Newborn and growing brains are more prone to Mn toxicity (32, 33). The upregulation of pro-inflammatory biomarkers, reactive nitrogen, and oxygen species because of exposures during crucial developmental stages can reverberate in adulthood and may result in the emergence of neurodegenerative diseases (34–36). As a result, it is hypothesized that primary prevention can be accomplished in early life to avert disorders in the future (37).
PUFAs (ALA & LA) and phytochemicals, including flavonoids, polyphenols, melatonin, resveratrol, etc., are abundant in walnuts. These and numerous other factors add to walnuts’ well-known health advantages, earning the local moniker “brain food.” According to several epidemiological research, eating more foods high in omega-3 fatty acids is strongly linked to a decreased prevalence of brain disorders (37–39). To our knowledge, no studies have been done on the possible neuroprotective effects of a walnut-enriched diet during pregnancy or preweaning against Mn-induced developmental neurotoxicity. Therefore, this study examined the effects of a maternal diet supplemented with African walnuts on changes in behaviour, cortico-hippocampal structure, and function brought on by manganese.
Understanding the unique functional abnormalities brought on by neurotoxicants like Mn is vital to devise efficient treatment and prevention strategies. An increasing body of evidence suggests that prenatal and early childhood neurobehavioral outcomes are negatively impacted by historical Mn exposure. According to these studies, developmental Mn exposure is associated with hyperactivity, impulsivity, rebellious behaviour, inattentiveness, and reduced fine motor abilities (40–44).
Like the control group, the offspring of dams maintained on WED scored significantly higher in correct spontaneous alternation performance percentage than those of dams treated with MnCl2 (Fig. 4.13). This observation suggests substantial effects of developmental Mn neurotoxicity on short-term spatial memory. Like the report by Hogas et al. (2011), significant deficits in spontaneous alternation percentage in rats administered high-dose manganese was observed (24). Furthermore, Schneider et al. (2015) found that manganese exposure resulted in impairments in spatial working memory with more substantial deficits in non-spatial working memory in macaque monkeys (45). Expressly, those whose mothers were maintained on WED after prenatal MnCl2 treatment (MnCl2//WED) showed significant improvement in alternating sequences compared to animals whose mothers were treated with MnCl2 alone, suggesting that WED forestalled alterations in spatial working memory induced by development Mn overexposure.
One of the rodent’s most significant behavioural paradigms to assess anxiety behaviour is the elevated plus-maze (EPM). Since rats’ natural propensity is to hide in the closed components of the EPM apparatus due to anxiety generated by a novel environment, it is commonly known that anxiolytic agents improve the frequency of entrances and the period spent in the EPM’s open arms (46, 47). In the current study, offspring of control and WED-maintained dams significantly frequented the open arm portion of the maze more than those whose mothers were treated with MnCl2 during early development. This latter observation suggests a higher level of anxiety or decreased impulsivity induced by developmental Mn neurotoxicity in offspring of these dams, likely by deficits in inhibition control. Hence, the prolonged time spent in the closed arms by offspring of Mn-treated rats. Notably, these findings contrast with those of Pappas et al. (1997) (48), who reported that neither the 2 mg/ml prenatal manganese-exposed rats nor the 10 mg/ml perinatal manganese-exposed rats differed from controls on the elevated plus apparatus, the Morris water maze, or the radial arm maze and Kern et al. (2010) (49), who found no behavioural effects following Mn exposure via the EPM test. These disparities in results are likely due to different exposure protocols and routes of administration (i.e., direct oral administration to the pups) vs transplacental and lactational transfer in the present study.
Nonetheless, prenatal exposure to WED significantly prevented postnatal Mn-induced anxiety. Similarly, concurrent WED with MnCl2 treatment significantly counteracted the anxiogenic effects of excess developmental Mn. Although there was no significant influence of postnatal WED on anxiety measures following prenatal exposure to MnCl2, there was an insignificant trend toward increased open-arms exploration, all indicative of an anxiolytic property of WED.
Just as important, several studies have revealed that Mn can exacerbate the effects of cytokines on the activation of both microglia and astrocytes that causes dramatic potentiation in the production of TNFα, IL-1β, ROS, and NOS2 expression (2, 25, 26, 28). Increased levels of these and other inflammatory genes have been measured in both rodent (50, 51) and nonhuman primate (52) studies, with deletion or inhibition of these pathways showing neuroprotection (51, 53). Notably, results from this current study revealed an Mn-correlated significant uptrend in the levels of inflammatory biomarkers (iNOS, TNF-α, IL-1β, and COX-2) in the PFC and hippocampus of rat offspring (Fig. 3). Mn levels in the blood and brain of developmentally exposed rats were significantly higher than in the offspring of the control and WED animals.
Interestingly, prenatal supplementation delayed treatment, and prophylactic/postnatal exposure of dams to WED significantly inhibited the overexpression of these pro-inflammatory cytokines in the PFC and hippocampus of rat offspring, respectively. Moreover, only concurrent treatment with WED showed no significant difference with the control and WED-only groups. While pre-treatment and post-treatment significantly reduced Mn levels compared to the MnCl2 group, there were still significant differences (p < 0.05) between these groups and the control and WED groups.
Additionally, studies indicate that the cholinergic system may be significantly involved in PD and Manganism through choline uptake, release, and acetyltransferase activity, even though it is not the primary target in Mn toxicity and several symptoms are primarily related to effects on the dopaminergic system (29). Specifically, MnCl2 significantly altered cortical and hippocampal cholinergic homeostasis in this study (Fig. 4a & b), which is consistent with the impairment in spatial working memory in this study’s Y-maze spontaneous alternation test and the raised levels of inflammatory biomarkers. Neurotransmitter metabolism disruption is correlated with Mn-induced behavioural abnormalities, such as motor incoordination or emotional and cognitive impairment, shown in both human and animal models (54). Different processes, such as neurotransmitter release inhibition, changes in neurotransmitter clearance from the synaptic cleft, or receptor modification, can lead to impaired neurotransmitter signalling (54). Acetylcholine (ACh) is an excitatory neurotransmitter that modulates important cognitive activities such as learning, memory, and movement in the central and peripheral nervous systems, all of which may be hampered due to Mn’s effects on cholinergic signalling (54, 55).
Still, WED was significantly effective in repressing Mn-induced upregulation of AChE in the cortex and hippocampus of rat offspring. This effect was observed at the studied exposure time points and is most likely potentiated by the synergy of African walnuts’ antioxidant and anti-inflammatory phytochemicals.
Conversely, developmental exposure to excess Mn caused a significant reduction in DNMT3a expression in the PFC and hippocampus of offspring of exposed dams (Fig. 5a & 6a, respectively). DNMT3a expression in offspring of dams on WED from gestation through weaning with simultaneous exposure to MnCl2 was modestly impacted compared to the offspring of dams treated with MnCl2 alone. There was no significant difference between this group and the control or WED group. This expression was comparable in both PFC and hippocampal tissues (Fig. 5a & 6a, respectively). Age-related decline in the protein levels of DNMT3a is reported to be reduced in the cortex and hippocampus of mice. It is linked to memory decline that can be salvaged by restoring DNMT3a levels (56).
In addition, prenatal dietary supplementation alone significantly prevented postnatal MnCl2-induced hypomethylation in the hippocampus but not in the PFC (Fig. 5a). Furthermore, postnatal WED supplementation after prenatal MnCl2 exposure did not significantly remediate DNMT3a expression in the PFC and hippocampus. While potential mitigation is apparent in both groups, it was statistically insignificant. A possible inference that could be drawn concerns the duration of exposure to WED.
Then again, MnCl2 correlated with the diminution of fold-change relative expression of H2AX in the PFC and hippocampus of rat offspring (Fig. 5b & 6b). Nevertheless, there were substantial decreases in this effect of MnCl2 on the hippocampus of rat offspring following prenatal supplementation with African walnuts (Fig. 6b). However, this result was not replicated in the PFC (Fig. 5b), where a modest effect was observed. Nonetheless, concurrent WED supplementation was significantly effective (p < 0.05) in counteracting neurotoxic cascades of MnCl2. This observation is quite interesting because several studies have reported that H2AX mutant mice, apart from being more sensitive to DNA damage, were uniquely vulnerable to mitochondrial injury in the brain, and mitochondrial defects constitute a significant source of impaired redox homeostasis, which are usually associated with age-related neurological diseases (57, 58). Therefore, WED appears involved in mitochondrial integrity and consequent maintenance of redox homeostasis.
Per the study’s goals and because mitochondrial abnormalities significantly cause redox dyshomeostasis, the mitochondrial dynamics gene OPA1 was assessed in each experimental group. Through the fusion process, OPA1 shapes and preserves mitochondrial morphology (59). Developmental MnCl2 exposure led to repression of the OPA1 gene in the PFC and hippocampus of rat offspring (Fig. 5c & 6c, respectively), which confirms the results for H2AX (Fig. 5b & 6b). Attendant maintenance on WED with manganese treatment throughout gestation and early postnatal life (WED + MnCl2) effectively restored OPA1 expression in PFC and hippocampus, potentially abrogating mitochondrial deficits and mitigating cell death.
Accordingly, MnCl2 exposure caused a significant diminution in the expression of the BDNF gene in the PFC and hippocampus (Fig. 5d & 6d, respectively). This observation was speculated because a study examining the effects of prenatal stress reported decreased BDNF expression, specifically in the hippocampus and amygdala (30, 60). In addition, mice deficient in TrkB, the BDNF receptor, show increased anxiety-like behaviour (61). Similarly, A study using overexpression and knockdown of BDNF in rats showed that BDNF overexpression could rescue depression-like behaviour in chronically stressed rats. In contrast, the knockdown of BDNF in the hippocampus, at least in young animals, produced a depression-like phenotype (62, 63). The repression of BDNF induced by MnCl2 might explain the perceived level of anxiety in this group, indicated by the low frequency of entry into the open arm (Fig. 1b). Nonetheless, WED significantly (p < 0.05) mitigated this effect of MnCl2 on BDNF expression in the PFC and hippocampus of rats.
Histological sections of rat offspring; control and WED-treated rats’ prefrontal cortices demonstrated the same cortical layers containing medium/large pyramidal and non-pyramidal cells scattered in a background formed by neuroglia cells and myelinated axons (Fig. 7). In contrast, sections of offspring of MnCl2-treated rats revealed shrunken pyramidal cells, vacuolated cytoplasm (spongiosis), perineural neuroglia, and ill-defined axons (Fig. 7, 8, & 9). Guilarte (2010) reported observing AD-like pathology and neurodegeneration in the frontal cortex of manganese-exposed nonhuman primates (64), and Lazrishvili et al. (2009) also noted alteration in the histoarchitecture of rat pups’ brains following subchronic poisoning with manganese chloride (65).