Impact of maternal dietary folic acid or choline dietary deficiencies on vascular function in young and middle-aged female mouse offspring after ischemic stroke

Abstract

Adequate maternal dietary levels of one-carbon metabolites, such as folic acid and choline, play an important role in the closure of the neural tube in utero; however, the impact of deficiencies in one-carbon (1C) metabolism on offspring neurological function after birth remain undefined. Stroke is one of the leading causes of death and disability globally. The aim of our study was to determine the impact of maternal 1C nutritional deficiencies on cerebral and peripheral blood flow after ischemic stroke in adult female offspring. In this study, female mice were placed on either control (CD)-, folic acid (FADD)-, or choline (ChDD)-deficient diets before pregnancy. Female offspring were weaned onto a CD for the duration of the study. Ischemic stroke was induced in offspring and after 6 wk cerebral and peripheral blood flow velocity was measured using ultrasound imaging. Our data showed that 11.5-mo-old female offspring from ChDD mothers had reduced blood flow in the posterior cerebral artery compared with controls. In peripheral blood flow velocity measurements, we report an aging effect. These results emphasize the importance of maternal 1C diet in early life neuro-programming on long-term vasculature health.

NEW & NOTEWORTHY We demonstrate that a maternal dietary deficiency in one-carbon (1C) metabolites result in reduced cerebral blood flow in adult female offspring after ischemic stroke, but the long-term effects are not present. This result points to the key role of the maternal diet in early life neuroprogramming, while emphasizing its effects on both fetal development and long-term cerebrovascular health.

Listen to this article’s corresponding podcast at https://ajpheart.podbean.com/e/impact-of-maternal-diet-on-offspring-blood-flow/.

INTRODUCTION

Maternal nutrition during pregnancy and lactation is recognized as a critical factor determining the health of offspring (1–4). The developmental origins of health and disease (DOHaD) theory suggest that prospective chronic diseases are programmed in utero (5, 6). In offspring, maternal dietary deficiencies have been associated with modified neural tube closure (7, 8) and neurocognitive development (9–12). Epidemiological studies have demonstrated the effect of maternal diet on lifelong cardiovascular and neurological function (13). Among nutritional cofactors, folates and choline are important players in healthy fetal neurodevelopment due to their involvement in the closure of the neural tube and are components of one-carbon (1C) metabolism (14). Folates and its chemically synthesized form folic acid are important for fetal neurodevelopment (15), as folate requirements during pregnancy are increased by five- to tenfold compared with nonpregnant women (16).

The link between maternal nutrition and fetal development is abundantly clear, but the long-term, postnatal, effects of maternal nutritional deficiencies on adult offspring are not clearly understood. In this study, we aim to improve our understanding of maternal 1C metabolite deficiencies’ impact on adult offspring ischemic stroke outcomes. Stroke is among the leading causes of death globally and its prevalence as a major health concern is predicted to increase, as the global population ages. In addition, the demographics of populations change, for example, there is a rise in the number of people affected by hypertension, diabetes, and obesity, all increasing risk factors for ischemic stroke (17, 18). One of the many reasons these problems exist is that the majority of preclinical studies are targeted only toward male subjects (19). Over 90% of preclinical studies use strictly male mice whereas all clinical studies use equal parts male and female participants (19, 20). This makes clinical pharmaceutical findings favor better outcomes in males (21, 22). Furthermore, maternal perturbations during pregnancy like stress negatively impact the neurodevelopment of males more than females (23). To address the gap in the literature, we investigated the impact of maternal nutritional deficiencies in folic acid or choline on cerebral and peripheral blood flow velocity after ischemic stroke in female offspring.

MATERIALS AND METHODS

Experimental Design

All animal experimentation was performed following approval by the Midwestern University Institutional Animal Care and Use Committee (Protocol No. 2983) in accordance with animal welfare and ARRIVE guidelines. Briefly, for the maternal cohort female and male mice (RRID: IMSR_JAX:000664) were purchased from Jackson Laboratories. The mice were acclimatized for 1 wk to controlled housing conditions with ad libitum access to food and water (Fig. 1). At 2 mo of age, females were randomized to control diet (CD, TD.190790, 2 mg/kg folic acid, 1,150 mg/kg choline bitartrate), commercially (Envigo) prepared folic acid-deficient diet (FADD, TD.01546, 0.3 mg/kg folic acid), or choline-deficient diet (ChDD, TD.06119, 300 mg/kg choline bitartrate) and maintained on these diets for 4 wk before mating, throughout pregnancy (3 wk) and lactation (3 wk) for a total of 10 wk (24–26). Females were group housed until after ischemic stroke when they were housed individually.

Figure 1.

Experimental timeline. Beginning at 2 mo of age female mice were fed either control diet (CD), folic acid-deficient diet (FADD), or choline-deficient diet (ChDD). The female mice were maintained on these diets throughout the pregnancy and lactation until the offspring were weaned. Once the offspring were weaned, they were fed the CD. Separate cohorts of female offspring at 2 or 10 mo of age had ischemic stroke induced via the photothrombosis (PT) model. At 3.5 (CD, n = 6; FADD, n = 7; ChDD, n = 6) and 11.5 (CD, n = 6; FADD, n = 6; ChDD, n = 6) mo of age all female mouse offspring underwent ultrasound imaging (U).

At 3 wk of age, newly born female pups were weaned and were maintained on the CD ad libitum for the duration of the experiment. Offspring were randomized to one of two cohorts undergoing photothrombotic stroke at either 2 or 10 mo of age, followed by ultrasound measurements.

Photothrombosis

When female offspring reached 2 or 10 mo of age, ischemia was induced using the photothrombosis model (27–34). Briefly, Rose Bengal dye (Sigma-Aldrich) 10 mg/kg of the photosensitive was injected intraperitoneally 5 min before irradiation. A 532-nm green laser (MGM20, 20–25 mW, Beta Electronics) was placed 3 cm above the animal and directed to the sensorimotor cortex (mediolateral ± 0.24 mm) for 15 min. At the completion of the study brain tissue was collected, sectioned, and stained to confirm ischemic damage (27–34).

Ultrasound Imaging

Approximately 45 days after ischemic stroke induction, in vivo ultrasound imaging of posterior cerebral artery (PCA) blood flow, coronary artery blood flow, and aortic and cardiac function were performed using Vevo 2100 ultrasound system (FUJIFILM, VisualSonics), the high-frequency/high-resolution ultrasound system used in this study was equipped with a 40-MHz transducer (MS550S) with a focal length of 7.0 mm, frame rate of 557 frames/s (single zone, 5.08 mm width, B-mode), and a maximum two-dimensional field of view of 14.1 × 15.0 mm with a spatial resolution of 90-µm lateral by 40-µm axial. As described in our previous published reports (35–37), mice were anesthetized in an induction chamber. Cardiac parameters, including stroke volume, ejection fraction, fractional shortening, and cardiac output, were calculated from the left ventricular (LV) parasternal short-axis M-mode view.

Aortic pulse wave velocity (PWV) was obtained from the B-mode, Doppler view of the aortic arch. The coronary artery peak flow in systole and diastole was measured from the long-axis, B-mode Doppler view. The PCA peak blood flow was measured using PW Doppler mode using a 24-MHz (MS250) transducer.

Statistics

Ultrasound data were performed and analyzed by two individuals who were blinded to experimental treatment groups using Vevo Lab ultrasound analysis software (FUJIFILM, VisualSonics). With GraphPad Prism 9.0, two-way ANOVA analysis was performed to assess maternal diet and age effects, and the number of animals per group was 5 to 7 per group. Significant main effects of two-way ANOVAs were followed up with Tukey’s honestly significant difference post hoc test to adjust for multiple comparisons. All data are presented as means ± SE. Statistical tests were performed using a significance level, P < 0.05.

RESULTS

Ischemic Volume Damage in Brain Tissue

To confirm that mice had an ischemic stroke, brain tissue was collected from all animals and damage was confirmed by staining sectioned tissue and quantifying damage volume using microscopy (data not shown) (34).

Cerebral Blood Flow

At 45 days postischemic stroke, cerebral blood flow was measured in the posterior cerebral artery using ultrasound. Representation of the posterior cerebral artery is shown in Fig. 2A. Maternal diet impacted blood flow velocity within the posterior cerebral artery (Fig. 2B; P = 0.009, n = 4–8/group). There was no effect of offspring age (P = 0.37, n = 4–8/group) and no interaction between both factors (P = 0.30, n = 4–8/group).

Figure 2.

The impact of maternal diet on posterior artery blood flow velocity, aorta, and coronary artery function. A: visual representation of mouse cerebral vasculature, posterior cerebral artery (PCA), and location of ischemic stroke. B–D: blood flow velocity in the PCA (B), aortic pulse wave velocity as an index of aortic wall stiffness (C), and ratio of systolic and diastolic coronary flow velocity (D) after ischemic stroke in 3.5- and 11.5-mo-old female offspring from mothers on control diet (CD), folic acid-deficient diet (FADD), or choline-deficient diet (ChDD). Scatter plot with means ± SE of 6 or 7 mice/group. **P < 0.01, Tukey’s pairwise comparison. Images in A were created using a licensed version of BioRender.com.

Measurements of Cardiac Function and Structure

Cardiac function and structure were evaluated at a 1.5-mo time point after ischemic stroke using ultrasound. Both maternal diet (P = 0.03, n = 4–8/group) and offspring age (P = 0.001) impacted average heart rate (Table 1). Aged (11.5 mo old) offspring from CD (P < 0.01), FADD (P < 0 .01), and ChDD (P < 0.01) mothers had higher heart rates compared with 3.5-mo-old controls (Table 2). Although exclusively offspring age affected ejection fraction (P = 0.01) and cardiac output (P < 0.0001), there were no pairwise differences (Table 2). There were interactions between maternal diet and offspring age for stroke volume (P = 0.03), and there was a difference between 3.5- and 11.5-mo-old FADD offspring (Table 2). There were no differences between groups for fractional shortening and internal diameter in systolic and diastolic measurements.

Table 1.

Impact of maternal diet on cerebral and peripheral blood flow after ischemic stroke in female offspring

	Offspring Age		P Values
	3.5 mo	11.5 mo	Diet	Age	Diet × Age
Average heart rate, beats/min
CD	494.3 ± 13.23	524.78 ± 8.82
FADD	504.5 ± 12.68	531.42 ± 4.02	0.0261*	0.0010**	0.1374
ChDD	513.0 ± 9.55	531.30 ± 6.02
Average stroke volume, µL
CD	24.04 ± 2.54	23.55 ± 4.55
FADD	28.67 ± 2.17	37.47 ± 5.56	0.2744	0.2319	0.0282*
ChDD	25.26 ± 2.06	38.24 ± 2.08
Average ejection fraction, %
CD	86.58 ± 4.24	84.88 ± 3.55
FADD	84.68 ± 3.09	73.10 ± 4.61	0.1638	0.009*	0.2929
ChDD	86.46 ± 2.30	74.05 ± 2.49
Average fractional shortening, %
CD	57.94 ± 6.28	53.65 ± 4.18
FADD	53.89 ± 3.67	53.92 ± 3.88	0.3924	0.1811	0.4998
ChDD	54.89 ± 3.13	43.70 ± 4.04
Average cardiac output, mL/min
CD	11.77 ± 1.04	20.32 ± 0.78
FADD	14.43 ± 1.08	19.91 ± 2.94	0.7197	<0.0001***	0.5400
ChDD	12.94 ± 1.03	20.28 ± 0.96
Left internal diameter in systole, mm
CD	0.90 ± 0.08	0.90 ± 0.10
FADD	1.13 ± 0.10	0.91 ± 0.05	0.2428	0.4138	0.3333
ChDD	0.95 ± 0.09	0.99 ± 0.12
Left internal diameter in diastole, mm
CD	1.37 ± 0.10	1.56 ± 0.12
FADD	1.71 ± 0.04	1.44 ± 0.18	0.08098	0.5384	0.4547
ChDD	1.61 ± 0.15	1.53 ± 0.12

Values are means ± SE; n = 5–7 mice/group. Maternal diets included control diet (CD), folic acid-deficient diet (FADD), and choline-deficient diet (ChDD). P values result from two-way ANOVA of cerebral and peripheral blood flow in 3.5- and 11.5-mo-old female mouse offspring.

^*P < 0.05; **P < 0.01; and ***P < 0.001 significant diet, age, or diet and age interaction.

Table 2.

Tukey’s HSD post hoc pairwise analysis of cardiac function in 3.5- and 11.5-mo-old female mouse offspring

Maternal Diet
3.5-mo offspring age	11.5-mo offspring age	P Value
Average heart rate
CD	CD	0.0061**
CD	FADD	0.0027**
CD	ChDD	0.0028**
FADD	CD	0.9755
FADD	FADD	0.8215
FADD	ChDD	0.8263
ChDD	CD	0.9290
ChDD	FADD	0.7478
ChDD	ChDD	0.7525
Average stroke volume
CD	CD	0.9985
CD	FADD	0.2006
CD	ChDD	0.9997
FADD	CD	>0.9999
FADD	FADD	0.0426*
FADD	ChDD	0.9331
ChDD	CD	0.8983
ChDD	FADD	0.6806
ChDD	ChDD	0.9978
CD	CD	0.9985
Average ejection fraction
CD	CD	0.9993
CD	FADD	0.1809
CD	ChDD	0.1476
FADD	CD	>0.9999
FADD	FADD	0.2741
FADD	ChDD	0.2275
ChDD	CD	0.9997
ChDD	FADD	0.2825
ChDD	ChDD	0.2493
Average cardiac output
CD	CD	0.0020*
CD	FADD	0.0057*
CD	ChDD	0.0036*
FADD	CD	0.0355*
FADD	FADD	0.0825
FADD	ChDD	0.0543
ChDD	CD	0.0245*
ChDD	FADD	0.0499*
ChDD	ChDD	0.0343*

Maternal diets included control diet (CD), folic acid-deficient diet (FADD), and choline-deficient diet (ChDD). HSD, honestly significant difference. P values are of 5 to 7 mice/group.

^*P < 0.05; **P < 0.01, post hoc pairwise comparison.

Aortic Pulse Wave Velocity

Aortic pulse wave velocity is a measure of aortic wall stiffness and was evaluated 1.5 mo after ischemic stroke using ultrasound. Offspring age impacted pulse wave velocity (Fig. 2C, P < 0.0001, n = 4–8/group), there were differences between 3.5- and 11.5-mo-old CD (P < 0.01) and ChDD (P < 0.01) offspring. There was no impact of maternal diet (P = 0.81) or interaction between maternal diet and offspring age (P = 0.15).

Measurements of Coronary Artery Function

Coronary artery function was measured using ultrasound 45 days after ischemic stroke using ultrasound. There was significant interaction between maternal diet and age for coronary artery velocity ratio (Fig. 2D, P = 0.02, n = 4–8/group). There was no impact of material diet (P = 0.38) or age (P = 0.95).

DISCUSSION

The DOHaD theory suggests that prospective chronic diseases are programmed in utero, giving rise to programming of offspring cardiovascular, metabolic, and neuroendocrine dysfunction (5, 6). Despite impressive evidence of the importance of the maternal environment for fetal growth and development in utero, there is not much information available on the impact on offspring as they age. Nutrition is a modifiable risk factor (38) and can also impact outcomes after ischemic stroke (27–30, 33, 34, 39). Using an experimental model of ischemic stroke, our study determined the impact of perinatal maternal dietary deficiencies in folic acid and choline on measures of cerebral, cardiac, aorta, and coronary in offspring, following ischemic injury. Our results demonstrate that maternal diet impacts cerebral blood in offspring after an ischemic stroke in female offspring. The effects of both maternal diet and offspring age, as well as interactions between these variables, were observed for numerous cardiac, aortic, and coronary artery functions after ischemic stroke in female offspring. Our blood flow measurements were taken in 3.5-mo-old mice, which correspond to a mature adult 20–30 yr of age, and 11.5-mo-old mice, which correspond to middle-aged humans, 38–47 yr of age according to the Jackson Laboratory (40).

The neurovascular unit (NVU) comprises a number of unique neuronal, glial, and endothelial cell types, and recent findings indicate unique cross talk between neurons and the cerebral vasculature (41). The NVU is responsible for the maintenance of a highly selective blood–brain barrier and cerebral homeostasis, as well as the control of cerebral blood flow (42). The impact of maternal diet on the NVU, modulating integrity of cerebral blood vessels and closure of the neural tube has been established (42). Our study adds to these investigations by assessing the response of blood flow within the PCA in both young and aged offspring. The contralesional PCA was selected as an index of cerebral blood flow because of its spatial and functional independence from the sensorimotor cortex targeted during photothrombotic stroke (43), and evident correlation to measures of the middle cerebral artery (44).

We did not observe the effect of maternal diet on cerebral blood flow in 11.5-mo-old offspring. This could be due to the well-investigated aging-associated changes in the structural and functional integrity of the vasculature (45). Therefore, we propose that the difference in effect between young and old female offspring is a result of aging. In addition, the presumed damage or endothelial dysfunction induced by the maternal choline- and folic acid-deficient diets is long lasting and may contribute to premature aging, generating a mathematically significant difference when compared with young, healthy controls, but only a minor difference when compared with senescent offspring displaying similar levels of vascular dysfunction. This result is supported by the interaction effect of diet and offspring age and requires further investigation.

The present study has demonstrated novel data in the field of maternal dietary deficiencies and blood flow after ischemic stroke in offspring. Our data have demonstrated that there are changes in vasculature of female offspring after ischemic stroke, because of maternal diet, this is the first study of its kind. Furthermore, our data point to the need for rodent models spanning a variety of ages for research in age-related diseases such as stroke and vascular dysfunction. We recognize that the exclusion of male subjects in this study may limit our ability to draw conclusions with respect to the impact of sex hormone in observed phenomenon. Future studies are needed to include male animals and design experiments that would also allow investigation of the role of paternal dietary effects. In addition, we plan to further age animals to ∼20 mo after ischemic stroke, as well as investigate the role of over supplementation on blood flow after stroke. A detailed analysis of angiogenesis after ischemic stroke might also be prudent.

In conclusion, maternal dietary deficiencies in folic acid and choline have unique roles in cerebral and peripheral blood flow after ischemic stroke in female offspring. Maternal nutrition during pregnancy and lactation has effects, even after infancy and childhood. Our work demonstrated that an age effect in animal models encourages further comprehensive longitudinal time-point studies that include older age animals.

DATA AVAILABILITY

N.M. Jadavji, corresponding author, may be contacted for all data requests related to this study.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grant R15HL145646 (to M. Esfandiarei) and American Heart Association Grant 20AIREA35050015 (to N.M. Jadavji).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

M.E. and N.M.J. conceived and designed research; R.F., B.G., and N.M.J. performed experiments; K.P., R.F., J.K., S.J., B.G., and N.M.J. analyzed data; K.P., B.G., M.E., and N.M.J. interpreted results of experiments; N.M.J. prepared figures; K.P. and N.M.J. drafted manuscript; M.E. and N.M.J. edited and revised manuscript; M.E. and N.M.J. approved final version of manuscript.