Mhoyra Fraser

Connexin hemichannel blockade improves outcomes in a model of fetal ischemia

Connexin hemichannels can open during ischemia, resulting in loss of membrane potential, calcium influx, and release of glutamate. In this study, we tested the hypothesis that opening of hemichannels after cerebral ischemia may contribute to delayed evolution of injury.

Growth hormone and prolactin regulate human neural stem cell regenerative activity.

We have previously shown that the growth hormone (GH)/prolactin (PRL) axis has a significant role in regulating neuroprotective and/or neurorestorative mechanisms in the brain and that these effects are mediated, at least partly, via actions on neural stem cells (NSCs). Here, using NSCs with properties of neurogenic radial glia derived from fetal human forebrains, we show that exogenously applied GH and PRL promote the proliferation of NSCs in the absence of epidermal growth factor or basic fibroblast growth factor. When applied to differentiating NSCs, they both induce neuronal progenitor proliferation, but only PRL has proliferative effects on glial progenitors. Both GH and PRL also promote NSC migration, particularly at higher concentrations. Since human GH activates both GH and PRL receptors, we hypothesized that at least some of these effects may be mediated via the latter. Migration studies using receptor-specific antagonists confirmed that GH signals via the PRL receptor promote migration. Mechanisms of receptor signaling in NSC proliferation, however, remain to be elucidated. In summary, GH and PRL have complex stimulatory and modulatory effects on NSC activity and as such may have a role in injury-related recovery processes in the brain.

Subclinical exposure to low-dose endotoxin impairs EEG maturation in preterm fetal sheep

Exposure to chorioamnionitis is strongly associated with neurodevelopmental disability after premature birth; however, it remains unclear whether subclinical infection affects functional EEG maturation. Chronically instrumented 103-104-day-old (0.7 gestational age: term 147 days) fetal sheep in utero were randomized to receive either gram-negative LPS by continuous low-dose infusion (100 ng iv over 24 h, followed by 250 ng/24 h for 4 days; n = 6) or the same volume of normal saline (n = 9). Arterial plasma cortisol, ACTH, and IL-6 were measured. The delta (0-3.9 Hz), theta (4-7.9 Hz), alpha (8-12.9 Hz), and beta (13-22 Hz) components of the EEG were determined by power spectral analysis. Brains were taken after 10 days for histopathology. There were no changes in blood gases, cardiovascular variables, or EEG power during LPS infusion, but a transient rise in plasma cortisol and IL-6 (P < 0.05). LPS infusion was associated with loss of the maturational increase to higher frequency activity, with reduced alpha and beta power, and greater delta power than saline controls from 6 to 10 days (P < 0.05). Histologically, LPS was associated with increased numbers of microglia and TNF-α-positive cells in the periventricular white matter and frontoparietal cortex, increased caspase-3-positive cells in white matter, but no loss of CNPase-positive oligodendrocytes, Nurr-1 subplate cells, or gyral complexity. These data suggest that low-dose endotoxin exposure can impair EEG maturation in preterm fetal sheep in association with neural inflammation but without hemodynamic disturbances or cortical injury.

Partial neural protection with prophylactic low-dose melatonin after asphyxia in preterm fetal sheep

Melatonin is a naturally occurring indolamine with mild antioxidant properties that is neuroprotective in perinatal animals. There is limited information on its effects on preterm brain injury. In this study, 23 chronically instrumented fetal sheep received 25 minutes of complete umbilical cord occlusion at 101 to 104 days gestation (term is 147 days). Melatonin was administered to the ewe 15 minutes before occlusion (0.1 mg/kg bolus followed by 0.1 mg/kg per hour for 6 hours, n=8), or the equivalent volume of vehicle (2% ethanol, n=7), or saline (n=8), or maternal saline plus sham occlusion (n=8). Sheep were killed after 7 days recovery in utero. Fetal blood pressure, heart rate, nuchal activity, and temperature were similar between groups. Vehicle infusion was associated with improved neuronal survival in the caudate nucleus, but greater neuronal loss in the regions of the hippocampus, with reduced proliferation and increased ameboid microglia in the white matter (P<0.05). Maternal melatonin infusion was associated with faster recovery of fetal EEG, prolonged reduction in carotid blood flow, similar neuronal survival to vehicle, improved numbers of mature oligodendrocytes, and reduced microglial activation in the white matter (P<0.05). Prophylactic maternal melatonin treatment is partially protective but its effects may be partly confounded by ethanol used to dissolve melatonin.

Global undernutrition during gestation influences learning during adult life

Intrauterine growth restriction can lead to significant long-term health consequences such as metabolic and cardiovascular disorders, but less is known about its effects on choice and behavioral adaptation in later life. Virgin Wistar rats were time mated and randomly assigned to receive either ad-libitum access to chow or 30% of that level of nutrition during pregnancy to generate growth-restricted offspring. At 60 days of age, 6 female offspring from each group were trained on concurrent variable-interval schedules. Sessions consisted of seven randomly arranged concurrent-schedule components, each with a different reinforcer ratio that varied from 27∶1 to 1∶27, and each component lasting for 10 reinforcer deliveries. Behavioral change across reinforcers in components, measured by sensitivity to reinforcement, was consistently lower for offspring of undernourished mothers, showing that their behavior was less adaptable to environmental change. These results provide direct experimental evidence for a link between prenatal environmental conditions and reduced behavioral adaptability—learning—in later life.

PROTEOLYTIC ACTIVITY DURING CORTICAL DEVELOPMENT IS DISTINCT FROM THAT INVOLVED IN HYPOXIC ISCHEMIC INJURY

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases involved in brain development and the etiology of adult cerebral injuries. In this study, we determined the MMP-2 and 9 responses following hypoxic ischemia (HI) injury in the developing brain. First, we characterized the developmental changes of MMP activity in the rat brain from embryonic day 18 (E18) to postnatal day 120 (P120). MMP-2 activity was high from E18 to P3 and decreased with age (P< or =0.001), while MMP-9 activity was not detectable. MMP-2 immunoreactivity was closely associated with differentiating cortical plate and subplate neurons. Next, we characterized the proteolytic changes after unilateral HI brain injury in 3- (P3) and 21- (P21) day-old rats. Zymography revealed that in the P21 rat brain, MMP-9 activity (150 and 92 kDa forms) was increased at 6 h and remained elevated 24 h post-injury in the ipsilateral injured hemisphere (P< or =0.001), whereas there was a gradual increase in MMP-2 (65 kDa) activity, reaching a peak at 5 days (P< or =0.001). Similarly, quantitative real time polymerase chain reaction (qRT-PCR) indicated significant elevations in MMP-9 and MMP-2 mRNA expression in the injured cortex (P< or =0.05) and hippocampus (P< or =0.05) at 1 and 5 days post-injury, respectively in the P21 rat brain. In the P3 rat brain, zymography results revealed that both pro (92 kDa) and cleaved (87 kDa) MMP-9 activities were upregulated in the ipsilateral injured hemisphere from 6 h to 1 day after injury (P< or =0.001). In contrast, cleaved MMP-2 (60 kDa) was only moderately upregulated at 6 h (P< or =0.01), while pro MMP-2 (65 kDa) levels were unaffected. MMP-9 mRNA expression was also increased at 6 h (P< or =0.05) following injury at P3, whereas MMP-2 expression remained unchanged compared with the uninjured contralateral hemisphere. Immunohistochemistry indicated that MMP-9 protein expression was localized predominantly to neurons and peri-vascular astrocytes in the affected regions at early time points, whereas MMP-2 was present on reactive astrocytes surrounding the infarct at later time points. Together, these results indicate that MMP-2 may be primarily associated with the development and differentiation of cortical plate neurons and wound recovery processes. Conversely, MMP-9 appeared to be associated with more acute processes during the period of lesion development.

Extracellular amino acids and lipid peroxidation products in periventricular white matter during and after cerebral ischemia in preterm fetal sheep

It is widely hypothesized that accumulation of excitatory amino acids, and oxygen free radicals during or after exposure to hypoxia–ischemia play a pivotal role in preterm periventricular white matter injury; however, there is limited evidence in the intact brain. In preterm fetal sheep (0.65 gestation; term 147 days) we found no significant increase in extracellular levels of excitatory amino acids measured by microdialysis in the periventricular white matter during cerebral ischemia induced by bilateral carotid occlusion. There was no significant change in 8‐isoprostane or malondialdehyde levels in the early phase of recovery after occlusion. In contrast, there was a significant delayed increase in most amino acids and in malondialdehyde (but not 8‐isoprostane) that was maximal approximately 2–3 days after occlusion. The increase in glutamate was significantly correlated with a secondary increase in cortical impedance, an index of cytotoxic edema, and with white matter damage 3 days post‐insult. In conclusion, no significant accumulation of cytotoxins was found within immature white matter during cerebral ischemia. Although a minority of fetuses showed a delayed increase in some cytotoxins, this occurred many days after ischemia, in association with secondary cytotoxic edema, strongly suggesting that these changes are mainly a consequence of evolving cell death.

Cortisol and ACTH responses to severe asphyxia in preterm fetal sheep

It has been hypothesized that the hypothalamic‐pituitary‐adrenal (HPA) axis is immature in the preterm fetus and that this compromises their ability to adapt to hypoxic stress; however, there are few direct data. We therefore examined the effects of asphyxia on HPA responses in chronically instrumented preterm fetal sheep (104 days of gestation; term is 147 days), allocated to a sham control group (n= 7) or 25 min of complete umbilical cord occlusion (n= 8), followed by recovery for 72 h. During umbilical cord occlusion there was a rapid rise in ACTH levels (230.4 ± 63.5 versus 14.1 ± 1.8 ng ml−1 in sham controls, 16‐fold) and cortisol levels (7.4 ± 4.9 versus 0.2 ± 0.1 ng ml−1, 31‐fold), with further increases after release of cord occlusion. ACTH levels were normalized by 24 h, while plasma cortisol levels returned to sham control values 72 h after asphyxia. Fetal arterial blood pressure was elevated in the first 36 h, with a marked increase in femoral vascular resistance, and correlated positively with cortisol levels after asphyxia (P= 0.05). In conclusion, the preterm fetus shows a brisk, substantial HPA response to severe hypoxia.

Acute on chronic exposure to endotoxin in preterm fetal sheep

Acute, high-dose exposure to endotoxin lipopolysaccharide (LPS) in preterm fetal sheep can trigger periventricular white matter lesions (PVL), in association with severe hypotension/hypoxemia and significant mortality. Intriguingly, however, chronic or repeated exposure to LPS can induce tachyphylaxis. We therefore tested the hypothesis that progressive, acute on chronic fetal infection would be associated with white matter injury with little fetal mortality. Chronically instrumented preterm (0.7 gestational age) fetal sheep were exposed to a continuous low-dose LPS infusion (100 ng over 24 h, followed by 250 ng/24 h for 96 h) or saline. Boluses of 1 μg LPS or saline were given at 48, 72, and 96 h; sheep were killed at day 10. Six of 11 fetal sheep exposed to saline infusion + LPS boluses died 4-7 h after the first bolus. In contrast, there was no fetal mortality after saline infusions alone (n = 9), low-dose LPS infusion + saline boluses (n = 5), or low-dose LPS + LPS boluses (n = 9). Low-dose LPS infusion + LPS boluses was associated with greater microglial induction than low-dose LPS + saline boluses but a similar area of periventricular white matter inflammation. One fetus developed severe focal white matter necrosis after LPS infusion + boluses. The acute cardiovascular compromise associated with high-dose, acute exposure to LPS is markedly attenuated by previous low-dose infusions, with limited apparent exacerbation of periventricular white matter injury compared with low-dose infusion alone.

Sustained sympathetic nervous system support of arterial blood pressure during repeated brief umbilical cord occlusions in near-term fetal sheep

Sympathetic nervous system (SNS)-mediated peripheral vasoconstriction plays a key role in initial maintenance of blood pressure during rapid-onset asphyxia in the mammalian fetus, but it is attenuated after the first few minutes. It is unclear whether the SNS response is sustained during the brief, but frequently repeated, episodes of asphyxia characteristic of labor. In the present study, 14 fetal sheep at 0.85 of gestation received either chemical sympathectomy with 6-hydroxydopamine (6-OHDA; n = 7) or sham injection (control; n = 7), followed 4-5 days later by repeated 2-min episodes of complete umbilical cord occlusion every 5 min for up to 4 h or until mean arterial blood pressure (MAP) fell to <20 mmHg for two successive occlusions. In controls, umbilical cord occlusions were associated with a rapid initial fall in fetal heart rate (FHR) and femoral blood flow (FBF), with initial hypertension, followed by progressive development of hypotension during ongoing occlusions. Sympathectomy was associated with attenuation of the initial rise in MAP during umbilical cord occlusion, and after the onset of hypotension, a markedly more rapid fall of MAP to the nadir, with a correspondingly slower fall in FBF (P < 0.05). In contrast, MAP and FHR between successive occlusions were higher after sympathectomy (P < 0.05). There was no significant difference in the number of occlusions before terminal hypotension (6-OHDA; 16.1 ± 2.2 vs. control; 18.7 ± 2.3). These data show that SNS activity provides ongoing support for fetal MAP during prolonged exposure to brief repeated asphyxia.