Brian Berg

Exaggerated neuroinflammation and sickness behavior in aged mice following activation of the peripheral innate immune system

Acute cognitive impairment (i.e., delirium) is common in elderly emergency department patients and frequently results from infections that are unrelated to the central nervous system. Since activation of the peripheral innate immune system induces brain microglia to produce inflammatory cytokines that are responsible for behavioral deficits, we investigated if aging exacerbated neuroinflammation and sickness behavior after peripheral injection of lipopolysaccharide (LPS). Microarray analysis revealed a transcriptional profile indicating the presence of primed or activated microglia and increased inflammation in the aged brain. Furthermore, aged mice had a unique gene expression profile in the brain after an intraperitoneal injection of LPS, and the LPS‐induced elevation in the brain inflammatory cytokines and oxidative stress was both exaggerated and prolonged compared with adults. Aged mice were anorectic longer and lost more weight than adults after peripheral LPS administration. Moreover, reductions in both locomotor and social behavior remained 24 h later in aged mice, when adults had fully recovered, and the exaggerated neuroinflammatory response in aged mice was not reliably paralleled by increased circulating cytokines in the periphery. Taken together, these data establish that activation of the peripheral innate immune system leads to exacerbated neuroinflammation in the aged as compared with adult mice. This dysregulated link between the peripheral and central innate immune system is likely to be involved in the severe behavioral deficits that frequently occur in older adults with systemic infections.

α-Tocopherol reduces lipopolysaccharide-induced peroxide radical formation and interleukin-6 secretion in primary murine microglia and in brain

The purpose of this study was to determine if alpha-tocopherol-a reactive oxygen species (ROS) scavenging agent-inhibits LPS-induced oxidative stress and interleukin-6 (IL-6) production in murine microglia and brain. LPS increased intracellular peroxides and IL-6 in a dose-dependent fashion in primary microglia. The LPS-induced increase in ROS and IL-6 was reduced by pretreatment of alpha-tocopherol. Intraperitoneal injection (i.p.) of LPS induced lipid peroxidation and IL-6 in brain. Importantly, three daily injections (20 mg) of alpha-tocopherol increased brain alpha-tocopherol and decreased LPS-induced lipid peroxidation and IL-6 in brain. Taken together, these data indicate that alpha-tocopherol can mitigate inflammatory cytokine production in the brain.

α-Tocopherol attenuates lipopolysaccharide-induced sickness behavior in mice

Antioxidants protect cells from oxidative damage and reduce lipopolysaccharide (LPS)-induced expression of inflammatory cytokines. Because inflammatory cytokines induce sickness behavior, we hypothesized that antioxidants, namely alpha-tocopherol (alpha-T) and selenium would inhibit sickness behavior caused by LPS. In an initial study, mice were injected intraperitoneal (i.p.) with vehicle, 2, or 20mg alpha-T for 3 consecutive days and then challenged with vehicle, 1, 10, or 100 microg of LPS. Sickness behavior was quantified by measuring social exploratory behavior. Vehicle pretreated mice injected with LPS showed a marked reduction in social behavior at 4h (p < .01). However, sickness behavior induced by the lowest dose of LPS was partially or completely blocked by 2 or 20mg alpha-T, respectively. alpha-T did not prevent sickness from higher doses of LPS. In a second study, mice were fed AIN93-M modified diets containing 10, 75, and 500 mg alpha-T/kg and 0.05, 0.15, and 2mg selenium/kg for 8 weeks and then challenged with saline or LPS (1 microg). The highest concentration of dietary alpha-T and selenium tended (p = .1) to reduce LPS-induced sickness behavior. Mice fed diets low in antioxidants had reduced plasma alpha-T levels and glutathione peroxidase activity (p = .08 and p < .01, respectively) and elevated liver thiobarbituric acid reactive substances (p < .001) 24h post LPS. Collectively, these data indicate that alpha-T improved the oxidative status after exposure to LPS, which may explain its ability to ameliorate symptoms of sickness.

α-Tocopherol attenuates NFκB activation and pro-inflammatory cytokine production in brain and improves recovery from lipopolysaccharide-induced sickness behavior

This study was conducted to determine if alpha-tocopherol facilitates recovery from lipopolysaccharide (LPS)-induced sickness behavior through a NFkappaB-dependent mechanism. In the first study, 3 daily intraperitoneal (i.p.) injections of alpha-tocopherol (20 mg) improved recovery from sickness behavior induced by i.p. injected LPS. Furthermore, alpha-tocopherol pretreatment attenuated LPS-activated NFkappaB and pro-inflammatory cytokine production in brain. In addition, inhibiting NFkappaB activity in the brain specifically by ICV injection of a NFkappaB decoy prior to LPS, significantly accelerated recovery from LPS-induced sickness behavior. Taken together, these data indicate alpha-tocopherol modulates sickness behavior and inflammatory cytokine production in the brain through an NFkappaB-dependent pathway.

Improved psychomotor performance in aged mice fed diet high in antioxidants is associated with reduced ex vivo brain interleukin-6 production.

Psychomotor performance is decreased in the aged. This study investigated the relationship between brain oxidative stress, interleukin-6 (IL-6) production by brain tissue ex vivo and psychomotor deficits during aging, and the effects of feeding an antioxidant-rich diet on ex vivo brain IL-6 production and motor function in aged mice. Male BALBc mice reared in SPF conditions and ranging in age from 3 to 24 months were studied. There was a precipitous decline in motor function after 12 months of age and an increase in brain lipid peroxidation and IL-6 production by coronal brain slices ex vivo. In another study, 12-month-old mice were fed diets formulated to provide a disparate range of antioxidants. At 18 months of age psychomotor coordination, motor learning, and ex vivo brain IL-6 production were evaluated. Mice fed an antioxidant-rich diet had improved psychomotor coordination compared to mice fed diet adequate or low in antioxidants. When mice were tested on successive days, only those fed adequate and high antioxidants exhibited motor learning. Analysis of IL-6 production by coronal brain slices indicated that as dietary antioxidants increased, IL-6 production decreased. Collectively, these data indicate that antioxidants improve psychomotor performance in aged mice, and suggest antioxidants may be useful for reducing brain IL-6 production, which has been shown to increase in aged mice.

Dietary Prebiotics, Milk Fat Globule Membrane, and Lactoferrin Affects Structural Neurodevelopment in the Young Piglet

Introduction Milk fat globule membrane (MFGM) and lactoferrin have been identified as two components that have potential to affect neurodevelopment. While concentrations of some MFGM constituents in infant formulas are within human milk range, they may not be present at optimal or clinically effective levels. However, lactoferrin levels of infant formulas are consistently reported to be lower than human milk. This study sought to provide a novel combination of prebiotics, bovine-derived MFGM, and lactoferrin and assess their influence on neurodevelopment. Methods Twenty-four male piglets were provided either TEST (n = 12) or CONT (n = 12) diet from 2 to 31 days of age. Piglets underwent spatial T-maze assessment starting at 17 days of age, were subjected to magnetic resonance imaging at 30 days of age, and were euthanized for tissue collection at 31 days of age. Results Diffusion tensor imaging revealed differences in radial (P = 0.032) and mean (P = 0.028) diffusivities in the internal capsule, where CONT piglets had higher rates of diffusion compared with TEST piglets. Voxel-based morphometry indicated larger (P < 0.05) differences in cortical gray and white matter concentrations, with CONT piglets having larger tissue clusters in these regions compared with TEST piglets. In the spatial T-maze assessment, CONT piglets exhibited shorter latency to choice compared with TEST piglets on day 2 of acquisition and days 3 and 4 of reversal. Conclusion Observed differences in microstructure maturation of the internal capsule and cortical tissue concentrations suggest that piglets provided TEST diet were more advanced developmentally than piglets provided CONT diet. Therefore, supplementation of infant formula with prebiotics, MFGM, and lactoferrin may support neurodevelopment in human infants.

Early life diets with prebiotics and bioactive milk fractions attenuate the impact of stress on learned helplessness behaviours and alter gene expression within neural circuits important for stress resistance

Manipulating gut microbes may improve mental health. Prebiotics are indigestible compounds that increase the growth and activity of health‐promoting microorganisms, yet few studies have examined how prebiotics affect CNS function. Using an acute inescapable stressor known to produce learned helplessness behaviours such as failure to escape and exaggerated fear, we tested whether early life supplementation of a blend of two prebiotics, galactooligosaccharide (GOS) and polydextrose (PDX), and the glycoprotein lactoferrin (LAC) would attenuate behavioural and biological responses to stress later in life. Juvenile, male F344 rats were fed diets containing either GOS and PDX alone, LAC alone, or GOS, PDX and LAC. All diets altered gut bacteria, while diets containing GOS and PDX increased Lactobacillus spp. After 4 weeks, rats were exposed to inescapable stress, and either immediately killed for blood and tissues, or assessed for learned helplessness 24 h later. Diets did not attenuate stress effects on spleen weight, corticosterone and blood glucose; however, all diets differentially attenuated stress‐induced learned helplessness. Notably, in situ hybridization revealed that all diets reduced stress‐evoked cfos mRNA in the dorsal raphe nucleus (DRN), a structure important for learned helplessness behaviours. In addition, GOS, PDX and LAC diet attenuated stress‐evoked decreases in mRNA for the 5‐HT1A autoreceptor in the DRN and increased basal BDNF mRNA within the prefrontal cortex. These data suggest early life diets containing prebiotics and/or LAC promote behavioural stress resistance and uniquely modulate gene expression in corresponding circuits.

Prebiotics and Bioactive Milk Fractions Affect Gut Development, Microbiota, and Neurotransmitter Expression in Piglets.

Objective: This study tested the hypothesis that the addition of prebiotics and 2 functional milk ingredients to infant formula would maintain normal growth and gut development, and modify microbiota composition and neurotransmitter gene expression in neonatal piglets. Methods: Two-day-old male piglets (n = 24) were fed formula (CONT) or formula with polydextrose (1.2 g/100 g diet), galactooligosaccharides (3.5 g/100 g diet), bovine lactoferrin (0.3 g/100 g diet), and milk fat globule membrane-10 (2.5 g/100 g diet) (TEST) for 30 days. On study day 31, intestinal samples, ileal and colonic contents, and feces were collected. Intestinal histomorphology, disaccharidase activity, serotonin (5’HT), vasoactive intestinal peptide (VIP), and tyrosine hydroxylase (TH) were measured. Gut microbiota composition was assessed by pyrosequencing of the V3–V5 regions of 16S rRNA and quantitative polymerase chain reaction. Results: Body weight of piglets on TEST was greater (P ⩽ 0.05) than CONT on days 17 to 30. Both groups displayed growth patterns within the range observed for sow-reared pigs. TEST piglets had greater jejunal lactase (P = 0.03) and higher (P = 0.003) ileal VIP expression. TEST piglets tended to have greater (P = 0.09) sucrase activity, longer (P = 0.08) ileal villi, and greater (P = 0.06) duodenal TH expression. Microbial communities of TEST piglets differed from CONT in ascending colon (AC, P = 0.001) and feces (P ⩽ 0.05). CONT piglets had greater relative abundances of Mogibacterium, Collinsella, Klebsiella, Escherichia/Shigella, Eubacterium, and Roseburia compared with TEST piglets in AC. In feces, CONT piglets harbored lower (P ⩽ 0.05) proportions of Parabacteroides, Clostridium IV, Lutispora, and Sutterella than TEST piglets. Conclusions: A mixture of bioactive ingredients improved weight gain and gut maturation, modulated colonic and fecal microbial composition, and reduced the proportions of opportunistic pathogens.

Delayed Initiation but Not Gradual Advancement of Enteral Formula Feeding Reduces the Incidence of Necrotizing Enterocolitis (NEC) in Preterm Pigs

Enteral formula feeding is a risk factor for necrotizing enterocolitis (NEC) in premature infants, yet studies are conflicting regarding the safest timing for introduction and advancement of feeds. Our aim was to test the effects of early vs. late initiation and abrupt vs. gradual advancement of enteral feeding of an intact vs. hydrolyzed protein formula on NEC incidence and severity in preterm pigs. In Experiment 1, preterm pigs received total parenteral nutrition (TPN) at birth with abrupt initiation of enteral formula feeds (50% full intake) on d of life (DOL) 2 (EA) or 5 (LA) while PN continued. Pigs were also fed formula containing either intact or hydrolyzed protein. In Experiment 2, preterm pigs received TPN at birth with enteral, hydrolyzed-protein formula feeds introduced on DOL 2 either abruptly (EA; 50% full feeds) or gradually (EG; 10–50% full feeds over 5 d) while PN continued. NEC incidence and severity were assessed based on macroscopic and histological scoring. In Experiment 1, NEC incidence (41% vs. 70%, P<0.05) and severity were reduced in LA vs. EA groups and LA was associated with a higher survival rate, daily weight gain and jejunum villus height. Piglets fed hydrolyzed vs. intact protein formula had lower stomach content weights and similar NEC incidence. In Experiment 2, NEC incidence and severity were not different between pigs the EG vs. EA group. Proinflammatory gene expression (IL-1β, IL-6 and S100A9) in the ileum was lower in both LA and EG vs. EA groups. In conclusion, delayed initiation but not gradual advancement of enteral feeding is protective against NEC in preterm pigs. Feeding hydrolyzed vs. intact protein formula improved gastric transit without affecting the NEC incidence.

Porcine Milk Oligosaccharides and Sialic Acid Concentrations Vary Throughout Lactation

Background Milk oligosaccharides (OSs) are bioactive components known to influence neonatal development. These compounds have specific physiological functions acting as prebiotics, immune system modulators, and enhancing intestine and brain development. Objectives The pig is a commonly used model for studying human nutrition, and there is interest in quantifying OS composition of porcine milk across lactation compared with human milk. In this study, we hypothesized that OS and sialic acid (SA) composition of porcine milk would be influenced by stage of lactation. Methods Up to 250 mL of milk were collected from seven sows at each of three time points: day 0 (colostrum), days 7–9 (mature), and days 17–19 (weaning). Colostrum was collected within 6 h of farrowing and 3-day intervals were used for mature and weaning milk to ensure representative sampling. Milk samples were analyzed for OS profiles by Nano-LC Chip–QTOF MS, OS concentrations via HPAEC-PAD, and SA (total and free) was assessed by enzymatic reaction fluorescence detection. Results Sixty unique OSs were identified in porcine milk. Neutral OSs were the most abundant at each lactation stage (69–81%), followed by acidic-sialylated OSs (16–29%) and neutral-fucosylated OSs (2–4%). As lactation progressed, acidic OSs decreased (P = 0.003), whereas neutral-fucosylated (P < 0.001) and neutral OSs (P = 0.003) increased throughout lactation. Six OSs were present in all samples analyzed across lactation [lacto-N-difucohexaose I (LNDFH-I), 2′-fucosyllactose (2′-FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-neohexaose (LNnH), α1-3,β-4-d-galactotriose (3-Hex), 3′-sialyllactose (3′-SL)], while LDFT was present only in colostrum samples. Analysis of individual OS concentrations indicated differences (P = 0.023) between days 0 and 7. Conversely, between days 7 and 18, OS concentrations remained stable with only LNnH (P < 0.001) and LNDFH-I (P = 0.002) decreasing over this period. Analysis of free SA indicated a decrease (P < 0.001) as lactation progressed, while bound (P < 0.001) and total (P < 0.001) SA increased across lactation. Conclusion Concentrations of OS differ between colostrum and mature milk in the pig, and SA concentrations shift from free to bound forms as lactation progresses. Our results suggest that although porcine milk OS concentration and the number of structures is lower than human milk, the OS profile appears to be closer to human milk rather than to bovine milk, based on previously published profiles.