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Neonatal encephalopathy (NE) is a leading cause of childhood death and disability in term infants. Treatment options for perinatal brain injury are limited and developing therapies that target multiple pathways within the pathophysiology of NE are of great interest. Pifithrin-µ (PFT-µ) is a drug with striking neuroprotective abilities in a preclinical model of hypoxia-ischemia (HI)-induced NE wherein cell death is a substantial cause of injury. Work from neurons and tumor cells reports that PFT-µ is able to inhibit p53 binding to the mitochondria, heat shock protein (HSP)-70 substrate binding and activation of the NF-kB pathway. The purpose of this study is to understand whether the neuroprotective effects of PFT-µ also include direct effects on microglia. We utilized the microglial cell line, BV2, and we studied the dose-dependent effect of PFT-µ on M1-like and M2-like phenotype using qRT-PCR and Western blotting, including the requirement for the presence of p53 or HSP-70 in these effects. We also assessed phagocytosis and the effects of PFT-µ on genes within metabolic pathways related to phenotype. We noted that PFT-µ robustly reduced the M1-like (lipopolysaccharide, LPS-induced) BV2 response, spared the LPS-induced phagocytic ability of BV2 and had no effect on the genes related to metabolism and that effects on phenotype were partially dependent on the presence of HSP-70 but not p53. This study demonstrates that the neuroprotective effects of PFT-µ in HI-induced NE may include an anti-inflammatory effect on microglia and adds to the evidence that this drug might be of clinical interest for the treatment of NE.

Toll-like receptors (TLRs) are members of the pattern recognition receptor family that detect components of foreign pathogens or endogenous molecules released in response to injury. Recent studies demonstrate that TLRs also have a functional role in regulating neuronal proliferation in the developing brain. This study investigated cellular expression of TLR3 using immunohistochemistry on human brain tissue. The tissue sections analysed contained anterior and lateral periventricular white matter from the frontal and parietal lobes in post-mortem neonatal cases with a postmenstrual age range of 23.6-31.4 weeks. In addition to preterm brains without overt pathology (control), preterm pathology cases with evidence of white matter injuries (WMI) were also examined. In order to identify TLR-positive cells, we utilized standard double-labelling immunofluorescence co-labelling techniques and confocal microscopy to compare co-expression of TLR3 with a neuronal marker (NeuN) or with glial markers (GFAP for astrocytes, Iba-1 for microglia and Olig2 for oligodendrocytes). We observed an increase in the neuronal (28 vs. 17%) and astroglial (38 vs. 21%) populations in the WMI group compared to controls in the anterior regions of the periventricular white matter in the frontal lobe. The increase in neurons and astrocytes in the WMI cases was associated with an increase in TLR3 immunoreactivity. This expression was significantly increased in the astroglia. The morphology of the TLR3 signal in the control cases was globular and restricted to the perinuclear region of the neurons and astrocytes, whilst in the cases of WMI, both neuronal, axonal and astroglial TLR3 expression was more diffuse (i.e., a different intracellular distribution) and could be detected along the extensions of the processes. This study demonstrates for the first time that neurons and glial cells in human neonatal periventricular white matter express TLR3 during development. The patterns of TLR3 expression were altered in the presence of WMI, which might influence normal developmental processes within the immature brain. Identifying changes in TLR3 expression during fetal development may be key to understanding the reduced volumes of grey matter and impaired cortical development seen in preterm infants.

Sick preterm and term newborns are highly vulnerable to neural injury, and thus there has been a major search for new, safe and efficacious neuroprotective interventions in recent decades. Preclinical studies are essential to select candidate drugs for clinical trials in humans. This article focuses on ‘negative’ preclinical studies, i.e. studies where significant differences cannot be detected. Such findings are critical to inform both clinical and preclinical investigators, but historically they have been difficult to publish. A significant amount of time and resources is lost when negative results or nonpromising therapeutics are replicated in separate laboratories because these negative results were not shared with the research community in an open and accessible format. In this article, we discuss approaches to strengthen conclusions from negative preclinical studies and, conversely, to reduce false-negative preclinical evaluations of potential therapeutic compounds. Without being exhaustive, we address three major issues in conducting and interpreting preclinical experiments, including: (a) the choice of animal models, (b) the experimental design, and (c) issues concerning statistical analyses of the experiments. This general introduction is followed by synopses of negative data obtained from studies of three potential therapeutics for perinatal brain injury: (1) the somatostatin analog octreotide, (2) an AMPA/kainate receptor antagonist, topiramate, and (3) a pyruvate derivative, ethyl pyruvate.

Interleukin (IL)-1 and IL-18 belong to the IL-1 family. IL-18 deficiency has been shown to confer moderate protection after hypoxia-ischemia (HI) in the immature brain, while the contribution of the two isoforms of IL-1 (IL-1alpha and IL-1beta) in neonatal HI brain injury has not been investigated previously. The aim of this study was to examine the contribution of the different members of the IL-1 family to neonatal HI damage. Unilateral HI was induced at postnatal day 9 in IL-1beta, IL-1beta18, and IL-1alphabeta knockout and wild-type mice and brain injury was evaluated 1 week later. IL-1beta18-deficient mice showed 17% reduction in brain injury, while no significant reduction in injury was detected between any of the other groups. These results indicate that IL-18, but not IL-1beta, or the combination of IL-1alpha and IL-1beta, is a contributor to HI injury in the immature brain.

We have previously shown that lipopolysaccharide (LPS) sensitizes the immature rat brain to subsequent hypoxic-ischemic (HI) injury; however, the underlying mechanisms remain unclear. In this study, we examined the role of glucose in the sensitizing effects of LPS and lipoteichoic acid (LTA) in combination with HI in 7-day-old rats. LPS/HI resulted in hypoglycemia which lasted 24 h and lactate levels were increased from 6 to 10 h after LPS administration. LPS/HI induced severe brain injury, which persisted 2 weeks after LPS/HI. Administration of glucose to LPS-treated animals with HI reduced brain injury in the cerebral cortex and hippocampus, while striatal damage remained. LTA/HI did not affect blood glucose, lactate or brain injury. In conclusion, enhanced blood glucose levels during HI after LPS administration conferred protection in some brain regions but not in the striatum, suggesting that alterations in glucose can only partly explain the sensitizing effect of LPS.

The aim of this study was to evaluate the involvement of mitochondrial membrane permeability transition (MPT) after hypoxia-ischemia (HI) in 7-day-old rats. [14C]2-deoxyglucose (DOG) was administered to controls, and at various time points after HI. MPT in the cerebral cortex was measured as entrapment of DOG-6-P in mitochondria. Another group of rats was treated with the MPT inhibitor cyclosporin A (CsA; 10–50 mg/kg i.p.) or vehicle before and after HI, and the effect on brain injury and mitochondrial respiration was evaluated. A significant increase in DOG-6-P entrapment in mitochondria indicated that MPT occurred in two phases: a primary MPT after 0–1.5 h and a secondary MPT after 6.5–8 h of reperfusion. However, CsA did not affect brain injury or mitochondrial respiration. The data suggest that MPT occurred after HI but does not provide evidence for its involvement in the development of injury.

Caspase-3 is a major effector protease in several apoptotic pathways, but its role in hypoxic-ischemic (HI) brain injury is incompletely understood. Cerebral HI was induced in 7-day-old rats by unilateral carotid artery ligation and exposure to 7.7% oxygen for 55 min. Caspase-3-like activity was significantly increased at 1 h (208%), peaked at 24 h (2,563%) and was still increased 6 days after HI (169%) in the ipsilateral cerebral cortex. Concomitantly, cleavage of the caspase-3 proform (31/33 kD) was detected on immunoblots, producing 29- and 17-kD fragments. Furthermore, significant degradation of the endogenous caspase-3 substrates inhibitor of caspase-activated DNase (DNA fragmentation factor 45), poly(ADP-ribose) polymerase and fodrin occurred. In conclusion, caspase-3 is activated extensively in the immature brain after HI. The subsequent cleavage of proteins involved in cellular homeostasis and repair may contribute to the process of brain injury.

In a model of perinatal hypoxic-ischemic brain damage, we examined the neuroprotective efficacy of posttreatment with the NMDA receptor antagonist MK-801 and the AMPA receptor antagonist NBQX. Unilateral brain damage developed in 95% of rat pups subjected to hypoxia-ischemia with a 27.8 ± 1.2% weight deficit of the damaged hemisphere. MK-801 in doses of 0.3 and 0.5 mg/kg i.p. reduced the brain damage by 61% (p < 0.001) and 43% (p < 0.001), respectively. A higher dose of MK-801 (0.75 mg/kg) did not offer neuroprotection. Treatment with NBQX (40 mg/kg) reduced the hemispheric lesion by 28% (p < 0.05). In conclusion, posttreatment with both NBQX and low doses of MK-801 reduced perinatal brain damage. The NMDA receptor antagonist offered stronger neuroprotection which is in agreement with a proposed NMDA receptor hyperactivity around postnatal day 7 in rats.

Perinatal brain damage is a major clinical problem. Recent studies suggest that excitatory amino acids (EAAs) may be important for the development of hypoxic-ischemic brain injury in the newborn. Experimental work demonstrates that the immature brain is hypersensitive to the toxic effects EAA ('excitotoxicity'), hypoxic-ischemia is accompanied by an extracellular overflow of EAAs and hypoxic-ischemic brain damage is reduced by EAA receptor antagonists. Clinical investigations demonstrate the presence of EAA receptors in vulnerable areas of the newborn human brain and the concentrations of EAAs in the cerebrospinal fluid are higher in asphyxiated than in control infants. Clinical studies are warranted to evaluate the importance of excitotoxicity for development of brain lesions after severe asphyxia.

A prospective study was conducted to determine the risk of placenta previa and unexplained antepartum hemorrhage after a previous cesarean section (CS). Of a total of 24,644 patients, 81 (0.33%) had a placenta previa which demanded abdominal delivery. The risk of placenta previa was 0.25% with an unscarred uterus and 1.22% in patients with one or more previous CS (the difference was statistically significant p less than 0.001). The corresponding figures for unexplained antepartum hemorrhage were 0.40% and 3.81%, respectively (p less than 0.001). Patients presenting with a placenta previa and a scarred uterus had a 16% risk of undergoing cesarean hysterectomy because of placenta accreta and severe hemorrhage compared to 3.6% in patients with placenta previa and an unscarred uterus. In conclusion, cesarean deliveries predispose to placenta previa, placenta accreta and antepartum hemorrhage during subsequent pregnancies. This relationship has to be considered in the cost-benefit equation for decision of route of delivery.