Laura Bennet

Which Neuroprotective Agents are Ready for Bench to Bedside Translation in the Newborn Infant

Neonatal encephalopathy caused by perinatal hypoxiaischemia in term newborn infants occurs in 1 to 3 per 1000 births1 and leads to high mortality and morbidity rates with life-long chronic disabilities.2,3 Although therapeutic hypothermia is a significant advance in the developed world and improves outcome,4,5 hypothermia offers just 11% reduction in risk of death or disability, from 58% to 47%. Therefore, there still is an urgent need for other treatment options. Further, there are currently no clinically established interventions that can be given antenatally to ameliorate brain injury after fetal distress. One of the major limitations to progress is what may be called “the curse of choice.” A large number of possible neuroprotective therapies have shown promise in pre-clinical studies.6,7 How should we select from them? There is no consensus at present on which drugs have a high chance of success for either antenatal or postnatal treatment. There are insufficient societal resources available to test them all. Thus, it is imperative to marshal finite resources and prioritize potential therapies for investigation. The authors believe that facilitating discussion of strategy and findings in “competing” laboratories is critical to facilitate efficient progress toward optimizing neuroprotection after hypoxia-ischemia. Few studies have examined possible interactions of medications with hypothermia and whether combination therapies augment neuroprotection. The timing of the administration of medications may be critical to optimize benefit and avoid neurotoxicity (eg, early acute treatments targeted at amelioration of the neurotoxic cascade compared with subacute treatment that can promote regeneration and repair). Intervention early on in the cascade of neural injury is likely to achieve more optimal neuroprotection8,9; however, there is frequently little warning of impending perinatal hypoxia-ischemia episodes. Sensitizing factors such as maternal pyrexia,10 maternal/fetal infection,11,12 and poor fetal growth13 are well recognized and contribute to the heterogeneity of the fetal response and outcome in neonatal encephalopathy. We include potential antenatal therapy medications in the scoring process; however, electronic fetal monitoring has a low positive predictive value (3%–18%) for identifying intrapartum asphyxia.14–18 At present, therefore, any antenatal intervention potentially involves treatment of many cases that do not need treatment in order to benefit a few at risk of brain injury. In January 2008, investigators from research institutions with a special interest in neuroprotection of the newborn appraised published evidence about medications that have been used in pre-clinical animal models, pilot clinical studies, or both as treatments for: (1) antenatal therapy for fetuses with a diagnosis of antenatal fetal distress at term; and (2) postnatal therapy of infants with moderate to severe neonatal encephalopathy. The aims of this study were to: (1) prioritize potential treatments for antenatal and postnatal therapy; and (2) provide a balanced reference for further discussions in the perinatal neuroscience community for future research and clinical translation of novel neuroprotective treatments of the newborn.

Insulin-Like Growth Factor-1 Reduces Postischemic White Matter Injury in Fetal Sheep

Insulin-like growth factor-1 (IGF-1) is known to be important for oligodendrocyte survival and myelination. In the current study, the authors examined the hypothesis that exogenous IGF-1 could reduce postischemic white matter injury. Bilateral brain injury was induced in near-term fetal sheep by 30 minutes of reversible carotid artery occlusion. Ninety minutes after ischemia, either vehicle (n = 8) or a single dose of 3 μg IGF-1 (n = 9) was infused intracerebroventricularly over 1 hour. White matter changes were assessed after 4 days recovery in the parasagittal intragyral white matter and underlying corona radiata. Proteolipid protein (PLP) mRNA staining was used to identify bioactive oligodendrocytes. Glial fibrillary acidic protein (GFAP) and isolectin B-4 immunoreactivity were used to label astrocytes and microglia, respectively. Myelin basic protein (MBP) density and the area of the intragyral white matter tracts were determined by image analysis. Insulin-like growth factor-1 treatment was associated with significantly reduced loss of oligodendrocytes in the intragyral white matter (P < 0.05), with improved MBP density (P < 0.05), reduced tissue swelling, and increased numbers of GFAP and isolectin B-4 positive cells compared with vehicle treatment. After ischemia there was a close association of PLP mRNA labeled cells with reactive astrocytes and macrophages/microglia. In conclusion, IGF-1 can prevent delayed, postischemic oligodendrocyte cell loss and associated demyelination.

The cardiovascular and cerebrovascular responses of the immature fetal sheep to acute umbilical cord occlusion.

1 In premature fetal sheep (89‐93 days gestation) we examined the fetal response to asphyxia induced by 30 min of complete umbilical cord occlusion. Fetuses were also studied during the first 3 days after asphyxia. We measured heart rate, blood pressure, carotid and femoral blood flows, vascular resistance, electroencephalographic activity and cerebral changes in haemoglobin concentration by near infrared spectroscopy (NIRS). 2 Fetuses tolerated 30 min of asphyxia and the cardiovascular response was characterized by three phases: initial redistribution of blood flow away from the periphery to maintain vital organ function, partial failure of this redistribution and near terminal cardiovascular collapse, with profound hypotension and cerebral and peripheral hypoperfusion. 3 Post‐asphyxia carotid blood flow and NIRS data demonstrated that between 3‐5 h there was a significant secondary reduction in cerebral blood flow, blood volume and oxygenation despite normal perfusion pressure and heart rate. There was also a secondary fall in femoral blood flow which persisted throughout recovery. 4 These data demonstrate that the immature fetus can survive a prolonged period of asphyxia, but paradoxically the capacity to survive exposes the fetus to profound hypotension and hypoperfusion. A secondary period of significant cerebral hypoperfusion and reduced oxygen delivery also occurred post‐asphyxia. These cardiovascular and cerebrovascular responses may contribute to the patterns of cerebral injury seen in the human preterm fetus.

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.

Cell therapy for neonatal hypoxia–ischemia and cerebral palsy

Perinatal hypoxic–ischemic brain injury remains a major cause of cerebral palsy. Although therapeutic hypothermia is now established to improve recovery from hypoxia–ischemia (HI) at term, many infants continue to survive with disability, and hypothermia has not yet been tested in preterm infants. There is increasing evidence from in vitro and in vivo preclinical studies that stem/progenitor cells may have multiple beneficial effects on outcome after hypoxic–ischemic injury. Stem/progenitor cells have shown great promise in animal studies in decreasing neurological impairment; however, the mechanisms of action of stem cells, and the optimal type, dose, and method of administration remain surprisingly unclear, and some studies have found no benefit. Although cell‐based interventions after completion of the majority of secondary cell death appear to have potential to improve functional outcome for neonates after HI, further rigorous testing in translational animal models is required before randomized controlled trials should be considered. ANN NEUROL 2012;

Insulin-Like Growth Factor (IGF)-1 Suppresses Oligodendrocyte Caspase-3 Activation and Increases Glial Proliferation after Ischemia in Near-Term Fetal Sheep

Insulin-like growth factor (IGF-1) markedly increases myelination and glial numbers in white matter after ischemia in near-term fetal sheep; however, it is unclear whether this is due to reduced cell loss or increased secondary proliferation. Brain injury was induced in near-term fetal sheep by 30 minutes of bilateral carotid artery occlusion. Ninety minutes after the occlusion, fetuses were given, intracerebroventricularly, either a single dose of IGF-1 (either 3 or 30 μg), or 3 μg followed by 3 μg over 24 hours (3 + 3 μg). White matter was assessed 4 days after reperfusion. Three micrograms, but not 30 μg of IGF-1 prevented loss of oligodendrocytes and myelin basic protein density (P < 0.001) compared to the vehicle-treated ischemia controls. No additional effect was observed in the 3 + 3 μg group. IGF-1 treatment was associated with reduced caspase-3 activation and increased glial proliferation in a similar dose-dependent manner. Caspase-3 was only expressed in oligodendrocytes that showed apoptotic morphology. Proliferating cell nuclear antigen co-localized with both oligodendrocytes and astrocytes and microglia. Thus, increased oligodendrocyte numbers after IGF-1 treatment is partly due to suppression of apoptosis, and partly to increased proliferation. In contrast, the increase in reactive glia was related only to proliferation. Speculatively, reactive glia may partly mediate IGF-1 white matter protection.

Window of Opportunity of Cerebral Hypothermia for Postischemic White Matter Injury in the Near-Term Fetal Sheep

Postresuscitation cerebral hypothermia is consistently neuroprotective in experimental preparations; however, its effects on white matter injury are poorly understood. Using a model of reversible cerebral ischemia in unanesthetized near-term fetal sheep, we examined the effects of cerebral hypothermia (fetal extradural temperature reduced from 39.4±0.1°C to between 30 and 33°C), induced at different times after reperfusion and continued for 72 hours after ischemia, on injury in the parasagittal white matter 5 days after ischemia. Cooling started within 90 minutes of reperfusion was associated with a significant increase in bioactive oligodendrocytes in the intragyral white matter compared with sham cooling (41±20 vs 18±11 per field, P < 0.05), increased myelin basic protein density and reduced expression of activated caspase-3 (14±12 vs 91±51, P < 0.05). Reactive microglia were profoundly suppressed compared with sham cooling (4±6 vs 38±18 per field, P < 0.05) with no effect on numbers of astrocytes. When cooling was delayed until 5.5 hours after reperfusion there was no significant effect on loss of oligodendrocytes (24±12 per field). In conclusion, hypothermia can effectively protect white matter after ischemia, but only if initiated early after the insult. Protection was closely associated with reduced expression of both activated caspase-3 and of reactive microglia.

The effect of cerebral hypothermia on white and grey matter injury induced by severe hypoxia in preterm fetal sheep

Prolonged, moderate cerebral hypothermia is consistently neuroprotective after experimental hypoxia–ischaemia; however, it has not been tested in the preterm brain. Preterm (0.7 gestation) fetal sheep received complete umbilical cord occlusion for 25 min followed by cerebral hypothermia (fetal extradural temperature reduced from 39.4 ± 0.3 to 29.5 ± 2.6°C) from 90 min to 70 h after the end of occlusion or sham cooling. Occlusion led to severe acidosis and profound hypotension, which recovered rapidly after release of occlusion. After 3 days recovery the EEG spectral frequency, but not total intensity, was increased in the hypothermia‐occlusion group compared with normothermia‐occlusion. Hypothermia was associated with a significant overall reduction in loss of immature oligodendrocytes in the periventricular white matter (P < 0.001), and neuronal loss in the hippocampus and basal ganglia (P < 0.001), with suppression of activated caspase‐3 and microglia (isolectin‐B4 positive). Proliferation was significantly reduced in periventricular white matter after occlusion (P < 0.05), but not improved after hypothermia. In conclusion, delayed, prolonged head cooling after a profound hypoxic insult in the preterm fetus was associated with a significant reduction in loss of neurons and immature oligodendroglia, with evidence of EEG and haemodynamic improvement after 3 days recovery, but also with a persisting reduction in proliferation of cells in the periventricular region. Further studies are required to evaluate the long‐term impact of cooling on brain growth and maturation.

Asphyxial brain injury--the role of the IGF system.

Transient neural injuries, such as asphyxia, can trigger considerable delayed neuronal death. Inappropriate induction of apoptosis is thought to play an important role in this process. Our studies have shown marked changes in the IGF system in the brain in response to these injuries with an induction of insulin growth factor (IGF)-1 and insulin growth factor binding protein (IGFBP)-2 and IGFBP-3 in glial cells in the region of injury. This suggests that the IGF-1 system may be an endogenous neuroprotective system. Earlier administration of IGF-1 - 2 h after injury reduced the phase of secondary neuronal loss suggesting that IGF-1 may well have therapeutic potential as a neuronal rescue agent. The action of IGF-1 appears to involve binding proteins, transport to the site of injury and the IGF-1 receptor and inhibition of apoptosis, but might also involve generation of GPE which itself appears to be neuroprotective. Together these results indicate considerable potential of these agents to treat stroke, perinatal asphyxia and other forms of acute brain injury.

Mechanisms of hypothermic neuroprotection

There is now compelling clinical evidence that prolonged, moderate cerebral hypothermia initiated within a few hours after severe hypoxia-ischemia and continued until resolution of the acute phase of delayed cell death can reduce subsequent neuronal loss and improve behavioral recovery in term infants and adults after cardiac arrest. Perhaps surprisingly, the specific mechanisms of hypothermic neuroprotection remain unclear, at least in part because hypothermia suppresses a broad range of potential injurious factors. In the present review we critically examine proposed mechanisms in relation to the known window of opportunity for effective protection with hypothermia. Better knowledge of the mechanisms of hypothermia is critical to help guide the rational development of future combination treatments to augment neuroprotection with hypothermia, and to identify those most likely to benefit from it.