Background
Pregnancy and gestation are a critical time for fetal development. Recent evidence suggests that fetal exposure to inflammatory cytokines can have long-lasting effects on postnatal physiology, sometimes lasting into adulthood [
1,
2]. During intrauterine infection/inflammation, proinflammatory cytokines and other mediators can cross the compromised blood–brain barrier and induce activation of microglia and signaling through astrocytes in the fetal brain, causing subsequent production of reactive oxygen species and cytokines that can lead to glutamate-induced excitotoxicity [
3‐
5]. In addition to the possibility of modulation of neurodevelopment, moderate maternal inflammation also leads to elevated production of serotonin in the placenta, which can disrupt fetal neurodevelopment of serotonin-dependent processes in the forebrain [
6].
Preterm children that are exposed to inflammation in utero are at a greater risk for neurological, emotional, and learning disorders [
7‐
9]. Additionally, maternal inflammation has been linked with increased prevalence of autism and experimental animal models mimicking maternal infection and inflammation result in autism-like phenotypes [
10,
11]. Similarly, elevated levels of maternal cytokines, in particular tumor necrosis factor (TNF)α, were associated with increasing odds of adult schizophrenia and other psychoses in their offspring [
12]. These results strongly suggest that in utero inflammation and postnatal cognitive abnormalities are causally linked.
Animal models of intrauterine inflammation include mice, rats, rabbits, and sheep [
13,
14]. One well-characterized rodent model of preterm birth that mimics human situation of local inflammatory response in the uterus and no overt infection in the dam is intrauterine lipopolysaccharide (LPS) injection [
13]. At high doses (250 μg/100 μl), LPS injection results in > 95% preterm birth and significant fetal brain injury [
1,
15]. Decreasing LPS doses (50 μg/100 μl), however, resulted in ~ 30% preterm births, yet with detectable levels of activated microglia as late as 60 days postnatal (DPN) [
2,
16]. In mice exposed to LPS in utero, the volume of the hippocampus, a structure important for learning and memory, is reduced. Whether hippocampal functions are altered in the survivors of preterm birth is currently unclear. In this study, we tested whether exposure to low-dose LPS in utero alters information transfer and storage by the hippocampus in adult survivor mice to understand the cellular mechanisms contributing to cognitive deficits in survivors of preterm birth.
Methods
Mouse model of intrauterine inflammation
All animal care and treatment procedures were approved by the Institutional Animal Care and Use Committee, and animals were handled according to the National Institutes of Health guidelines. An established model of intrauterine inflammation was utilized for these studies [
15]. Briefly, timed pregnant CD-1 outbred mice were obtained from Charles River Laboratories (Wilmington, MA). Intrauterine injections of 100 μl of LPS (from
Escherichia coli, 055: B5, Sigma-Aldrich, St. Louis, MO) at a dose of 50 μg in 100 μL of phosphate-buffered saline (PBS) were administered on embryonic day 17 (E17) of a 19-day gestation period in four independent experiments. Control dams for these experiments received the same volume of intrauterine injection of vehicle. In total, 11 dams were injected with PBS with all litters surviving and 43 dams were injected with LPS with 16 litters surviving. For survival surgery, pregnant mice were anesthetized using isoflurane, and a mini-laparotomy was then performed in the lower abdomen for intrauterine injections. Live pups were separated by sex, and only males were utilized for these studies. While sex could play an important role in the long-term effects of intrauterine inflammation [
2], we concentrated on males only in the current study to avoid possible effects of estrus cycle in which circulating hormones could affect hippocampal function [
17] as well as potential sex difference in microglia during development [
18].
Immunohistochemistry
At 45 DPN, after the animals were euthanized, 1× PBS was perfused transcardially, followed by 4% paraformaldehyde (PFA). The brain from one animal from each dam was dissected and post-fixed in PFA overnight. The next day, specimens were washed with PBS extensively and immersed in 30% sucrose until saturation, followed by cryosection at 20-μm thickness. Sections were incubated overnight at 4 °C with rabbit anti-Iba-1 (Wako, Richmond, VA) to identify microglia. Donkey anti-rabbit Dylight 568 (Abcam, Cambridge, MA) was applied as the secondary antibody. The sections were further counter-stained with DAPI (Roche, Indianapolis, IN) to identify cell nuclei. Images were obtained using an Axioplan 2 Imaging system (Carl Zeiss, Thornwood, NY) at the bregma level from − 1.34 to − 1.70 mm. Quantitative analysis of Iba-1 expression cell numbers and area percentage within CA3–CA1 was performed using ImageJ 1.37V (NIH). Each cell was identified as the positive expression (red) in cytoplasm and DAPI (blue) in nucleus. The percentage of Iba-1 expression area was calculated by positive expression area (cell bodies and branches) divided by CA3–CA1 area. The average number of both hippocampi in each hemisphere represented the section counted. The average number of five sections represented the specimen counted.
Hippocampal slice preparation
Mice were anesthetized with isoflurane and rapidly decapitated. The brain was removed, and 300-μm slices from the middle of the hippocampus were cut using a vibrating microtome (VT1000S; Leica Instrument, Leitz) as the brain was immersed in an ice-cold sucrose substituted artificial CSF (aCSF) of the following composition (in mM): 119 NaCl, 26 NaHCO3, 2.5 KCl, 1 NaH2PO4, 1 MgCl2, 2 CaCl2, and 25 dextrose (oxygenated with a carbogen mixture of 95% O2 and 5% CO2). Slices were held in oxygenated aCSF at 35 °C for 30 min and then at room temperature (22–24 °C) for at least 1 h before recording.
Electrophysiology
All recordings were made at room temperature. Hippocampal slices were visualized using a fixed-stage, upright microscope (Axio Examiner or Leica DMLFS) equipped with infrared differential interference contrast optics. The recording chamber was continuously perfused with oxygenated aCSF flowing at a rate of 1–2 ml/min. Recording electrodes were pulled from borosilicate pipettes (Sutter Instruments) and had tip resistances of 2–3.5 MΩ when filled with aCSF for extracellular field recordings. Glass stimulating electrodes of approximate resistance of 1 MΩ were filled with aCSF, connected to a Digitimer constant current stimulus isolation unit (AutoMate Scientific, Berkeley, CA), and placed in the middle of the CA1 stratum radiatum to stimulate the CA3 axon collaterals. The CA3 axons were severed to eliminate recurrent excitation within the CA3 subfield. The stimulating and recording electrodes were placed in the middle portion of the CA1 stratum radiatum (approximately equal distance from stratum pyramidal and stratum lacunosum moleculare). Stimulus duration was 0.1–0.2 ms allowing for clear separation of fiber volley (FV) from the preceding stimulus artifact. Long-term potentiation (LTP) of CA3–CA1 excitatory synapses was induced by stimulating CA3 axons with 3 sets of 100 stimuli delivered at 50 Hz.
Recordings were obtained using a Multiclamp 700B amplifier (Molecular Devices) or GeneClamp 500 amplifier (Axon Instruments). Signals were filtered at 3 kHz, digitized using a Digidata 1440A interface (Molecular Devices) at 10 kHz, and transferred to a computer using pClamp10 software (Molecular Devices) or an ITC-16 (Instrutech Corp., NY) and a computer using Patchmaster (Heka Instruments).
All experiments were performed in the presence of picrotoxin (100 μM) and CGP55845 (2 μM; both from Tocris Bioscience) to suppress inhibitory synaptic transmission.
Data and statistical analyses
Voltage traces were analyzed using custom macros written in Igor Pro (WaveMetrics). Statistical analysis was done using R version 3.3.2 and the geepack package [
19]. To account for correlation among hippocampal slices taken from the same animal, we constructed simple GEE models of field excitatory postsynaptic potential (fEPSP) input–output (I/O) relationship, FV–stimulus intensity, paired pulse ratio, and long-term synaptic plasticity with exchangeable correlation structures and, to accommodate the small number of mice relative to the number of slices, a jackknife variance estimator, with intrauterine exposure to LPS as the explanatory variable. We compared Iba-1 expression cell numbers and area percentage using Student’s
t test with unequal variance.
Availability of data and materials
All data generated or analyzed during this study are included in this published article. Custom macros written in Igor Pro (WaveMetrics) are available on request (JM).
Discussion
Children born preterm due to exposure to intrauterine infection or inflammation are at greater risk for developing acute fetal brain injury as well as adverse neurological outcomes including cognitive, motor, and behavioral disabilities such as autism [
13,
26‐
28]. Using a murine model of inflammation and perinatal brain injury, we demonstrated that offspring of pregnant mice exposed to LPS displayed increased synaptic strength, due in part to an increase in the probability of glutamate release from the presynaptic CA3 axon terminals, as evidenced by the reduced PPR ratio. LPS-exposed mice also had lower levels of LTP compared to control mice. This finding could be due to disrupted AMPA receptor trafficking in CA1 pyramidal neurons—a similar cellular change observed in ischemia-induced impairment of LTP [
29]. Synaptic dysfunctions and impaired synaptic plasticity at the hippocampal synapses are commonly observed in animal models of disease or toxic substance exposure that exhibit cognitive deficits, including fragile X syndrome [
30], Alzheimer’s disease [
31], accelerated aging [
32], and perinatal/acute exposure to lead and polychlorinated biphenyls [
33] or marijuana [
34]. Thus, the novel findings from the present studies showing changes in hippocampal synaptic transmission and plasticity offer a plausible explanation for the cognitive and behavioral deficits observed in survivors of preterm birth.
The cellular and molecular mechanisms of inflammation-induced fetal brain injury are not fully understood but are likely to involve proinflammatory chemokine and cytokine signaling. During intrauterine inflammation, infectious pathogens likely activate toll-like receptors (TLR) on the surface of cells in the decidua and placental membranes, resulting in the production of proinflammatory cytokines that can cross the compromised blood–brain barrier into the fetal brain where they activate microglia, the primary defense mechanism in the brain [
4,
5]. This process then initiates a cascade of events that leads to increase in proinflammatory cytokines IL-1, IL-6, and TNFα that remain elevated after birth [
35‐
37]. Evidence from adult inflammation models shows that microglia are activated and produce the proinflammatory cytokine TNFα that signals through astrocytes to irreversibly alter synaptic transmission and impair cognition [
38‐
40]. The precise mechanism by which TNFα causes increased synaptic transmission has been studied extensively and may include both pre- and post-synaptic effects that involve retrograde signaling of prostaglandins and nitric oxide [
41‐
43] and synaptic scaling [
40]. Importantly, activation of microglia and release of TNFα and the subsequent signaling through astrocytes is the key event leading to behavioral comorbidities as a result of chronic inflammation. Indeed, chronic administration of the microglial/macrophage activation inhibitor minocycline to the inflamed animal both lowered the level of TNFα in the hippocampus and completely abolished the effect of peripheral inflammation which induced changes in synaptic transmission and synaptic plasticity [
44].
Interestingly, peripheral inflammation is a feature of many adult neurodegenerative diseases and is often associated with marked behavioral changes, including mood disorders, fatigue, cognitive and memory dysfunction, and sleep disturbances. Moreover, the inflammation is capable of aggravating other neurological and neuropsychiatric conditions, including seizure disorders, major depression, Alzheimer’s disease, multiple sclerosis, Parkinson’s disease, and autism [
45]. The changes in synaptic transmission in adult inflammation models are similar to those observed here with prenatal inflammation. It is likely that the persistent activation of microglia 45 DPN in survivors of LPS-treated mice in utero contributes to these changes. Whether they are mediated by TNFα signaling is yet to be determined.
This study not only demonstrates the effects of inflammation on brain function but also reveals a unique long-lasting component of the effect of inflammation into adulthood when experienced during a critical period of development. In addition to having implications for neurological effects from peripheral inflammation, these findings extend our understanding of the cognitive deficits in those born preterm as well as other disorders such as autism and schizophrenia.
Conclusion
Using a murine model of inflammation and perinatal brain injury, we demonstrated that offspring of pregnant mice exposed to LPS displayed altered hippocampal excitatory synaptic function. Synaptic transmission at CA3–CA1 synapses was increased due in part to an increase in the probability of glutamate release from the presynaptic CA3 axon terminals. Importantly, LPS-exposed mice also had lower levels of LTP compared to control mice. These novel findings offer a plausible explanation for the cognitive and behavioral deficits observed in survivors of preterm birth caused by intrauterine inflammation.
Acknowledgements
This work was supported by research funding: NIH and Maternal Fetal Medicine Fund to JM and Sheikh Bugshan Fund and Integrated Research Center for Fetal Medicine Fund to IB.
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