sow nursing piglets Jevtic/iStock/Thinkstock

N&H TOPLINE: Research looks at how prenatal interventions affect brain development

Choline and prebiotic research uses piglets as translational model for human growth and development.

Pediatric nutrition research has shown the important effects of early-life nutrition on a baby’s development — especially the gastrointestinal tract — and more recent research indicates that nutrition may also have an influence on an infant’s brain as it develops.

For nearly a decade, researchers at the University of Illinois have studied the piglet as a translational model to understand which aspects of early brain development are affected by nutrition interventions. Most recently, the Piglet Nutrition & Cognition Laboratory at the University of Illinois studied maternal choline supplementation on piglet brain development as well as prebiotics included in infant formulas.

Choline intake during pregnancy can influence infant metabolism and brain development, according to a series of studies the lab conducted. Although the role of choline in neurodevelopment has been studied before in rodents, the new research, done with pigs, has more relevance to humans.

"We know the pig is a good model for humans because they have the same nutrient requirements, similar metabolic function and also have very similar brain development, following the same growth trajectories," said Austin Mudd, a doctoral student in the Neuroscience Program at the University of Illinois. "The pig is bridging the gap between the mechanistic work we see in rodents and that higher-level cognitive function that they're looking at in humans."

Choline, found in liver, eggs, wheat germ and other foods, is an essential nutrient in human and animal diets. It's required to make cell membranes, neurotransmitters and myelin, the fatty sheath surrounding nerve cells. Most adults, including pregnant women, don't consume enough choline.

"In the U.S., 90% of us don't meet our choline requirement," Mudd said. "The most recent data say pregnant women, who should consume 450 mg per day, may not even be reaching 300 mg."

To understand how choline deficiency affects the developing brain during and after pregnancy, Mudd and his collaborators gave pregnant sows choline-deficient or choline-sufficient diets through the second half of their pregnancies. After weaning, piglets were fed choline-deficient or choline-sufficient milk replacer for 30 days. Then, the month-old piglets were scanned by magnetic resonance imaging (MRI).

Mudd analyzed the MRI images of the piglet brains in terms of volume and makeup. The first analysis, reported in a 2016 article in Nutritional Neuroscience, compared the volumes of 19 brain regions in piglets that had received deficient or sufficient choline prenatally and postnatally.

The second analysis, published last week in Current Developments in Nutrition, corrected for volume differences to isolate differences in the gray and white matter concentration in the piglets' brains.

"In pigs from choline-deficient moms, their brains were about 10% smaller overall," Mudd said, adding that 11 of the 19 regions were significantly smaller in choline-deficient brains.

When Mudd corrected for these volume differences to look specifically at gray and white matter concentration, the story was the same: Piglets whose mothers consumed sufficient choline during pregnancy had higher concentrations of gray and white matter in the brain's cortical regions.

Gray matter is made up of primarily the neurons themselves, while white matter comprises the material that connects neurons and bridges different parts of the brain.

"In our first paper, we saw that the left and right cortex were larger in the choline-sufficient pigs," he said. "After our second analysis, that makes sense. If you have a greater density of gray matter in the cortex, it is likely that brain region will have a greater volume as well."

Decades' worth of research has shown that particular nutrients play specific roles in the neurodevelopmental process. The use of MRI technology has allowed researchers to identify the global influence of a specific nutrient on particular aspects of brain development. In an earlier study, the researchers found that another nutrient — iron — influenced aspects of both gray and white matter development.

"Our research shows that choline, like iron, does not appear to be specific to one part of brain development; it's important for all of it," Mudd said.

All of these results were for piglets born to choline-deficient mothers. However, as part of the study, some of the pigs from choline-deficient mothers were given adequate amounts of choline after birth to see if it was enough to offset the deficiency during pregnancy.

The short answer is no -- at least not for the brain. "Postnatal supplementation cannot correct for prenatal deficiency. It has to occur during development. We can't recover that after the fact," said Ryan Dilger, associate professor in the University of Illinois department of animal sciences, division of nutritional sciences and neuroscience program.

However, another graduate researcher who collaborated on these papers led an earlier study on the same piglets in 2015. That study, led by Caitlyn Getty, showed lower brain weights in piglets from choline-deficient mothers, but her study focused more on the overall health and metabolism of the piglets. From that perspective, postnatal choline intake was most important, particularly for liver and kidney function.

The research, taken together, suggests a cellular-level mechanism, backing up a 2013 study that concluded that children born to mothers whose choline intake was well below the recommendation have lower academic outcomes by age seven.

"We know that the structural alteration is there, but it may not manifest in ways we can see until later in life. That's why it's important to think about this during gestation, because the changes are occurring then," Mudd said.

Infant formula

Nearly every American who has become a parent in the last decade has heard the slogan that “breast milk is best” and has likely been encouraged to offer breast milk to newborns. Among other things, breast milk contains natural sources of prebiotics, which are small, indigestible fiber molecules that promote the growth of good bacteria in the baby’s gut.

Yet, for many families, breastfeeding is difficult or impossible. Fortunately, modern infant formulas are getting closer to the real thing, and University of Illinois researchers are contributing to the effort. In a recent study, researchers worked with piglets to show that prebiotics included in infant formula can enhance memory and exploratory behavior.

“When we provide prebiotics in formula, our results confirm that we can not only benefit gut health, which is known, but we can also influence brain development,” Dilger said. “We can actually change the way piglets learn and remember by influencing bacteria in the colon.”

In this study, the team concentrated solely on the effects of prebiotics.

Starting on the second day of life, piglets were given a cow’s milk-based infant formula supplemented with polydextrose (PDX), a synthetic carbohydrate with prebiotic activity, and galactooligosaccharide (GOS), a naturally occurring prebiotic. When the piglets were 25 days old, Stephen Fleming, a doctoral student in the neuroscience program, took them through several learning, memory and stress tests. After 33 days, blood, brain and intestinal tissues were collected for analysis.

The test for learning and memory gave piglets a chance to play with dog toys: one they’d seen before, and one brand-new toy. If they spent more time with the new toy, that was an indication that the piglet recognized it as new and preferred it. This “novel object recognition” test improves on the classic maze tests commonly used in rodent studies.

Pigs fed PDX and GOS spent more time playing with new objects than pigs who didn’t receive the prebiotic supplements. The preference for novel objects — an indication of natural curiosity — is a sign of healthy brain development and points to positive developments in learning and memory, the researchers said.

When prebiotics are working the way they should, good bacteria increase in abundance. One way to tell is by looking at metabolic end products — volatile fatty acids (VFAs) — excreted by bacteria during digestion of prebiotic fibers.

Dilger noted that VFAs “are a global indicator for whether prebiotics had an effect on the overall population of bacteria. For example, we might want to see an increase in Lactobacillus and other beneficial bacteria that produce butyrate.” VFA concentrations in the colon, blood and brain were changed in pigs receiving PDX and GOS compared with control pigs.

Recent evidence suggests that bacterial VFAs could be getting into the blood and traveling to the brain, where they could potentially affect mood and behavior.

“We found that, yes, VFAs are absorbed in the blood of pigs that were fed PDX/GOS, and, yes, they do get into the brain,” Fleming explained. “When we looked at the relationship between these VFAs and the results of our behavior tests, there did not appear to be a clear connection.”

Another surprise was a decrease in serotonin levels in the brains of pigs fed the prebiotic. Fleming said, “When you hear 'less serotonin,' there’s an immediate reaction to say, ‘Well, that’s bad.’” Yet,  those pigs didn’t show greater anxiety than control pigs during a stress test or poorer performance when given a learning and memory test.

The researchers hypothesized that the prebiotics may alter levels of tryptophan, the amino acid precursor to serotonin, but they noted that it’s too early to say.

Although more work is needed to tackle remaining questions, the study adds to the growing body of research suggesting a strong and potentially modifiable link between the gut and the brain.

“There are so many ways we can alter the composition of the microbiota, and they can have very strong benefits. Promoting good ‘gut health’ remains a strong focus in the field of nutrition,” Dilger said.

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