Mingju Cao

Fetal microglial phenotype in vitro carries memory of prior in vivo exposure to inflammation

Objective: Neuroinflammation in utero may result in life-long neurological disabilities. The molecular mechanisms whereby microglia contribute to this response remain incompletely understood. Methods: Lipopolysaccharide (LPS) or saline were administered intravenously to non-anesthetized chronically instrumented near-term fetal sheep to model fetal inflammation in vivo. Microglia were then isolated from in vivo LPS and saline (naïve) exposed animals. To mimic the second hit of neuroinflammation, these microglia were then re-exposed to LPS in vitro. Cytokine responses were measured in vivo and subsequently in vitro in the primary microglia cultures derived from these animals. We sequenced the whole transcriptome of naïve and second hit microglia and profiled their genetic expression to define molecular pathways disrupted during neuroinflammation. Results: In vivo LPS exposure resulted in IL-6 increase in fetal plasma 3 h post LPS exposure. Even though not histologically apparent, microglia acquired a pro-inflammatory phenotype in vivo that was sustained and amplified in vitro upon second hit LPS exposure as measured by IL-1β response in vitro and RNAseq analyses. While NFKB and Jak-Stat inflammatory pathways were up regulated in naïve microglia, heme oxygenase 1 (HMOX1) and Fructose-1,6-bisphosphatase (FBP) genes were uniquely differentially expressed in the second hit microglia. Compared to the microglia exposed to LPS in vitro only, the transcriptome of the in vivo LPS pre-exposed microglia showed a diminished differential gene expression in inflammatory and metabolic pathways prior and upon re-exposure to LPS in vitro. Notably, this desensitization response was also observed in histone deacetylases (HDAC) 1, 2, 4, and 6. Microglial calreticulin/LRP genes implicated in microglia-neuronal communication relevant for the neuronal development were up regulated in second hit microglia. Discussion: We identified a unique HMOX1down and FBPup phenotype of microglia exposed to the double-hit suggesting interplay of inflammatory and metabolic pathways. Our findings suggest that epigenetic mechanisms mediate this immunological and metabolic memory of the prior inflammatory insult relevant to neuronal development and provide new therapeutic targets for early postnatal intervention to prevent brain injury.

Does heart rate variability reflect the systemic inflammatory response in a fetal sheep model of lipopolysaccharide-induced sepsis?

Fetal inflammatory response occurs during chorioamnionitis, a frequent and often subclinical inflammation associated with increased risk for brain injury and life-lasting neurologic deficits. No means of early detection exist. We hypothesized that systemic fetal inflammation without septic shock will be reflected in alterations of fetal heart rate (FHR) variability (fHRV) distinguishing baseline versus inflammatory response states. In chronically instrumented near-term fetal sheep (n = 24), we induced an inflammatory response with lipopolysaccharide (LPS) injected intravenously (n = 14). Ten additional fetuses served as controls. We measured fetal plasma inflammatory cytokine IL-6 at baseline, 1, 3, 6, 24 and 48 h. 44 fHRV measures were determined continuously every 5 min using continuous individualized multi-organ variability analysis (CIMVA). CIMVA creates an fHRV measures matrix across five signal-analytical domains, thus describing complementary properties of fHRV. Using principal component analysis (PCA), a widely used technique for dimensionality reduction, we derived and quantitatively compared the CIMVA fHRV PCA signatures of inflammatory response in LPS and control groups. In the LPS group, IL-6 peaked at 3 h. In parallel, PCA-derived fHRV composite measures revealed a significant difference between LPS and control group at different time points. For the LPS group, a sharp increase compared to baseline levels was observed between 3 h and 6 h, and then abating to baseline levels, thus tracking closely the IL-6 inflammatory profile. This pattern was not observed in the control group. We also show that a preselection of fHRV measures prior to the PCA can potentially increase the difference between LPS and control groups, as early as 1 h post LPS injection. We propose a fHRV composite measure that correlates well with levels of inflammation and tracks well its temporal profile. Our results highlight the potential role of HRV to study and monitor the inflammatory response non-invasively over time.

Can Monitoring Fetal Intestinal Inflammation Using Heart Rate Variability Analysis Signal Incipient Necrotizing Enterocolitis of the Neonate

Objective: Necrotizing enterocolitis of the neonate is an acute inflammatory intestinal disease that can cause necrosis and sepsis. Chorioamnionitis is a risk factor of necrotizing enterocolitis. The gut represents the biggest vagus-innervated organ. Vagal activity can be measured via fetal heart rate variability. We hypothesized that fetal heart rate variability can detect fetuses with incipient gut inflammation. Design: Prospective animal study. Setting: University research laboratory. Subjects: Chronically instrumented near-term fetal sheep (n = 21). Measurements and Main Results: Animals were surgically instrumented with vascular catheters and electrocardiogram to allow manipulation and recording from nonanesthetized animals. In 14 fetal sheep, inflammation was induced with lipopolysaccharide (IV) to mimic chorioamnionitis. Fetal arterial blood samples were drawn at selected time points over 54 hours post lipopolysaccharide for blood gas and cytokines (interleukin-6 and tumor necrosis factor-&agr; enzymelinked immunosorbent assay). Fetal heart rateV was quantified throughout the experiment. The time-matched fetal heart rate variability measures were correlated to the levels of interleukin-6 and tumor necrosis factor-&agr;. Upon necropsy, ionized calcium binding adaptor molecule 1+ (Iba1+), CD11c+ (M1), CD206+ (M2 macrophages), and occludin (leakiness marker) immunofluorescence in the terminal ileum was quantified along with regional Iba1+ signal in the brain (microglia). Interleukin-6 peaked at 3 hours post lipopolysaccharide accompanied by mild cardiovascular signs of sepsis. At 54 hours, we identified an increase in Iba1+ and, specifically, M1 macrophages in the ileum accompanied by increased leakiness, with no change in Iba1 signal in the brain. Preceding this change on tissue level, at 24 hours, a subset of nine fetal heart rate variability measures correlated exclusively to the Iba+ markers of ileal, but not brain, inflammation. An additional fetal heart rate variability measure, mean of the differences of R-R intervals, correlated uniquely to M1 ileum macrophages increasing due to lipopolysaccharide. Conclusions: We identified a unique subset of fetal heart rate variability measures reflecting 1.5 days ahead of time the levels of macrophage activation and increased leakiness in terminal ileum. We propose that such subset of fetal heart rate variability measures reflects brain-gut communication via the vagus nerve. Detecting such noninvasively obtainable organ-specific fetal heart rate variability signature of inflammation would alarm neonatologists about neonates at risk of developing necrotizing enterocolitis and sepsis. Clinical validation studies are required.

Instrumentation of Near-term Fetal Sheep for Multivariate Chronic Non-anesthetized Recordings

The chronically instrumented pregnant sheep has been used as a model of human fetal development and responses to pathophysiologic stimuli such as endotoxins, bacteria, umbilical cord occlusions, hypoxia and various pharmacological treatments. The life-saving clinical practices of glucocorticoid treatment in fetuses at risk of premature birth and the therapeutic hypothermia have been developed in this model. This is due to the unique amenability of the non-anesthetized fetal sheep to the surgical placement and maintenance of catheters and electrodes, allowing repetitive blood sampling, substance injection, recording of bioelectrical activity, application of electric stimulation and in vivo organ imaging. Here we describe the surgical instrumentation procedure required to achieve a stable chronically instrumented non-anesthetized fetal sheep model including characterization of the post-operative recovery from blood gas, metabolic and inflammation standpoints.

Temporal Patterns in Sheep Fetal Heart Rate Variability Correlate to Systemic Cytokine Inflammatory Response: A Methodological Exploration of Monitoring Potential Using Complex Signals Bioinformatics

Fetal inflammation is associated with increased risk for postnatal organ injuries. No means of early detection exist. We hypothesized that systemic fetal inflammation leads to distinct alterations of fetal heart rate variability (fHRV). We tested this hypothesis deploying a novel series of approaches from complex signals bioinformatics. In chronically instrumented near-term fetal sheep, we induced an inflammatory response with lipopolysaccharide (LPS) injected intravenously (n = 10) observing it over 54 hours; seven additional fetuses served as controls. Fifty-one fHRV measures were determined continuously every 5 minutes using Continuous Individualized Multi-organ Variability Analysis (CIMVA). CIMVA creates an fHRV measures matrix across five signal-analytical domains, thus describing complementary properties of fHRV. We implemented, validated and tested methodology to obtain a subset of CIMVA fHRV measures that matched best the temporal profile of the inflammatory cytokine IL-6. In the LPS group, IL-6 peaked at 3 hours. For the LPS, but not control group, a sharp increase in standardized difference in variability with respect to baseline levels was observed between 3 h and 6 h abating to baseline levels, thus tracking closely the IL-6 inflammatory profile. We derived fHRV inflammatory index (FII) consisting of 15 fHRV measures reflecting the fetal inflammatory response with prediction accuracy of 90%. Hierarchical clustering validated the selection of 14 out of 15 fHRV measures comprising FII. We developed methodology to identify a distinctive subset of fHRV measures that tracks inflammation over time. The broader potential of this bioinformatics approach is discussed to detect physiological responses encoded in HRV measures.

Adaptive shut‐down of EEG activity predicts critical acidemia in the near‐term ovine fetus

In fetal sheep, the electrocorticogram (ECOG) recorded directly from the cortex during repetitive heart rate (FHR) decelerations induced by umbilical cord occlusions (UCO) predictably correlates with worsening hypoxic‐acidemia. In human fetal monitoring during labor, the equivalent electroencephalogram (EEG) can be recorded noninvasively from the scalp. We tested the hypothesis that combined fetal EEG – FHR monitoring allows for early detection of worsening hypoxic‐acidemia similar to that shown for ECOG‐FHR monitoring. Near‐term fetal sheep (n = 9) were chronically instrumented with arterial and venous catheters, ECG, ECOG, and EEG electrodes and umbilical cord occluder, followed by 4 days of recovery. Repetitive UCOs of 1 min duration and increasing strength (with regard to the degree of reduction in umbilical blood flow) were induced each 2.5 min until pH dropped to <7.00. Repetitive UCOs led to marked acidosis (arterial pH 7.35 ± 0.01 to 7.00 ± 0.03). At pH of 7.22 ± 0.03 (range 7.32–7.07), and 45 ± 9 min (range 1 h 33 min–20 min) prior to attaining pH < 7.00, both ECOG and EEG amplitudes began to decrease ~fourfold during each FHR deceleration in a synchronized manner. Confirming our hypothesis, these findings support fetal EEG as a useful adjunct to FHR monitoring during human labor for early detection of incipient fetal acidemia.

RNAseq profiling of primary microglia and astrocyte cultures in near-term ovine fetus: a glial in vivo-in vitro multi-hit paradigm in large mammalian brain

BACKGROUND The chronically instrumented fetal sheep is a widely used animal model to study fetal brain development in health and disease, but no methods exist yet to interrogate dedicated brain cell populations to identify their molecular and genomic phenotype. For example, the molecular mechanisms whereby microglia or astrocytes contribute to inflammation in the brain remain incompletely understood. NEW METHOD Here we present a protocol to derive primary pure microglial or astrocyte cultures from near-term fetal sheep brain, after the animals have been chronically instrumented and studied in vivo. Next, we present the implementation of whole transcriptome sequencing (RNAseq) pipeline to deeper elucidate the phenotype of such primary sheep brain glial cultures. RESULTS We validate the new primary cultures method for cell purity and test the function of the glial cells on protein (IL-1β) and transcriptome (RNAseq) levels in response to a lipopolysaccharide (LPS) challenge in vitro. COMPARISON WITH EXISTING METHODS This method represents the first implementation of pure microglial or astrocytes cultures in fetal sheep brain. CONCLUSIONS The presented approach opens new possibilities for testing not only supernatant protein levels in response to an in vitro challenge, but also to evaluate changes in the transcriptome of glial cells derived from a large mammalian brain bearing high resemblance to the human brain. Moreover, the presented approach lends itself to modeling the complex multi-hit paradigms of antenatal and perinatal cerebral insults in vivo and in vitro.

The Ovine Fetal and Placental Inflammatory Response to Umbilical Cord Occlusions With Worsening Acidosis

We hypothesized that repetitive umbilical cord occlusions (UCOs) leading to severe acidemia will stimulate a placental and thereby fetal inflammatory response which will be exacerbated by chronic hypoxemia and low-grade bacterial infection. Chronically instrumented fetal sheep served as controls or underwent repetitive UCOs for up to 4 hours or until fetal arterial pH was <7.00. Normoxic-UCO and hypoxic-UCO fetuses had arterial O2 saturation pre-UCOs of >55% and <55%, respectively, while lipopolysaccharide (LPS)-UCO fetuses received LPS intra-amniotic (2 mg/h) starting 1 hour pre-UCOs. Fetal plasma and amniotic fluid were sampled for interleukin (IL) 6 and IL-1β. Animals were euthanized at 48 hours of recovery with placental cotyledons processed for measurement of macrophage, neutrophil, and mast cell counts. Repetitive UCOs resulted in severe fetal acidemia with pH approaching 7.00 for all 3 UCO groups. Neutrophils, while unchanged within the cotyledon fetal and intermediate zones, were ∼2-fold higher within the zona intima for all 3 UCO groups. However, no differences were observed in macrophage counts among the treatment groups and no cotyledon mast cells were seen. Fetal plasma and amniotic fluid cytokines remained little changed post-UCOs and/or at 1 and 48 hours of recovery in the normoxic-UCO and hypoxic-UCO groups but increased several fold in the LPS-UCO group with IL-6 plasma values at 1 hour recovery highly correlated with the nadir pH attained (r = −.97). As such, repetitive UCOs with severe acidemia can induce a placental inflammatory response and more so with simulated low-grade infection and likely contributing to cytokine release in the umbilical circulation.

Sculpting the Sculptors: Methods for Studying the Fetal Cholinergic Signaling on Systems and Cellular Scales

The non-neuronal, immunological effects of the cholinergic signaling are exerted on the systems scale of observation via the vagus nerve and on the cellular scale via α7 nicotinic acetylcholine receptor (nAChR) signaling in myeloid cells of the periphery or brains microglia and astrocytes. The developmental effects of such multi-scale signaling can be conceived of as an example of psychoneuroimmunological (PNI) homeokinesis and, while reported in the literature, are not yet systematically well studied. To be better understood, the intricacy of the multi-scale interactions requires relevant preclinical animal models. Chronically instrumented non-anesthetized fetal sheep model comes with a strong track record of bench-to-bed translation and a large body of evidence for its strong resemblance to and relevance for human physiology on various scales of organization. Recently, there has been growing interest in pleiotropic effects of vagus nerve stimulation (VNS) on various organ systems such as innate immunity, metabolism, and emotion with implications for programming of PNI phenotype. Here we describe the procedures required to record and manipulate the vagus nerve activity in this large pregnant mammalian organism. Extending this in vivo model to in vitro, on the cellular scale, we present the method to manipulate the cholinergic signaling in ovine fetal microglia and astrocytes and analyze their responses on protein and RNA levels. Together these models can provide multi-scale-level mechanistic insights into the effects of cholinergic signaling on PNI phenotype.

α7 nicotinic acetylcholine receptor signalling modulates the inflammatory and iron homeostasis in fetal brain microglia

Neuroinflammation in utero may result in life-long neurological disabilities. Microglia play a pivotal role, but the mechanisms are poorly understood. No early postnatal treatment strategies exist to enhance neuroprotective potential of microglia. We hypothesized that agonism on α7 nicotinic acetylcholine receptor (α7nAChR) in fetal microglia will augment their neuroprotective transcriptome profile, while the antagonistic stimulation of α7nAChR will achieve the opposite. Using an in vivo - in vitro model of developmental programming of neuroinflammation induced by lipopolysaccharide (LPS), we validated this hypothesis in primary fetal sheep microglia cultures re-exposed to LPS in presence of a selective α7nAChR agonist or antagonist. Our RNAseq and protein level findings show that a pro-inflammatory microglial phenotype acquired in vitro by LPS stimulation is reversed with α7nAChR agonistic stimulation. Conversely, antagonistic α7nAChR stimulation potentiates the pro-inflammatory microglial phenotype. Surprisingly, under conditions of LPS double-hit an interference of a postulated α7nAChR - ferroportin signaling pathway may impede this mechanism. These results suggest a therapeutic potential of α7nAChR agonists in early re-programming of microglia in neonates exposed to in utero inflammation via an endogenous cerebral cholinergic anti-inflammatory pathway. Future studies will assess the role of interactions between inflammation-triggered microglial iron sequestering and α7nAChR signaling in neurodevelopment.