Jhodie R. Duncan

White Matter Injury after Repeated Endotoxin Exposure in the Preterm Ovine Fetus

Intrauterine infection has been linked to neurologic injury in preterm infants. However, a reproducible model of white matter injury in the preterm fetus in a long gestation species that can be monitored in utero is currently unavailable. Thus, our objective was to determine the effects of bacterial endotoxin (lipopolysaccharide, LPS) on physiologic and inflammatory responses and brain structure in the preterm ovine fetus. At 0.7 of gestation, six catheterized fetuses received three to five intravenous injections of LPS (1 μg/kg) over 5 d; seven fetuses served as controls. Fetal responses were monitored and brain tissue examined 10–11 d after the initial LPS injection. After LPS on d 1 and 2, fetuses became transiently hypoxemic and hypotensive and blood IL-6 levels were increased, but these responses were smaller or absent after subsequent LPS exposures. Neural injury was observed in all LPS-exposed fetuses, most prominently in the cerebral white matter. Injury ranged from diffuse subcortical damage to periventricular leukomalacia, and in the brainstem the cross-sectional area of the corticospinal tract was reduced by 30%. Thus, repeated exposure of the preterm ovine fetus to LPS causes neuropathology resembling that of cerebral palsy and provides a robust model for exploring the etiology, prevention, and treatment of white matter damage.

Programming effects of short prenatal exposure to cortisol

Recent studies have linked fetal exposure to a suboptimal intrauterine environment with adult hypertension. The aims of this study were twofold: 1) to see whether cortisol treatment administered to the ewe for 2 days at 27 days of gestation (term ~150 days) resulted in high blood pressure in offspring; 2) to study the effect of the same treatment on gene expression in the brain at 130 days of gestation and in lambs at 2 months of age. Mean arterial pressure was significantly higher in the adult female and male offspring of sheep treated with cortisol than in the control group (females: 89±2 mmHg vs. 81±2; P<0.05 and males: 102±4 mmHg vs. 91±3; P<0.05). Prenatal cortisol treatment led to up‐regulation of angiotensinogen, AT1, MR, and GR mRNA in the hippocampus in fetuses at 130 days of gestation but not in the animals at 2 months of age. This is the first evidence that short prenatal exposure to cortisol programmed high blood pressure in the adult female and male offspring of sheep. Altered gene expression in the hippocampus could have a significant effect on the development of the hippocampus, and on postnatal behavior.—Dodic, M., Hantzis, V., Duncan, J., Rees, S., Koukoulas, I., Johnson, K., Wintour, E. M., Moritz, K. Programming effects of short prenatal exposure to cortisol. FASEB J. 16, 1017–1026 (2002)

Chronic Exposure to Intra-Amniotic Lipopolysaccharide Affects the Ovine Fetal Brain

Objective: Fetal brain injury is associated with chorioamnionitis, which is often present without signs of overt infection of fetal compromise. We aimed to determine if prolonged exposure to intrauterine inflammation caused by intra-amniotic infusion of lipopolysaccharide (LPS) would affect the fetal brain. Methods: At 80 days of pregnancy ewes bearing singletons had osmotic pumps implanted intraamniotically to infuse Escherichia coli LPS (055:B5; n = 8) or saline (n = 7) for 28 days. At delivery (110 days), umbilical arterial blood and chorioamnion were assessed for inflammation; cytokine concentrations (interleukin [IL]-6 and IL-8) in amniotic fluid and fetal and maternal plasma were measured. The fetal cerebral hemispheres were examined for gross anatomical changes and the number of activated microglia/macrophages, astrocytes, and oligodendrocytes estimated after immunohistochemical staining. Results: Intra-amniotic administration of LPS caused chorioamnionitis, fetal leucocytosis, and a moderate to extensive infiltration of activated microglia/macrophages in the subcortical white matter in six of eight fetuses; the remaining two fetuses were less affected. Within these focal regions of damage there was an attenuation of astrocytic processes, axonal injury, and a reduction in the number of 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) immunoreactive oligodendrocytes in areas of extensive focal damage. In control fetuses there was mild (#/7) or no infiltration of activated microglia/macrophages in the subcortical white matter. Overall the infiltration of activated microglia/macrophages in the white matter was significantly greater in LPS-exposed fetuses compared to controls. In regions devoid of injury, the number of oligodendrocytes and astrocytes was not different between groups, nor was there a difference in the volume of cerebral white matter or density of blood vessels within the white matter. Amniotic fluid IL-6 and IL-6, and maternal plasma IL-8 concentrations were significantly increased by LPS infusion. Conclusions: An increase in inflammatory cells and axonal disruption in the subcortical white matter of the fetal brain can accompany chorioamnionitis induced by intra-amniotic administration of LPS, but cystic lesions do not occur. Thus, the effect on the fetal brain is milder than that reported from animal models of acute fetal/intrauterine infection.

Chronic endotoxin exposure causes brain injury in the ovine fetus in the absence of hypoxemia.

Objective: Intrauterine infection has been linked to brain injury in human infants, although the mechanisms are not fully understood. We recently showed that repeated acute exposure of preterm fetal sheep to bacterial endotoxin (lipopolysaccharide [LPS]) results in fetal hypoxemia, hypotension, increased systematic proinflammatory cytokines, and brain damage, including white matter injury. However, it is not clear whether this injury is caused by reduced cerebral oxygen delivery or inflammatory pathways independent of hypoxia. The aim of the present study was to determine the effects on the fetal brain and placenta of a chronic intrauterine inflammatory state, induced by LPS infusion into the fetal circulation, a model that did not cause hypoxia. Methods: At 0.65 of term, eight catheterized fetal sheep received intravenous infusions of LPS (5 to 15 μg) over 5 days; control fetuses received saline. Fetal physiologic responses were monitored throughout the infusion. Fetal brain and placental tissues were examined histologically 6 days after the conclusion of the infusion. Results: LPS infusions did not result in physiologically significant alterations to fetal blood gases or mean arterial pressure; however, plasma proinflammatory cytokine levels were elevated. Following LPS exposure there was no difference in fetal body or brain weights (P >.05); placental weight was reduced (P <.05), consistent with reduced placentome cross-sectional area (P <.05). In the cerebral hemispheres subcortical white matter injury was present in six LPS-exposed fetuses and included axonal damage, microgliosis, oligodendrycyte injury, and increased β amyloid precursor protein (β-APP) expression. Conclusions: Chronic, systemic exposure of the fetus to LPS resulted in fetal brain damage in the absence of hypoxemia or hypotension, although the resulting injury was less severe than following repeated acute exposure.

The fetal brainstem is relatively spared from injury following intrauterine hypoxemia

Our aim was to test the hypothesis that the fetal brainstem is relatively spared, compared to other brain regions, from hypoxia-induced damage. We have used established experimental models of acute and chronic intrauterine compromise in sheep to mimic conditions that can arise in human pregnancy. The acute insult was 12 h of placental insufficiency induced by restricted utero-placental blood flow at 90 days of gestation (term approximately 147 days). Five weeks after this insult (n=7 fetuses) there was no overt damage to the brainstem nor were there alterations to the blood vessel morphology, volume of the medulla or of medullary nuclei compared to controls (n=8). This regimen is known to have significant effects on the forebrain and cerebellum. The chronic insult was induced in five fetuses via embolisation of the umbilico-placental circulation from 120 to 140 days of gestation. An additional three fetuses were found to be spontaneously hypoxemic (SH) immediately after surgery. At 140 days, in brainstems of all chronically hypoxemic fetuses compared to controls (n=8), there was an increase (P<0.05) in the percentage of neuropil occupied by blood vessels and abnormal myelin in the most severely SH fetus but no other morphological or neurochemical alterations. This regimen is known to cause marked damage to the cerebral hemispheres and to a lesser extent to the cerebellum. We suggest that the absence of marked structural or neurochemical alterations in the brainstem is most likely due to the maintenance of oxygen delivery to the brainstem during fetal hypoxemia.

The role of metabotropic glutamate receptors in addiction: Evidence from preclinical models

Addiction is a chronic disorder characterised by repeated bouts of drug taking, abstinence and relapse. The addicted state may be in part due to drug-induced neuroadaptations in the mesocorticolimbic and corticostriatal pathways. Recently focus has been on the role of aberrant glutamate transmission and its contribution to the hierarchical control over these systems. This review will expand our current knowledge of the most recent advances that have been made in preclinical animal models that provide evidence that implicate metabotropic glutamate receptors (mGluRs) in contributing to the neuroadaptations pertinent to addiction, as well as the role of Homer proteins in regulating these responses. The recent discovery of receptor mosaics will be discussed which add an additional dimension to the complexity of understanding the mechanism of glutamate mediated behaviours. Finally this review introduces a new area related to glutamatergic responses, namely microRNAs, that may become pivotal in directing our future understanding of how to best target intervention strategies to prevent addictive behaviours.

Adolescent toluene inhalation in rats affects white matter maturation with the potential for recovery following abstinence.

Inhalant misuse is common during adolescence, with ongoing chronic misuse associated with neurobiological and cognitive abnormalities. While human imaging studies consistently report white matter abnormalities among long-term inhalant users, longitudinal studies have been lacking with limited data available regarding the progressive nature of such abnormalities, including the potential for recovery following periods of sustained abstinence. We exposed adolescent male Wistar rats (postnatal day 27) to chronic intermittent inhaled toluene (3,000 ppm) for 1 hour/day, 3 times/week for 8 weeks to model abuse patterns observed in adolescent and young adult human users. This dosing regimen resulted in a significant retardation in weight gain during the exposure period (p<0.05). In parallel, we performed longitudinal magnetic resonance imaging (T2-weighted) and diffusion tensor imaging prior to exposure, and after 4 and 8 weeks, to examine the integrity of white matter tracts, including the anterior commissure and corpus callosum. We also conducted imaging after 8 weeks of abstinence to assess for potential recovery. Chronic intermittent toluene exposure during adolescence and early adulthood resulted in white matter abnormalities, including a decrease in axial (p<0.05) and radial (p<0.05) diffusivity. These abnormalities appeared region-specific, occurring in the anterior commissure but not the corpus callosum and were not present until after at least 4 weeks of exposure. Toluene-induced effects on both body weight and white matter parameters recovered following abstinence. Behaviourally, we observed a progressive decrease in rearing activity following toluene exposure but no difference in motor function, suggesting cognitive function may be more sensitive to the effects of toluene. Furthermore, deficits in rearing were present by 4 weeks suggesting that toluene may affect behaviour prior to detectable white matter abnormalities. Consequently, exposure to inhalants that contain toluene during adolescence and early adulthood appear to differentially affect white matter maturation and behavioural outcomes, although recovery can occur following abstinence.

The mGlu5 receptor antagonist MTEP attenuates opiate self-administration and cue-induced opiate-seeking behaviour in mice

The mGlu5 receptor (mGluR5) has been implicated in the rewarding effect of various drugs of abuse and drug-seeking behaviour. In the present study we investigated the impact of antagonism of mGluR5 with the selective negative allosteric, modulator 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) on operant self-administration of morphine as well as cue-induced drug-seeking in adult CD1 mice. Administration of MTEP (20 mg/kg, i.p.) attenuated operant responding for morphine (0.1 mg/kg/infusion) and cue-induced morphine-seeking after a period of forced abstinence. Collectively, these data implicate mGluR5 in the reinforcing effects of opiates and support the proposition that mGluR5 is a potential therapeutic target for treatment of drug addiction.

mGlu5 and adenosine A2A receptor interactions regulate the conditioned effects of cocaine

Adenosine A2A receptors and metabotropic glutamate type 5 (mGlu5) receptors are co-localized in the striatum and can functionally interact to regulate drug-seeking. We further explored this interaction using antagonism of mGlu5 receptors with 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP) in combination with genetic deletion of A2A receptors. The conditioned rewarding and locomotor-activating properties of cocaine were evaluated via conditioned place preference (CPP). Vehicle-treated mice of both genotypes expressed a CPP to cocaine while MTEP abolished cocaine CPP in wild-type, but not A2A knockout, mice. These results were mirrored when conditioned hyperactivity was assessed. In contrast, MTEP attenuated the acute locomotor-activating properties of cocaine similarly in both genotypes. These data provide evidence for a functional interaction between adenosine A2A and mGlu5 receptors in mediating the conditioned effects of cocaine but not direct cocaine-induced hyperactivity. This functional interaction is supported by modulation of 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolol[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol ([125I]ZM241385) binding to the A2A receptor by MTEP.

Activation of NF‐κB transcription factor in the preterm ovine brain and placenta after acute LPS exposure

Intrauterine infection may be causally related to inflammation and injury of the fetal brain, however the mechanisms by which this occurs are unclear. We have investigated whether nuclear factor (NF)‐κB, a transcription factor for proinflammatory cytokines, is activated in the fetal brain after acute LPS‐exposure. At 95 days of gestation (term = ∼147 days), 5 fetuses received a single intravenous bolus dose of LPS (1 μg/kg); 6 fetuses served as controls. Fetal blood samples were taken hourly for 6 hr post LPS‐exposure to assess physiological status. Ewes and fetuses were then euthanased, placental and brain tissue examined histologically, and NF‐κB activation assessed in several regions of the fetal brain using an electromobility shift assay (EMSA). Oxidative stress was measured using lipid peroxidation and 8‐isoprostane biochemical assays and brain cytokine concentrations analysed by enzyme linked immunosorbent assay (ELISA). LPS‐exposed fetuses (relative to controls) were hypoxemic and the haematocrit and lactate levels had increased. In the brains of LPS‐exposed fetuses compared to controls, NF‐κB binding activity was elevated in the hippocampus and the thalamus/basal ganglia; 8‐isoprostane levels were elevated overall (P < 0.05) in the parietal/occipital/temporal lobes and thalamus/basal ganglia. TNF‐α and IL‐6 concentrations were not elevated, however, there was a tendency for an elevation of IFN‐γ concentrations in the thalamus/basal ganglia. IFN‐γ concentration was elevated (P < 0.05) in the plasma 4 hr after LPS‐exposure. In the placenta, NF‐κB binding activity was increased (P < 0.05). We conclude that acute systemic administration of LPS leads to increased binding activity of NF‐κB subunits in specific regions of the fetal brain and in the placenta, but that there is no clear‐cut relationship between this elevation and vulnerability to endotoxic damage.