Adverse neural effects of delayed, intermittent treatment with rEPO after asphyxia in preterm fetal sheep.

KEY POINTS: We have previously shown that high-dose constant infusion of recombinant human erythropoietin (rEPO) from 30 min to 72 h after asphyxia in preterm fetal sheep reduced histological injury and improved electrophysiological recovery. This study shows that a high-dose infusion of rEPO from 6 to 72 h after asphyxia did not improve EEG recovery, oligodendrocyte and neuronal survival at 1 week post-asphyxia. Of concern, intermittent rEPO boluses started 6 h after asphyxia were associated with impaired EEG recovery and bilateral cystic injury of temporal lobe intragyral white matter. Intermittent boluses of rEPO were associated with significantly increased cerebral vascular resistance and hypoperfusion, particularly after the first dose, but did not affect seizures, suggesting mismatch between perfusion and brain activity. ABSTRACT: Recombinant human erythropoietin (rEPO) is a promising treatment for hypoxic-ischaemic brain injury. Disappointingly, a large randomized controlled trial in preterm infants found that prophylactic, repeated high-dose rEPO boluses started within 24 h of birth did not improve neurodevelopmental outcomes. We examined whether initiation of a continuous infusion of rEPO at the end of the latent phase after hypoxic-ischaemia (HI) might improve outcomes compared with intermittent bolus injections. Chronically instrumented preterm (0.7 gestation) fetal sheep received sham asphyxia or asphyxia induced by complete umbilical cord occlusion for 25 min. Six hours after asphyxia, fetuses received either a continuous infusion of rEPO (loading dose 2000 IU, infusion at 520 IU/h) from 6 to 72 h post-asphyxia or intravenous saline or 5000 IU rEPO, with repeated doses every 48 h for 5 days. Continuous infusion of rEPO did not improve EEG recovery, oligodendrocyte and neuronal survival at 1 week post-asphyxia. By contrast, intermittent rEPO boluses were associated with impaired EEG recovery and bilateral cystic injury of temporal lobe intragyral white matter in 6/8 fetuses. These studies demonstrate for the first time that initiation of intermittent rEPO boluses 6 h after HI, at a dose comparable with recent clinical trials, exacerbated neural injury. These data reinforce the importance of early initiation of many potential neuroprotective therapies.

Window of opportunity for human amnion epithelial stem cells to attenuate astrogliosis after umbilical cord occlusion in preterm fetal sheep.

There is increasing evidence that administration of many types of stem cells, including human amnion epithelial cells (hAECs), can reduce hypoxic-ischemic injury, including in the perinatal brain. However, the therapeutic window for single dose treatment is not known. We compared the effects of early and delayed intracerebroventricular administration of hAECs in fetal sheep at 0.7 gestation on brain injury induced by 25 minutes of complete umbilical cord occlusion (UCO) or sham occlusion. Fetuses received either 1 × 106 hAECs or vehicle alone, as an infusion over 1 hour, either 2 or 24 hours after UCO. Fetuses were killed for brain histology at 7 days post-UCO. hAEC infusion at both 2 and 24 hours had dramatic anti-inflammatory and anti-gliotic effects, including significantly attenuating the increase in microglia after UCO in the white and gray matter and the number of astrocytes in the white matter. Both protocols partially improved myelination, but had no effect on total or immature/mature numbers of oligodendrocytes. Neuronal survival in the hippocampus was increased by hAEC infusion at either 2 or 24 hours, whereas only hAECs at 24 hours were associated with improved neuronal survival in the striatum and thalamus. Neither protocol improved recovery of electroencephalographic (EEG) power. These data suggest that a single infusion of hAECs is anti-inflammatory, anti-gliotic, and neuroprotective in preterm fetal sheep when given up to 24 hours after hypoxia-ischemia, but was associated with limited white matter protection after 7 days recovery and no improvement in the recovery of EEG power.

Connexin Hemichannel Mimetic Peptide Attenuates Cortical Interneuron Loss and Perineuronal Net Disruption Following Cerebral Ischemia in Near-Term Fetal Sheep.

Perinatal hypoxia-ischemia is associated with disruption of cortical gamma-aminobutyric acid (GABA)ergic interneurons and their surrounding perineuronal nets, which may contribute to persisting neurological deficits. Blockade of connexin43 hemichannels using a mimetic peptide can alleviate seizures and injury after hypoxia-ischemia. In this study, we tested the hypothesis that connexin43 hemichannel blockade improves the integrity of cortical interneurons and perineuronal nets. Term-equivalent fetal sheep received 30 min of bilateral carotid artery occlusion, recovery for 90 min, followed by a 25-h intracerebroventricular infusion of vehicle or a mimetic peptide that blocks connexin hemichannels or by a sham ischemia + vehicle infusion. Brain tissues were stained for interneuronal markers or perineuronal nets. Cerebral ischemia was associated with loss of cortical interneurons and perineuronal nets. The mimetic peptide infusion reduced loss of glutamic acid decarboxylase-, calretinin-, and parvalbumin-expressing interneurons and perineuronal nets. The interneuron and perineuronal net densities were negatively correlated with total seizure burden after ischemia. These data suggest that the opening of connexin43 hemichannels after perinatal hypoxia-ischemia causes loss of cortical interneurons and perineuronal nets and that this exacerbates seizures. Connexin43 hemichannel blockade may be an effective strategy to attenuate seizures and may improve long-term neurological outcomes after perinatal hypoxia-ischemia.

Tumor necrosis factor inhibition attenuates white matter gliosis after systemic inflammation in preterm fetal sheep.

BACKGROUND: Increased circulating levels of tumor necrosis factor (TNF) are associated with greater risk of impaired neurodevelopment after preterm birth. In this study, we tested the hypothesis that systemic TNF inhibition, using the soluble TNF receptor Etanercept, would attenuate neuroinflammation in preterm fetal sheep exposed to lipopolysaccharide (LPS). METHODS: Chronically instrumented preterm fetal sheep at 0.7 of gestation were randomly assigned to receive saline (control; n = 7), LPS infusion (100 ng/kg i.v. over 24 h then 250 ng/kg/24 h for 96 h plus 1 µg LPS boluses at 48, 72, and 96 h, to induce inflammation; n = 8) or LPS plus two i.v. infusions of Etanercept (2 doses, 5 mg/kg infused over 30 min, 48 h apart) started immediately before LPS-exposure (n = 8). Sheep were killed 10 days after starting infusions, for histology. RESULTS: LPS boluses were associated with increased circulating TNF, interleukin (IL)-6 and IL-10, electroencephalogram (EEG) suppression, hypotension, tachycardia, and increased carotid artery perfusion (P < 0.05 vs. control). In the periventricular and intragyral white matter, LPS exposure increased gliosis, TNF-positive cells, total oligodendrocytes, and cell proliferation (P < 0.05 vs control), but did not affect myelin expression or numbers of neurons in the cortex and subcortical regions. Etanercept delayed the rise in circulating IL-6, prolonged the increase in IL-10 (P < 0.05 vs. LPS), and attenuated EEG suppression, hypotension, and tachycardia after LPS boluses. Histologically, Etanercept normalized LPS-induced gliosis, and increase in TNF-positive cells, proliferation, and total oligodendrocytes. CONCLUSION: TNF inhibition markedly attenuated white matter gliosis but did not affect mature oligodendrocytes after prolonged systemic inflammation in preterm fetal sheep. Further studies of long-term brain maturation are now needed.

Protective effects of delayed intraventricular TLR7 agonist administration on cerebral white and gray matter following asphyxia in the preterm fetal sheep.

Preterm brain injury is highly associated with inflammation, which is likely related in part to sterile responses to hypoxia-ischemia. We have recently shown that neuroprotection with inflammatory pre-conditioning in the immature brain is associated with induction of toll-like receptor 7 (TLR7). We therefore tested the hypothesis that central administration of a synthetic TLR7 agonist, gardiquimod (GDQ), after severe hypoxia-ischemia in preterm-equivalent fetal sheep would improve white and gray matter recovery. Fetal sheep at 0.7 of gestation received sham asphyxia or asphyxia induced by umbilical cord occlusion for 25 minutes, followed by a continuous intracerebroventricular infusion of GDQ or vehicle from 1 to 4 hours (total dose 1.8 mg/kg). Sheep were killed 72 hours after asphyxia for histology. GDQ significantly improved survival of immature and mature oligodendrocytes (2',3'-cyclic-nucleotide 3'-phosphodiesterase, CNPase) and total oligodendrocytes (oligodendrocyte transcription factor 2, Olig-2) within the periventricular and intragyral white matter. There were reduced numbers of cells showing cleaved caspase-3 positive apoptosis and astrogliosis (glial fibrillary acidic protein, GFAP) in both white matter regions. Neuronal survival was increased in the dentate gyrus, caudate and medial thalamic nucleus. Central infusion of GDQ was associated with a robust increase in fetal plasma concentrations of the anti-inflammatory cytokines, interferon-ß (IFN-ß) and interleukin-10 (IL-10), with no significant change in the concentration of the pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α). In conclusion, delayed administration of the TLR7 agonist, GDQ, after severe hypoxia-ischemia in the developing brain markedly ameliorated white and gray matter damage, in association with upregulation of anti-inflammatory cytokines. These data strongly support the hypothesis that modulation of secondary inflammation may be a viable therapeutic target for injury of the preterm brain.

Delayed intranasal infusion of human amnion epithelial cells improves white matter maturation after asphyxia in preterm fetal sheep.

Perinatal hypoxic-ischemic (HI) brain injury remains highly associated with neurodevelopmental disability after preterm birth. There is increasing evidence that disability is linked with impaired white matter maturation, but there is no specific treatment. In this study, we evaluated whether, in preterm fetal sheep, delayed intranasal infusion of human amnion epithelial cells (hAECs) given 1, 3 and 10 days after severe HI, induced by umbilical cord occlusion for 25 min, can restore white matter maturation or reduce delayed cell loss. After 21 days recovery, asphyxia was associated with reduced electroencephalographic (EEG) maturation, brain weight and cortical area, impaired maturation of oligodendrocytes (OLs), no significant loss of total OLs but a marked reduction in immature/mature OLs and reduced myelination. Intranasal infusion of hAECs was associated with improved brain weight and restoration of immature/mature OLs and fractional area of myelin basic protein, with reduced microglia and astrogliosis. Cortical EEG frequency distribution was partially improved, with reduced loss of cortical area, and attenuated cleaved-caspase-3 expression and microgliosis. Neuronal survival in deep grey matter nuclei was improved, with reduced microglia, astrogliosis and cleaved-caspase-3-positive apoptosis. These findings suggest that delayed intranasal hAEC administration has potential to alleviate chronic dysmaturation after perinatal HI.

Chronic inflammation and impaired development of the preterm brain.

The preterm newborn is at significant risk of neural injury and impaired neurodevelopment. Infants with mild or no evidence of injury may also be at risk of altered brain development, with evidence impaired cell maturation. The underlying causes are multifactorial and include exposure of both the fetus and newborn to hypoxia-ischemia, inflammation (chorioamnionitis) and infection, adverse maternal lifestyle choices (smoking, drug and alcohol use, diet) and obesity, as well as the significant demand that adaptation to post-natal life places on immature organs. Further, many fetuses and infants may have combinations of these events, and repeated (multi-hit) events that may induce tolerance to injury or sensitize to greater injury. Currently there are no treatments to prevent preterm injury or impaired neurodevelopment. However, inflammation is a common pathway for many of these insults, and clinical and experimental evidence demonstrates that acute and chronic inflammation is associated with impaired brain development. This review examines our current knowledge about the relationship between inflammation and preterm brain development, and the potential for stem cell therapy to provide neuroprotection and neurorepair through reducing inflammation and release of trophic factors, which promote cell maturation and repair.

LDL receptor deficiency results in decreased cell proliferation and presynaptic bouton density in the murine hippocampus.

An aberrant cholesterol metabolism in the brain may contribute to the pathogenesis of Alzheimer's disease (AD). The LDL receptor (LDLR) regulates plasma cholesterol levels and recently we and others obtained evidence that it is also involved in regulating brain cholesterol homeostasis. Moreover, we found that LDLR-deficient mice display impaired spatial memory. Because cholesterol, in part derived from cellular uptake via LDLR, is required for peripheral cell proliferation and growth, we examined the effect of absence of the LDLR on hippocampal proliferation and the density of synaptic connections. Mice deficient for the LDLR displayed a reduced number of proliferating (BrdU-labeled) cells in the hippocampus as compared to wild type control mice. In addition, the number of synaptophysin-immunoreactive presynaptic boutons in the hippocampal CA1 and the dentate gyrus (DG) areas, but not in cortical areas, was lower in the LDLR-knockout mice than in the control mice. In vitro experiments showed that LDLR activity is increased when cell growth is enhanced by the addition of N2 supplement. This further supports a role for the LDLR in the outgrowth of neurites. These findings support the notion that, similar to its role in the periphery, the LDLR is important for the cellular uptake of cholesterol in the brain and that disturbance of this process affects neuronal plasticity.