Maternal dietary zinc supplementation prevents aberrant behaviour in an object recognition task in mice offspring exposed to LPS in early pregnancy

Abstract

Maternal infection during pregnancy is associated with an increased risk of neurodevelopmental damage. While the mechanism is unclear accumulating evidence suggests that the maternal inflammatory response may be responsible. Metallothionein (MT) is a zinc (Zn)-binding protein that when induced in the mother’s liver during the acute phase response has been found to cause a fetal Zn deficiency. Infection-mediated fetal Zn deficiency in early pregnancy has been shown to cause teratogenicity which can be prevented by dietary Zn supplementation throughout pregnancy. This study examined whether cognitive impairments can be caused by lipopolysaccharide (LPS) administration early in pregnancy and whether dietary Zn supplementation can ameliorate these changes. Maternal inflammation induced by LPS at gestation day (GD) 8 did not affect spatial learning or memory of adult mice offspring in a water cross-maze escape task. However, in an object recognition task, where control mice demonstrated good visual recognition memory by exploring a novel object more than a familiar object, LPS-treated offspring demonstrated abnormal perseverant exploration towards the familiar object that cannot be explained in full by impaired object recognition memory. In comparison, offspring of mice from dams given LPS and dietary Zn supplementation displayed normal object recognition task performance. Microarray analysis on the brain of GD 12 fetuses did not identify any differentially expressed genes between treatment groups. This study demonstrates that LPS administration in early pregnancy can cause an anomaly in object recognition that can be measured in adult offspring. This aberrant behaviour can be prevented by dietary Zn supplementation during pregnancy, thus providing a nutritional strategy to limit neurodevelopmental damage caused by infections early in pregnancy.

Introduction

Epidemiological studies have associated infections during pregnancy with the pathogenesis of cerebral palsy [1], [2], schizophrenia [3], [4], [5], [6], [7], non-genetic forms of autism [8], and mental retardation [9]. Studies in animals have confirmed that both bacterial and viral infections in utero can cause a spectrum of neuropathological and behavioural abnormalities in offspring [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]. Although it remains unclear how a broad range of infectious agents might trigger neurodevelopment anomalies, accumulating evidence suggest that the damage may be due to the maternal immune response rather than the infectious agent itself [13], [14], [15], [16], [17], [18], [19]. Pro-inflammatory cytokines are the main mediators of the maternal response to infection [20]. Studies have focused upon the potential of maternal and fetal-derived cytokines (IL-6, IL-1-β and TNF-α) to inhibit brain development however their causative role still remains unclear [19], [21], [22]. Secondary products of the maternal immune response may also influence the developing brain. Early in infection, inflammatory cytokines mediate a complex change in acute-phase reactants in the host’s liver. Induction of the zinc (Zn)-binding protein, metallothionein (MT), is a component of this acute response which results in a well characterized maternal hypozincaemia [23], [24] and a subsequent fetal Zn deficiency [25], [26], [27], [28], [29]. Teratogenicity and abnormal neurodevelopment are associated with fetal Zn deficiency in rodents and humans [25], [26], [27], [28], [29], [30], [31], [33], [34], [35], [36] and this has been linked to MT-induction by a diverse range of toxicants including inflammatory mediators.

Daston and co-workers [25], [26], [27], [28] were the first to show that teratogenicity by a number of substances including urethane, α-hedrin, ethanol and TNF-α was related to induction of hepatic MT and linked to changes in maternal-fetal Zn distribution. Our group has since demonstrated in a mouse model that induction of MT in the mother’s liver results in a redistribution of whole body zinc that causes short-term fetal Zn deficiency [29], [30], [31], [32]. In infection, inflammatory mediators stimulate MT synthesis in the mother’s liver, which in turn sequesters Zn from the maternal blood thus competing with that available to the fetus. No specific storage sites of Zn exist in the body, therefore serum Zn concentrations must be maintained to ensure an adequate supply to the fetus for normal fetal development. It is well recognized that a dietary deficiency of Zn is teratogenic [33], [34] and causes neurological abnormalities with alterations in both brain structure and function in humans [34], [35], [36]. The mechanism of damage is unknown but likely to be complex as Zn is required for many proteins that are critically important in the processes that underlie development of the fetus including neurodevelopment (e.g. DNA and protein synthesis, mitosis and cell division) [23], [24].

In our studies we have used lipopolysaccharide (LPS), the bacterial cell wall on gram-negative bacteria, to evoke a maternal immune response during pregnancy. We have shown that early in pregnancy on gestational day (GD) 8 in the mouse, LPS-mediated induction of MT caused a sharp fall in plasma zinc which resulted in 8-times as many birth abnormalities per litter than in controls [30]. We have further demonstrated that LPS-mediated teratology is not observed in MT-knockout mice and that plasma zinc levels do not fall after prenatal LPS-treatment, implicating MT and Zn deficiency in the teratogenic process. In addition, we have found that LPS-induced birth abnormalities in wild type mice can be prevented by injecting Zn subcutaneously at the time of LPS treatment [30] or by supplementing the mother’s diet with Zn throughout pregnancy [32]. Although the mechanism by which Zn supplementation protects against birth abnormalities is unknown, we have shown that it can limit the fall in plasma Zn in the mother during the period of MT induction, where it can fall by as much as 40% after LPS exposure on GD 8 [30]. The question now arises as to whether Zn deficiency plays a causative role in abnormal neurodevelopment due to prenatal infection and whether dietary Zn supplementation throughout pregnancy is protective.

Behavioural anomalies [10], [11], [12], [13], [14], [15], [16], [17], [18] and molecular and cellular changes [18], [37] in brain regions typically involved in cognition have been observed in rodent offspring born to mother’s exposed to a variety infectious agents. Studies using either the viral mimic, PolyI:C or bacterial endotoxin have confirmed the importance of the maternal cytokine-associated inflammatory response in neurodevelopmental damage and behavioural pathology in later life [for review see 38]. The timing of maternal immune challenge in pregnancy has been found to alter the cytokine response and the pathological consequences in brain and behaviour [12]. From studies mainly using PolyI:C, the most vulnerable period where infection has the greatest impact on the developing brain appears to be in early rather than late pregnancy [12], [38]. Little is known about the effect of LPS challenge in early pregnancy and whether it causes a similar pathological profile to PolyI:C, as each are recognized by specific toll-like receptors [39], [40] and consequently are likely to produce different cytokine responses and neurodevelopmental outcomes.

Ethanol like many inflammatory mediators is a potent inducer of liver MT with a consequent reduction in plasma Zn [27], [28]. We and others have shown that prenatal ethanol exposure on GD 8 causes impairments in spatial memory and object recognition memory in offspring well before the major structures involved in memory processing are formed [41], [42], [43], [44]. Similar spatial memory impairments have been found in adult offspring prenatally exposed to PolyI:C on GD 9 [reviewed by 38]. Most interestingly our studies have shown that dietary zinc supplementation throughout pregnancy can prevent the ethanol-mediated cognitive impairments [42]. Given that LPS-administration in early pregnancy causes a higher MT response and a greater fall in plasma Zn than with ethanol [30], [45], we hypothesize that this will also result in neurodevelopmental damage. Such damage could be reflected by differential expression of major genes involved in processes such as neurodevelopment, inflammatory mediators or defense mechanisms. The aim of the present study is to determine whether LPS administration early in pregnancy affects cognition in offspring or changes in gene expression in the fetal brain. We also determine whether dietary zinc supplementation can prevent any LPS-related changes.