Acute in utero exposure to lipopolysaccharide induces inflammation in the pre- and postnatal brain and alters the glial cytoarchitecture in the developing amygdala

All procedures were carried out in accordance with Republic of Ireland Department of Health and Children Licenses. The procedures used in this study had the approval of the Institutional Animal Care and Use Committee and were carried out in accordance with the European Council Directive of November 24, 1986 (86/609/EEC). Pregnant C57BL/6J mice were used and confirmation of a vaginal plug was designated as embryonic day zero (E0) (n = 4/group/time point). All pregnant dams were treated at embryonic day 12 (E12) and were randomly assigned to receive a single intraperitoneal (i.p.) injection of either lipopolysaccharide from Escherichia coli 026:B6 (LPS; 50 μg/kg; Sigma-Aldrich, Ireland) or 100 μl 0.9% saline vehicle. Male and female offspring were used for all experiments. The first day of birth was termed P0. Developmental stages were divided into prenatal/neonatal (E12, E16, E18, and P0) and postnatal (P7, P14, and P40) stages. All animals were housed on 12-h light/dark cycle (light on 0800 h) at constant temperature of 21 ± 2 °C with food and water available ad libitum.

Pregnant dams were anesthetized using isoflurane and decapitated. A midline incision was made into the peritoneal cavity to expose embryos. Control and LPS offspring were sacrificed at seven ages: E12, E16, E18, and P0 (prenatal/neonatal) and P7, P14, and P40 (postnatal). One- and 7-day-old mice were anesthetized by hypothermia, while 14- to 40-day-old mice were anesthetized using isoflurane and promptly decapitated. For molecular analysis, whole brains were removed (E12: 4-h post-, E16: 4-day post-, and E18: 6-day postinjection, and P0: neonatal (first day of birth)) or the amygdalar regions dissected out (P7, P14, and P40), snap-frozen with liquid nitrogen, and stored at − 80 °C until qPCR was carried out. For histological analysis, embryos were dissected out and immersion-fixed in 4% paraformaldehyde (PFA) overnight followed by 30% sucrose (E18 and P0 brains were removed from the skull). Offspring at P7, P14, and P40 were deeply anesthetized and transcardially perfused with 0.05 M PBS followed by 4% PFA in 0.05 M PBS. Brains were removed from the skull and postfixed in 4% PFA overnight followed by 30% sucrose. The litters were split and assigned for molecular or histological assessment.

Coronal 40-μm-thick sections (one-in-six series) were sectioned using a cryostat (Leica Microsystem GmbH, Wetzlar, Germany). The first and second series of sections were used for immunohistochemistry (IHC). The third series was Nissl-stained with 0.1% Cresyl Violet (CV) to identify structural alterations and neuronal damage. Processed sections were dehydrated, cleared in histolene, and coverslipped using DPX (Sigma-Aldrich, Wicklow, Ireland). The first and second series of sections were processed for immunohistochemistry using specific antisera against glial fibrillary acidic protein (GFAP) (1:2000, ab53554 goat polyclonal GFAP antibody Abcam, Cambridge, UK) and ionized calcium-binding adaptor molecule 1 (Iba-1) (1:1000, ab5076 goat polyclonal Iba-1 antibody Abcam, Cambridge, UK), respectively. Bound antibody was detected using a biotinylated secondary antibody and the avidin-biotin-peroxidase complex (ABC) method (Vector laboratories, Peterborough, UK). Immunohistochemical staining was visualized using 3′,3′-diaminobenzidine (Sigma Aldrich, Wicklow, Ireland) as a chromogen.

The amygdala region was delineated in postnatal animals using the mouse atlas. Iba-1 staining was used to examine the morphology of microglial cells. For each animal case, four sections were quantified and the number of total number of Iba-1-positive cells was counted with aid of ImageJ software. Iba-1-positive cells were divided into activated and resting and calculated as a percentage of total microglia present in that region. Iba-1-positive cells were counted in the postnatal amygdala and subdivided into ramified versus amoeboid-like morphology [40]. The quantification is displayed as total (%) of cells counted in the region. GFAP staining was analyzed as an indication of astrogliosis and inflammation in the amygdala in MIA offspring in comparison to saline-treated controls. GFAP-positive immunostaining per region was scored (n = 4 per treatment/group) with slight modifications according to O’Loughlin et al. [30] by performing background correction with the aid of ImageJ software. Density of GFAP-positive cells and variation in the staining intensity (somata and cellular processes) were included into scoring in a blinded manner as − = none (0% stained), + = very few cells (< 10% of area stained), ++ = few cells (10–25% of area stained), +++ = moderate number of cells (25–50% of area stained), and ++++ = numerous cells (> 50% stained).

Volumes of postnatal amygdalar regions were assessed according to the Cavalieri principle [16]. Amygdalar nuclei were delineated in Nissl-stained sections using a mouse atlas [13]. Consecutive sections (section sampling fraction (ssf) 1:6, sections 120 μm apart from each other) were sampled in saline- and LPS-injected offspring at three ages: P7, P14, and P40. Volumes were calculated using the formula: V = ∑P _i · a(p) ·d, where P _i is the sum of hit point counts in all sections, a(p) is the area associated with each point, and d is the section spacing. In the amygdala, the volumes of the lateral (LA), basolateral (BLA), and central (Ce) nuclei were measured in consecutive sections, commencing at Bregma − 0.70 to − 2.30 mm according to the mouse atlas [13]. Total numbers of neurons were estimated in Nissl-stained amygdalar nuclei using the optical dissector method [16]. Neurons were distinguished from astrocytes and oligodendrocytes based on cell and nuclear size, amount of nuclear heterochromatin, and organization of nucleolus [9]. Consecutive sections (1 in 6 series; 120 μm apart) throughout the rostro-caudal extent of the amygdala were used in saline- and LPS-injected offspring at three ages: P7, P14, and P40, (Bregma − 0.70 to − 2.30 mm) [13]. An Olympus BX 40 microscope (×100 objective) was used to count the neurons. An unbiased counting frame with inclusion and exclusion lines was superimposed, and neurons that lay between the boundaries were counted. The focal plane was moved down the z-axis using a digimatic micrometer that was attached to the stage of the microscope to measure the z-axis depth. The following equation was used: E(N) = Nv X Vref, as previously described in detail elsewhere [41].

Total RNA was extracted from brain/amygdala samples using Qiagen RNeasy Plus Lipid mini kit as per manufacturer’s guidelines (Qiagen, Crawley, UK). Next, cDNA was generated from total RNA using a High Capacity Reverse Transcriptase Kit (Applied Biosystems, Warrington, UK). All real-time quantitative PCR were performed using StepOne™ Real-Time PCR system (Applied Biosystems) with TaqMan® Gene expression Master Mix (Applied Biosystems) and the specific TaqMan® Gene Expression Assay for the following targets: IL-β (Mm00434228_m1), TNFα (Mm00443258_m1), IL-6 (Mm00446190_m1), TLR-2 (Mm00442346_m1), TLR-4 (Mm00445273_m1), COX-2 (Mm00478374_m1), iNOS (Mm00440502_m1), CD36 (Mm00432403_m1), and CCL2 (Mm00441242_m1) (Applied Biosystems). All samples were run in triplicate. The mRNA expression levels between samples were normalized using β-Actin (Mm02619580_g1) endogenous control (VIC dye labeled, Applied Biosystems™). The quantification of mRNA expression in the LPS treatment groups relative to the saline control (fold change) was done using the 2^−∆∆C _T method [39].

Statistical analyses were carried out using GraphPad Prism software version 7. Data were represented as mean values ± the standard error of the mean (SEM). For histology quantification, Student’s t test was used to compare parametric data of two treatment groups. Gene expression profiles were analyzed with one-way ANOVA followed by Tukey’s multiple-comparison post hoc test. Differences were considered to be significant only for ***p < 0.001, **p < 0.01, and *p < 0.05. There were four pregnant mice (n = 4) in each treatment group/time point.

We first investigated the effect MIA on inflammatory cytokine expression at different stages of development. The amygdala nuclei originate from different domains of the embryonic brain maturating at different developmental stages [25], and the size of the amygdala was too small to be dissected out reliably in the pre- and neonatal brain. Therefore, we divided the developmental ages into two groups (prenatal/neonatal and postnatal). Whole brain lysates were analyzed at E12 (4-h postinjection), E16, E18, and P0 in prenatal/neonatal stages, and the amygdala was dissected under the microscope at postnatal stages P7, P14, and P40 (Fig. 1a). One-way ANOVA for the main factor age showed a significant effect for IL-1β (F(_{4, 15}) = 5.97, p = 0.010, TNFα (F(_{4, 15}) = 4.93, p = 0.0186), IL-6 (F(_{4, 15}) = 3.178, p = 0.05), and IL-10 (F(_{4, 15}) = 7.713, p = 0.0042) in the prenatal/neonatal development stages. Likewise, a significant age effect was observed postnatally for IL-1β (F(_{3, 12}) = 8.42, p = 0.0028), but no significant differences were recorded for IL-6 (F(_{3, 12}) = 2.27, p = 0.1325), TNFα (F(_{3, 12}) = 2.013 p = 0.1660), or IL-10 (F(_{3, 12}) = 0.982, p = 0.4337). Data showed there was a rapid response to MIA-LPS with mRNA induction of several pro-inflammatory genes including IL-1β and TNFα. Specifically, MIA significantly increased mRNA expression of IL-1β at E12 by sixfold as indicated by post hoc analyses (Fig. 1b; *p = 0.011). Expression levels were also significantly higher at E12 in comparison to P0 (Fig. 1b; *p = 0.011). In the postnatal amygdala, IL-1β was increased approximately sixfold at P7 with levels decreasing but remaining significantly elevated at P40 (Fig. 1c; **p = 0.0032). IL-1β was also higher at P7 in comparisons to P14 (**p = 0.0095) and P40 (*p = 0.0156). IL-6, an important neuro-immune regulator, was increased about threefold after MIA on the first postnatal day P0 (Fig. 1b; *p = 0.05). In the postnatal amygdala, IL-6 was elevated across all ages (P7 to P40), which however did not reach significance (Fig. 1c). TNFα, another pro-inflammatory cytokine, was elevated across prenatal ages with significant increases at E16 (*p = 0.019) and at E18 (*p = 0.044) in comparison to saline-treated controls, but was not significantly increased in the postnatal amygdala (Fig. 1b, c). IL-10 is a known anti-inflammatory cytokine, and its expression was significantly increased at E16 in the prenatal brain in comparison to saline-treated controls (Fig. 1b; **p = 0.003). IL-10 mRNA levels were higher at E16 in comparison to E12 (*p = 0.0103), E18 (*p = 0.014), and P0 (**p = 0.0057) but remained unaffected at postnatal ages as indicated by post hoc analyses. Taken together, the data shows that offspring from MIA-induced dams had an elevated inflammatory response in the CNS from 4 h postinjection to early adolescence at P40.

Toll-like receptors (TLRs) are known to mediate immune responses and induce inflammatory cascades. TLR-4 is thought to mediate bacterial responses in such that LPS is a ligand of this receptor. TLR-2 is also involved in LPS-induced TLR-4 signaling and thus inflammation [21]. Therefore, we investigated the effect of LPS-induced MIA on TLR expression across stages of amygdalar development (Fig. 2a, c). One-way ANOVA for the main factor age showed a significant effect for TLR-2 (F(_{4, 15}) = 13.24, p < 0.0001) and TLR-4 (F(_{4, 15}) = 18.3, p < 0.0001) in the prenatal/neonatal development stages. Likewise, a significant age effect was observed postnatally for TLR-2 (F(_{3, 12}) = 6.45, p = 0.0075) and TLR-4 (F(_{3, 12}) = 3.354, p = 0.05). In post hoc analyses, TLR-2 was significantly increased at E16 relative to saline-treated controls (***p = 0.0002) and to LPS-treated offspring at E12 (***p = 0.0002), E18 (*p = 0.013), and P0 (***p = 0.0004). In the postnatal amygdala, TLR-2 expression was significantly increased at P40 in comparisons to saline-treated controls (**p = 0.007) and to P14 (*p = 0.0232). TLR-4 mRNA expression was significantly increased at E18 in comparison to saline treatment (****p < 0.0001) as well as to E12 (****p < 0.0001), E16 (***p = 0.0008), and P0 (**p = 0.0022), the prenatal/neonatal stages. In the postnatal amygdala, TLR-4 expression was increased at P40 in comparison to saline-treated controls and was unaffected over other postnatal ages examined (*p = 0.050). One-way ANOVA for the main factor age showed a significant effect for COX-2 (F(_{4, 15}) = 3.263, p = 0.0464) and CD36 (F(_{4, 15}) = 4.257, p = 0.0203) but did not record significant differences for iNOS (F(_{4, 15}) = 0.279, p = 0.8862) or CCL2 (F(_{4, 15}) = 1.403, p = 0.2872). Likewise, a significant age effect was observed postnatally for COX-2 (F(_{3, 11}) = 12.74, p = 0.0007), iNOS (F(_{3, 11}) = 3.676, p = 0.0470), and CCL2 (F(_{3, 12}) = 5.909, p = 0.0103), but no significant differences were noted for CD36 expression (F(_{3, 12}) = 1.277, p = 0.3265). In post hoc analyses, mRNA expression of the prostaglandin-synthetizing enzyme cyclooxygenase COX-2 was significantly increased at P40 in comparison to saline-treated controls (Fig. 2b, d **p = 0.0012) and to offspring treated with LPS at P7 (***p = 0.0009) and P14 (*p = 0.0281). CD36, a scavenger receptor expressed by activated microglia/macrophages in the CNS, is implicated in inflammatory responses [37]. CD36 mRNA expression was significantly increased at E16 in comparison to saline-treated controls (*p = 0.024) and higher at E16 versus P0 (*p = 0.035) but remained unaffected in the postnatal amygdala (Fig. 2b, d). Chemokine (c-c motif) ligand 2 (CCL2) was increased but did not reach significance in the prenatal/neonatal ages studied (Fig. 2b, d). Post hoc analyses showed that CCL2 expression was significantly elevated at P40 in comparison to P14 (Fig. 2d, **p = 0.006). The inducible form of nitric oxide (NO) synthase, iNOS, was significantly upregulated in the postnatal amygdala at P40 in comparison to saline-treated controls (Fig. 2d *p = 0.042). Together with the enhanced mRNA levels observed for COX-2, this elevation in iNOS mRNA expression in MIA offspring may indicate a heightened inflammatory response in the postnatal amygdala (Fig. 2b, d).

We next sought to determine if there were morphological alterations in microglia and astrocytes over the same time course after MIA induction in the prenatal brain (E12-P0) (Fig. 4) and postnatal amygdala (P7-P40) (Figs. 3 and 4). Morphological analysis of Iba-1 immunoreactivity (ir) revealed that microglia had larger cell bodies and thicker shortened processes consistent with a de-ramified morphological profile in LPS groups. During neurodevelopment, microglia have an amoeboid morphology consistent with their phagocytic processes and synaptic pruning; however, after MIA insult, these cells exhibit an amoeboid phenotype with an activated status. This was evident in the postnatal ages, as semi-quantitative analysis showed that the predominant microglial phenotype was amoeboid with an “activated” phenotype in offspring from MIA dams. At P7, there was a significant reduction in ramified-like “resting” microglia (***p < 0.001), whereas microglial cells were predominantly amoeboid after MIA (*p < 0.05). This trend was also significantly different at P40 and more robust (Fig. 4b, f; *p < 0.05, ***p < 0.001). GFAP-ir, a marker for astrocytes that is upregulated in inflammation and cell damage, showed moderate astrogliosis in the amygdalar region with bushy-like morphology and overlapping processes in MIA offspring in comparison to saline-treated controls (Fig. 4). In addition, GFAP-ir was more intense in the amygdalar regions with increasing age and treatment. Labeled astrocytes indicative of astrogliosis were prominent along the external capsule and also in the amygdalar region in MIA offspring, in particular at P14 (Fig. 4j). Semi-quantitative scoring of GFAP-ir revealed the highest GFAP staining at P14 in MIA offspring in comparison to controls (Fig. 4j).

After examining the cytokine profiles in the CNS after MIA, next we wanted to investigate the cytoarchitecture of the postnatal amygdala (Fig. 5). It is known that increased cytokine expression may have negative effects on brain structure [28]. Here, we investigated the cytoarchitecture of the postnatal amygdala after E12 maternal injection at P7, P14, and P40 in both treatment groups with the aid of Nissl stain and classification of cell types according to [9] (Fig. 5). Volumetric analysis revealed that the central nucleus (Ce) was significantly smaller at P14 after maternal LPS treatment (*p < 0.05). Moreover, total cell counts using Nissl stain showed a significantly lower number of the total neuron population (**p < 0.01).