Investigation of the effect of intrauterine inflammation and infection on fetal brain injury using human and animal models

In recent years, increased focus has been placed on the role of intrauterine infection and inflammation in the pathogenesis of fetal brain injury leading to neurodevelopmental disorders such as cerebral palsy. At present, the mechanisms by which inflammatory processes during pregnancy cause this effect on the fetus are poorly understood. Our previous work has indicated an association between experimentally-induced intrauterine infection, increased proinflammatory cytokines, and increased white matter injury in the guinea pig fetus. In order to further elucidate the pathways by which inflammation in the maternal system or the fetal membranes leads to fetal impairment, a number of studies investigating aspects of the disease process have been performed. These studies represent a body of work encompassing novel research and results in a number of human and animal studies. Using a guinea pig model of inflammation, increased amniotic fluid proinflammatory cytokines and fetal brain injury were found after a maternal inflammatory response was initiated using endotoxin. In order to more closely monitor the fetal response to chorioamnionitis, a model using the chronically catheterized fetal ovine was carried out. This study demonstrated the adverse effects on fetal white matter after intrauterine exposure to bacterial inoculation, though the physiological parameters of the fetus were relatively stable throughout the experimental protocol, even when challenged with intermittent hypoxic episodes. The placenta is an important mediator between mother and fetus during gestation, though its role in the inflammatory process is largely undefined. Studies on the placental role in the inflammatory process were undertaken, and the limited ability of proinflammatory cytokines and endotoxin to cross the placenta are detailed herein. Neurodevelopmental disorders can be monitored in animal models in order to determine effective disease models for characterization of injury and use in therapeutic strategies. Our characterizations of postnatal behaviour in the guinea pig model using motility monitoring and spatial memory testing have shown small but significant differences in pups exposed to inflammatory processes in utero. The data presented herein contributes a breadth of knowledge to the ongoing elucidation of the pathways by which fetal brain injury occurs. Determining the pathway of damage will lead to discovery of diagnostic criteria, while determining the vulnerabilities of the developing fetus is essential in formulating therapeutic options.

Higher brain neurons succumb to acute stroke-like insult while lower brain neurons strongly resist

Pyramidal neurons (PyNs) in ‘higher’ brain are highly susceptible to acute stroke injury yet ‘lower’ brain regions better survive global ischemia, presumably because of better residual blood flow. Here we show that projection neurons in ‘lower’ brain regions of hypothalamus and brainstem intrinsically resist acute stroke-like injury independent of blood flow in the brain slice. In contrast `higher` projection neurons in neocortex, hippocampus, striatum and thalamus are highly susceptible. In live brain slices from rat deprived of oxygen and glucose (OGD), we imaged anoxic depolarization (AD) as it propagates through these regions. AD, the initial electrophysiological event of stroke, is a depolarizing front that drains residual energy in compromised gray matter. The extent of AD reliably determines ensuing damage in higher brain, but using whole-cell recordings we found that all CNS neurons do not generate a robust AD. Higher neurons generate strong AD and show no functional recovery in contrast to neurons in hypothalamus and brainstem that generate a weak and gradual AD. Most dramatically, lower neurons recover their membrane potential, input resistance and spike amplitude when oxygen and glucose is restored, while higher neurons do not. Following OGD, new recordings could be acquired in all lower (but not higher) brain regions, with some neurons even withstanding multiple OGD exposure. Two-photon laser scanning microscopy confirmed neuroprotection in lower, but not higher gray matter. Specifically pyramidal neurons swell and lose their dendritic spines post-OGD, whereas neurons in hypothalamus and brainstem display no such injury. Exposure to the Na+/K+ ATPase inhibitor ouabain (100 μM), induces depolarization similar to OGD in all cell types tested. Moreover, elevated [K+]o evokes spreading depression (SD), a milder version of AD, in higher brain but not hypothalamus or brainstem so weak AD correlates with the inability to generate SD. In summary, overriding the Na+/K+ pump using OGD, ouabain or elevated [K+]o evokes steep and robust depolarization of higher gray matter. We show that this important regional difference can be largely accounted for by the intrinsic properties of the resident neurons and that Na+/K+ ATPase pump efficiency is a major determining factor generating strong or weak spreading depolarizations.

Glycemic State Regulates Brain-Derived Neurotrophic Factor Responsiveness of Neurons in the Paraventricular Nucleus of the Hypothalamus

Brain derived neurotrophic factor (BDNF) is a member of the family of neurotrophins and binds to the tropomyosin-related kinase B (TrkB) receptor. Like other neurotrophic factors, BDNF is involved in the development and differentiation of neurons. Recently, studies have suggested important roles for BDNF in the regulation of energy homeostasis. The paraventricular nucleus (PVN) is critical for normal energy balance contains high levels of both BDNF and TrkB mRNA. Studies have shown that microinjections of BDNF into the PVN increase energy expenditure, suggesting BDNF plays a role in energy homeostasis through direct actions in this hypothalamic nucleus. We used male Sprague-Dawley rats to perform whole-cell current-clamp experiments from PVN neurons in slice preparation. BDNF was bath applied at a concentration of 2nM and caused depolarizations in 54% of neurons (n = 25; mean change in membrane potential: 8.9 ± 1.2 mV), hyperpolarizations in 23% (n = 11; mean change in membrane potential: -6.7 ± 1.4 mV), while the remaining cells tested were unaffected. Previous studies showing effects of BDNF on γ-aminobutyric acid type A (GABAA) mediated neurotransmission in PVN led us to examine if these BDNF-mediated changes in membrane potential were maintained in the presence of tetrodotoxin (TTX) sodium channel blocker (N = 9; 56% depolarized, 22% hyperpolarized, 22% non-responders) and bicuculline (GABAA antagonist) (N = 12; 42% depolarized, 17% hyperpolarized, 41% non-responders), supporting the conclusion that these effects on membrane potential were postsynaptic. We also evaluated the effects of BDNF on these neurons across varying physiologically relevant extracellular glucose concentrations. At 10 mM 23% (n = 11; mean: -6.7 ± 1.4 mV) of PVN neurons hyperpolarized in response to BDNF treatment, whereas at 0.2 mM glucose, 71% showed hyperpolarizing effects (n = 12; mean: -6.3 ± 2.8 mV). Our findings reveal that BDNF has direct impacts on PVN neurons and that these neurons are capable of integrating multiple sources of metabolically relevant input. Our analysis regarding glucose concentrations and their effects on these neurons’ response to other metabolic signals emphasizes the importance of using physiologically relevant conditions for study of central pathways involved in the regulation of energy homeostasis.