4 weeks or after one to two rounds of pregnancies. Data from the liver and plasma were analyzed using one-way ANOVAs and statistical and graphing software. Any significant ANOVA diet effects with Tukey&#39;s pairwise comparisons, as well as pairwise comparisons between control diet and folic acid or choline-deficient diets.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66827/66827fig01.jpg" /> 
Figure 1: Body weight of female mice maintained on one-carbon sufficient and deficient diets. Body weights of female mice prior to starting specially formulated diets (baseline) and then weekly weights for 4 weeks on control (CD), as well as folic acid (FADD) and choline (ChDD) deficient diets. Data is presented as mean + standard error of the mean. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66827/66827fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66827/66827fig02.jpg" /> 
Figure 2: One-carbon metabolism and related biological roles (methylation and nucleotide synthesis). Enzymes are indicated in black circles. Abbreviations: BHMT, betaine homocysteine methyltransferase; CHDH, choline dehydrogenase; COMT, catechol-O-methyltransferase; DHFR, dihydrofolate reductase; dTMP, deoxythymidine monophosphate; dTTP, deoxythymidine triphosphate; dUMP, deoxyuridine monophosphate; dUTP, deoxyuridine triphosphate; FR, folate receptor; MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; MTRR, methionine synthase reductase; RFC, reduced folate carrier; SAM, S-adenosylmethionine; SAH S-adenosylhomocysteine; and TS, thymidylate synthase. This figure is modified from Jadavjiet al.18. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66827/66827fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: List of micro- and macro-nutrient contents in one-carbon metabolism control, folic acid, choline-deficient diets. The folic acid-deficient diet contains 0.3 mg/kg of folic acid. The choline-deficient diet contains 300 mg/kg of choline bitrate. Values are listed in the table in red highlights within the brackets. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66827/Table 1.xlsx" target="_blank">Please click here to download this table.</a>
Table 2: Maternal one-carbon metabolite concentrations in isolated liver tissue. This data is adapted from Hurley et al.4. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66827/Table 2.xlsx" target="_blank">Please click here to download this table.</a>
Table 3: Maternal one-carbon metabolite concentrations in isolated plasma. This data is adapted from Hurley et al.4. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66827/Table 3.xlsx" target="_blank">Please click here to download this table.</a>

Author Spotlight: Studying the Impact of Maternal Dietary Deficiencies on Long-Term Offspring Health Outcomes

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.

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

Maternal nutrition during pregnancy and lactation plays an important role in the neurodevelopment of offspring. One-carbon (1C) metabolism, which centers ...

modifying-levels-maternal-dietary-folic-acid-or-choline-to-study

Nanyang Technological University

Research

JoVE Journal

Medicine

Neuroscience

348 Views.  Midwestern University. We zijn geïnteresseerd in het begrijpen hoe maternale voedingstekorten aan foliumzuur of choline tijdens zwangerschap en borstvoeding de gezondheidsresultaten van het nageslacht op de lange termijn beïnvloeden. We weten dat foliumzuur erg belangrijk is voor de sluiting van de neurale buis in de baarmoeder, maar mijn laboratorium is geïnteresseerd in welke rollen het speelt na de geboorte. We lezen onderzoeken die aantonen dat de voeding van de moeder tijde...

Video: Auteur in de schijnwerpers: onderzoek naar de impact van maternale voedingstekorten op de gezondheidsresultaten van nakomelingen op de lange termijn

Instrumentation of Near-term Fetal Sheep for Multivariate Chronic Non-anesthetized Recordings

The chronically instrumented pregnant sheep has been used as a model of human fetal development and responses to pathophysiologic stimuli such as endotoxins, bacteria, umbilical cord occlusions, hypoxia and various pharmacological treatments. The life-saving clinical practices of glucocorticoid treatment in fetuses at risk of premature birth and the therapeutic hypothermia have been developed in this model. This is due to the unique amenability of the non-anesthetized fetal sheep to the surgical placement and maintenance of catheters and electrodes, allowing repetitive blood sampling, substance injection, recording of bioelectrical activity, application of electric stimulation and in vivo organ imaging. Here we describe the surgical instrumentation procedure required to achieve a stable chronically instrumented non-anesthetized fetal sheep model including characterization of the post-operative recovery from blood gas, metabolic and inflammation standpoints.

The chronically instrumented non-anesthetized fetal sheep model is used to study human fetal development in health and disease, because it permits surgical placement and maintenance of catheters and electrodes, repetitive blood sampling, substance injection, recording of bioelectrical activity, and in vivo imaging. We describe the procedures required to establish this model.

The chronically instrumented pregnant sheep has been used as a model of human fetal development and responses to pathophysiologic stimuli such as ...

Developmental Biology

Moderate Prenatal Alcohol Exposure and Quantification of Social Behavior in Adult Rats

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several independent laboratories have demonstrated robust alterations in the social behavior of rodents exposed to alcohol during brain development across a wide range of exposure durations, timing, doses, and ages at the time of behavioral quantification. Prior work from this laboratory has identified reliable alterations in specific forms of social interaction following moderate prenatal alcohol exposure (PAE) in the rat that persist well into adulthood, including increased wrestling and decreased investigation. These behavioral alterations have been useful in identifying neural circuits altered by moderate PAE1, and may hold importance for progressing toward a more complete understanding of the neural bases of PAE-related alterations in social behavior. This paper describes procedures for performing moderate PAE in which rat dams voluntarily consume ethanol or saccharin (control) throughout gestation, and measurement of social behaviors in adult offspring.

The goal of the protocol presented here is to describe procedures to expose rats to moderate levels of alcohol during prenatal brain development and to quantify resulting alterations in social behavior during adulthood.

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several ...

Behavior

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Simultaneously Harvesting the Brain and Serum from the Murine Fetus

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

Author Spotlight: Collecting the Brain and Serum from the Same Mice Fetus to Study Brain Tumor Development

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, ...

Genetics

Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.

We demonstrate the construction of alcohol vapor chambers using readily available materials that simultaneously house 6 mouse cages. We further describe their use in a mouse model of fetal alcohol exposure equivalent to all 3&#160;trimesters of human pregnancy. This paradigm exposes animals during gestation and postnatal days 1-12.

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal ...

Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic Poly(I:C)

Maternal immune activation (MIA) model is increasingly well appreciated as a rodent model for the environmental risk factor of various psychiatric disorders. Numerous studies have demonstrated that MIA model is able to show face, construct, and predictive validity that are relevant to autism and schizophrenia. To model MIA, investigators often use viral mimic polyinosinic:polycytidylic acid (poly(I:C)) to activate the immune system in pregnant rodents. Generally, the offspring from immune activated dam exhibit behavioral abnormalities and physiological alterations that are associated with autism and schizophrenia. However, poly(I:C) injection with different dosages and at different time points could lead to different outcomes by perturbing brain development at different stages. Here we provide a detailed method of inducing MIA by intraperitoneal (i.p.) injection of 20 mg/kg poly(I:C) at mid-gestational embryonic 12.5 days (E12.5). This method has been shown to induce acute inflammatory response in the maternal-placental-fetal axis, which ultimately results in the brain perturbations and behavioral phenotypes that are associated with autism and schizophrenia.

Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic PolyI:C

Maternal immune activation (MIA) is a model for an environmental risk factor of autism and schizophrenia. The goal of this article is to provide a step-by-step procedure of how to induce MIA in the pregnant mice in order to enhance the reproducibility of this model.

Maternal immune activation (MIA) model is increasingly well appreciated as a rodent model for the environmental risk factor of various psychiatric ...

Immunology and Infection

Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is well-defined, and it is relatively easy to achieve precise and timely exposure via multiple methods. Moreover, the process of heart development in chicken embryos is similar to mammals, also resulting in a four-chambered heart, making it a valuable alternative model in the assessment of developmental cardiotoxicities. In our lab, the chicken embryo model is routinely used in the assessment of developmental cardiotoxicities following exposure to various environmental pollutants, including per- and polyfluoroalkyl substances (PFAS), particulate matter (PMs), diesel exhaust (DE) and nano materials. The exposure time can be freely selected based on the need, from the beginning of development (embryonic day 0, ED0) all the way to the day prior to hatch. The major exposure methods include air-cell injection, direct microinjection, and air-cell inhalation (originally developed in our lab), and the currently available endpoints include cardiac function (electrocardiography), morphology (histological assessments) and molecular biological assessments (immunohistochemistry, qRT-PCR, western blotting, etc.). Of course, the chicken embryo model has its own limitations, such as limited availability of antibodies. Nevertheless, with more laboratories starting to utilize this model, it can be used to make significant contributions to the study of developmental cardiotoxicities.

Chicken embryos, as a classical developmental model, are used in our lab to assess developmental cardiotoxicities following exposure to various environmental contaminants. Exposure methods and morphological/functional assessment methods established are described in this manuscript.

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

ICSI-ET Mouse Model to Study Long-Term Impacts on Glucose Metabolism

Monitoring Blood Glucose in Mouse Offspring After Intracytoplasmic Sperm Injection

Human lifespan is considerably long, while mouse models can simulate the entire human lifespan in a relatively short period, with one year of mouse life roughly equivalent to 40 human years. Intracytoplasmic sperm injection (ICSI) is a commonly used assisted reproductive technology in clinical practice. However, given its relatively recent emergence about 30 years ago, the long-term effects of this technique on human development remain unclear. In this study, we established the ICSI combined with embryo transfer (ET) method using a mouse model. The results demonstrated that normal mouse sperm, after undergoing in vitro culture and subsequent ICSI, exhibited a fertilization rate of 89.57% and a two-cell rate of 87.38%. Following ET, the birth rate of offspring was approximately 42.50%. Furthermore, as the mice aged, fluctuations in glucose metabolism levels were observed, which may be associated with the application of the ICSI technique. These findings signify that the mouse ICSI-ET technique provides a valuable platform for evaluating the impact of sperm abnormalities on embryo development and their long-term effects on offspring health, particularly concerning glucose metabolism. This study provides important insights for further research on the potential effects of the ICSI technique on human development, emphasizing the necessity for in-depth investigation into the long-term implications of this technology.

Author Spotlight: Exploring the Long-Term Health Impacts of Intracytoplasmic Sperm Injection on Offspring

Here, we present a protocol to establish an intracytoplasmic sperm injection (ICSI)-embryo transfer (ET) mouse model, allowing us to observe age-related changes in glucose metabolism that may be attributed to ICSI, providing insights into its potential long-term impacts on human development.

Human lifespan is considerably long, while mouse models can simulate the entire human lifespan in a relatively short period, with one year of mouse life ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

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

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