Therapeutic Hypothermia Attenuates Cortical Interneuron Loss after Cerebral Ischemia in Near-Term Fetal Sheep.

Therapeutic hypothermia significantly improves outcomes after neonatal hypoxic-ischemic (HI) encephalopathy but is only partially protective. There is evidence that cortical inhibitory interneuron circuits are particularly vulnerable to HI and that loss of interneurons may be an important contributor to long-term neurological dysfunction in these infants. In the present study, we examined the hypothesis that the duration of hypothermia has differential effects on interneuron survival after HI. Near-term fetal sheep received sham ischemia or cerebral ischemia for 30 min, followed by cerebral hypothermia from 3 h after ischemia end and continued up to 48 h, 72 h, or 120 h recovery. Sheep were euthanized after 7 days for histology. Hypothermia up to 48 h recovery resulted in moderate neuroprotection of glutamate decarboxylase (GAD)+ and parvalbumin+ interneurons but did not improve survival of calbindin+ cells. Hypothermia up to 72 h recovery was associated with significantly increased survival of all three interneuron phenotypes compared with sham controls. By contrast, while hypothermia up to 120 h recovery did not further improve (or impair) GAD+ or parvalbumin+ neuronal survival compared with hypothermia up to 72 h, it was associated with decreased survival of calbindin+ interneurons. Finally, protection of parvalbumin+ and GAD+ interneurons, but not calbindin+ interneurons, with hypothermia was associated with improved recovery of electroencephalographic (EEG) power and frequency by day 7 after HI. The present study demonstrates differential effects of increasing the duration of hypothermia on interneuron survival after HI in near-term fetal sheep. These findings may contribute to the apparent preclinical and clinical lack of benefit of very prolonged hypothermia.

Persistent cortical and white matter inflammation after therapeutic hypothermia for ischemia in near-term fetal sheep.

BACKGROUND: Therapeutic hypothermia significantly improves outcomes after moderate-severe hypoxic-ischemic encephalopathy (HIE), but it is partially effective. Although hypothermia is consistently associated with reduced microgliosis, it is still unclear whether it normalizes microglial morphology and phenotype. METHODS: Near-term fetal sheep (n = 24) were randomized to sham control, ischemia-normothermia, or ischemia-hypothermia. Brain sections were immunohistochemically labeled to assess neurons, microglia and their interactions with neurons, astrocytes, myelination, and gitter cells (microglia with cytoplasmic lipid granules) 7 days after cerebral ischemia. Lesions were defined as areas with complete loss of cells. RNAscope® was used to assess microglial phenotype markers CD86 and CD206. RESULTS: Ischemia-normothermia was associated with severe loss of neurons and myelin (p < 0.05), with extensive lesions, astrogliosis and microgliosis with a high proportion of gitter cells (p < 0.05). Microglial wrapping of neurons was present in both the ischemia groups. Hypothermia improved neuronal survival, suppressed lesions, gitter cells and gliosis (p < 0.05), and attenuated the reduction of myelin area fraction. The "M1" marker CD86 and "M2" marker CD206 were upregulated after ischemia. Hypothermia partially suppressed CD86 in the cortex only (p < 0.05), but did not affect CD206. CONCLUSIONS: Hypothermia prevented lesions after cerebral ischemia, but only partially suppressed microglial wrapping and M1 marker expression. These data support the hypothesis that persistent upregulation of injurious microglial activity may contribute to partial neuroprotection after hypothermia, and that immunomodulation after rewarming may be an important therapeutic target.

Targeting Persistent Neuroinflammation after Hypoxic-Ischemic Encephalopathy-Is Exendin-4 the Answer?

Hypoxic-ischemic encephalopathy is brain injury resulting from the loss of oxygen and blood supply around the time of birth. It is associated with a high risk of death or disability. The only approved treatment is therapeutic hypothermia. Therapeutic hypothermia has consistently been shown to significantly reduce the risk of death and disability in infants with hypoxic-ischemic encephalopathy. However, approximately 29% of infants treated with therapeutic hypothermia still develop disability. Recent preclinical and clinical studies have shown that there is still persistent neuroinflammation even after treating with therapeutic hypothermia, which may contribute to the deficits seen in infants despite treatment. This suggests that potentially targeting this persistent neuroinflammation would have an additive benefit in addition to therapeutic hypothermia. A potential additive treatment is Exendin-4, which is a glucagon-like peptide 1 receptor agonist. Preclinical data from various in vitro and in vivo disease models have shown that Exendin-4 has anti-inflammatory, mitochondrial protective, anti-apoptotic, anti-oxidative and neurotrophic effects. Although preclinical studies of the effect of Exendin-4 in perinatal hypoxic-ischemic brain injury are limited, a seminal study in neonatal mice showed that Exendin-4 had promising neuroprotective effects. Further studies on Exendin-4 neuroprotection for perinatal hypoxic-ischemic brain injury, including in large animal translational models are warranted to better understand its safety, window of opportunity and effectiveness as an adjunct with therapeutic hypothermia.

Opioid-Sparing Anesthesia: Gabapentin and Postoperative Pain.

PURPOSE: Nonopioid analgesics are commonly used to augment or replace opioids in the perioperative setting. Perianesthesia nurses must consider timing and appropriateness when administering these medications to patients in the preoperative area or the postanesthesia care unit, particularly when other medications with sedative effects are being given. Gabapentin, originally proposed as an anticonvulsant medication, promotes analgesia and reduces risk for postoperative nausea and vomiting. This review examines the effect of gabapentin on postoperative pain. DESIGN: A systematic review. METHODS: CINAHL, PubMed, and Cochrane Review databases were searched to find a total of 93 sources that examined gabapentin and postoperative pain. After applying inclusion and exclusion criteria, four randomized controlled trials (RCT) were reviewed. Postoperative pain within the 24 hours of surgery was measured as the primary outcome using the visual analog scale in all sources FINDINGS: Three of the four reviewed RCTs determined gabapentin was both statistically and clinically significant in reducing postoperative pain, and all four sources showed a reduction in opioid consumption during the immediate postoperative period, which promoted patient satisfaction. CONCLUSIONS: Further study of gabapentin and postoperative pain is needed employing rigorous and robust methodology and diversity of the sample selections.

Transient effects of forebrain ischemia on fetal heart rate variability in fetal sheep.

Fetal heart rate variability (FHRV) is a key index of antenatal and intrapartum fetal well-being. FHRV is well established to be mediated by both arms of the autonomic nervous system, but it remains unknown whether higher centers in the forebrain contribute to FHRV. We tested the hypothesis that selective forebrain ischemia would impair the generation of FHRV. Sixteen chronically instrumented near-term fetal sheep were subjected to either forebrain ischemia induced by bilateral carotid occlusion or sham-ischemia for 30 min. Time, frequency, and nonlinear measures of FHRV were assessed during and for seven days after ischemia. Ischemia was associated with profound suppression of electroencephalographic (EEG) power, which remained suppressed throughout the recovery period (P < 0.001). During the first 5 min of ischemia, multiple time and frequency domain measures were increased (all P < 0.05) before returning back to sham levels. A delayed increase in sample entropy was observed during ischemia (P < 0.05). For the first 3 h after ischemia, there was moderate suppression of two measures of FHRV (very-low frequency power and the standard deviation of RR-intervals, both P < 0.05) and increased sample entropy (P < 0.05). Thereafter, all measures of FHRV returned to control levels. In conclusion, profound forebrain ischemia sufficient to lead to severe neural injury had only transient effect on multiple measures of FHRV. These findings suggest that the forebrain makes a limited contribution to FHRV. FHRV therefore primarily originates in the hindbrain and is unlikely to provide meaningful information on forebrain neurodevelopment or metabolism.

Treating Seizures after Hypoxic-Ischemic Encephalopathy-Current Controversies and Future Directions.

Seizures are common in newborn infants with hypoxic-ischemic encephalopathy and are highly associated with adverse neurodevelopmental outcomes. The impact of seizure activity on the developing brain and the most effective way to manage these seizures remain surprisingly poorly understood, particularly in the era of therapeutic hypothermia. Critically, the extent to which seizures exacerbate brain injury or merely reflect the underlying evolution of injury is unclear. Current anticonvulsants, such as phenobarbital and phenytoin have poor efficacy and preclinical studies suggest that most anticonvulsants are associated with adverse effects on the developing brain. Levetiracetam seems to have less potential neurotoxic effects than other anticonvulsants but may not be more effective. Given that therapeutic hypothermia itself has significant anticonvulsant effects, randomized controlled trials of anticonvulsants combined with therapeutic hypothermia, are required to properly determine the safety and efficacy of these drugs. Small clinical studies suggest that prophylactic phenobarbital administration may improve neurodevelopmental outcomes compared to delayed administration; however, larger high-quality studies are required to confirm this. In conclusion, there is a distinct lack of high-quality evidence for whether and to what extent neonatal seizures exacerbate brain damage after hypoxia-ischemia and how best to manage them in the era of therapeutic hypothermia.

Protection of axonal integrity with 48 or 72 h of cerebral hypothermia in near-term fetal sheep.

BACKGROUND: Therapeutic hypothermia is partially protective for neonatal hypoxic-ischemic encephalopathy (HIE). Damage to the white matter tracts is highly associated with adverse outcomes after HIE, but the effectiveness and optimal duration of hypothermia to attenuate axonal injury are unclear. METHODS: Near-term fetal sheep were randomized to sham control or cerebral ischemia for 30 min with normothermia or cerebral hypothermia from 3 to either 48 or 72 h. Sheep were killed after 7 days. SMI-312-labeled axons and myelin basic protein were quantified in the intragyral white matter of the first and second parasagittal gyri. RESULTS: Ischemia was associated with reduced axonal and myelin area fraction (p < 0.05); loss of axonal and myelin linearity (p < 0.05); and thin, sparse axons, with spheroids, compared to dense, linear morphology in sham controls and associated with induction of microglia in an amoeboid morphology. Both ischemia-48 h hypothermia and ischemia-72 h hypothermia improved axonal area fraction and linearity (p < 0.05), although abnormal morphological features were seen in a subset. Microglial induction was partially suppressed by ischemia-48 h hypothermia, with a ramified morphology. CONCLUSIONS: These data suggest that therapeutic hypothermia can alleviate post-ischemic axonopathy, in part by suppressing secondary inflammation.

Combination treatments with therapeutic hypothermia for hypoxic-ischemic neuroprotection.

Therapeutic hypothermia is now proven to reduce death or disability in term and near-term born infants with moderate to severe hypoxic-ischemic encephalopathy. Nevertheless, many infants still survive with disability, despite treatment with hypothermia. Recent preclinical and clinical studies suggest that current protocols for therapeutic hypothermia are near-optimal. The obvious strategy, in addition to improving early initiation of therapeutic hypothermia after birth, is to combine hypothermia with other neuroprotective agents. We review evidence that the mechanisms of action of many promising agents overlap with the anti-excitotoxic, anti-apoptotic, and anti-inflammatory mechanisms of hypothermia, leading to a lack of benefit from combination treatment. Moreover, even apparently beneficial combinations have failed to translate in clinical trials. These considerations highlight the need for preclinical studies to test clinically realistic protocols of timing and duration of treatment, before committing to large randomized controlled trials.

How do we reach the goal of personalized medicine for neuroprotection in neonatal hypoxic-ischemic encephalopathy?

Therapeutic hypothermia is now standard of care for neonates with hypoxic-ischemic encephalopathy (HIE) in high income countries (HIC). Conversely, compelling trial evidence suggests that hypothermia is ineffective, and may be deleterious, in low- and middle-income countries (LMIC), likely reflecting the lower proportion of infants who had sentinel events at birth, suggesting that injury had advanced to a stage when hypothermia is no longer effective. Although hypothermia significantly reduced the risk of death and disability in HICs, many infants survived with disability and in principle may benefit from targeted add-on neuroprotective or neurorestorative therapies. The present review will assess biomarkers that could be used to personalize treatment for babies with HIE - to determine first whether an individual infant is likely to respond to hypothermia, and second, whether additional treatments may be beneficial.

Blockade of connexin hemichannels with tonabersat protects against mild hypoxic ischemic brain injury in neonatal rats.

BACKGROUND AND PURPOSE: There is growing evidence that infants with mild hypoxic-ischemic (HI) encephalopathy have increased risk of brain injury and adverse neurodevelopmental outcomes. Currently, there is no approved treatment for these infants. It was previously shown that blocking connexin 43 hemichannels is neuroprotective in models of moderate to severe HI injury. However, it is yet to be established whether these channels play a role in the evolution of mild HI brain injury, and whether blocking these channels after mild HI is neuroprotective. METHODS: HI was induced in postnatal day 10 rats of both sexes by right carotid artery ligation followed by 80 min of hypoxia in 8% oxygen. Pups receiving HI were randomised to receive intraperitoneal injections of either saline, vehicle (2-hydroxypropyl-beta-cyclodextrin polyethylene glycol-400), or tonabersat (2 mg/kg), at 60 min, 24 h, and 48 h after hypoxia. Seven days after HI, brains were harvested for measurement of volume loss and histological analysis. RESULTS: HI resulted in a significant reduction in hemispheric, hippocampal, and white matter volumes, which were significantly attenuated after treatment with tonabersat. HI was also associated with a significant reduction in numbers of neurons in the CA1 and CA3 hippocampal regions, a reduction in the numbers of oligodendrocytes in the corpus callosum, and an increase in the number of astrocytes in both regions, which were significantly attenuated by tonabersat treatment. There were no differences in rectal temperatures between tonabersat- and vehicle-treated rat pups. CONCLUSIONS: Blockade of connexin hemichannels with tonabersat significantly reduced mild HI injury in the hippocampus and white matter, without causing hypothermia.

Deep Learning for Generalized EEG Seizure Detection after Hypoxia-Ischemia-Preclinical Validation.

Brain maturity and many clinical treatments such as therapeutic hypothermia (TH) can significantly influence the morphology of neonatal EEG seizures after hypoxia-ischemia (HI), and so there is a need for generalized automatic seizure identification. This study validates efficacy of advanced deep-learning pattern classifiers based on a convolutional neural network (CNN) for seizure detection after HI in fetal sheep and determines the effects of maturation and brain cooling on their accuracy. The cohorts included HI-normothermia term (n = 7), HI-hypothermia term (n = 14), sham-normothermia term (n = 5), and HI-normothermia preterm (n = 14) groups, with a total of >17,300 h of recordings. Algorithms were trained and tested using leave-one-out cross-validation and k-fold cross-validation approaches. The accuracy of the term-trained seizure detectors was consistently excellent for HI-normothermia preterm data (accuracy = 99.5%, area under curve (AUC) = 99.2%). Conversely, when the HI-normothermia preterm data were used in training, the performance on HI-normothermia term and HI-hypothermia term data fell (accuracy = 98.6%, AUC = 96.5% and accuracy = 96.9%, AUC = 89.6%, respectively). Findings suggest that HI-normothermia preterm seizures do not contain all the spectral features seen at term. Nevertheless, an average 5-fold cross-validated accuracy of 99.7% (AUC = 99.4%) was achieved from all seizure detectors. This significant advancement highlights the reliability of the proposed deep-learning algorithms in identifying clinically translatable post-HI stereotypic seizures in 256Hz recordings, regardless of maturity and with minimal impact from hypothermia.

Connexins, Pannexins and Gap Junctions in Perinatal Brain Injury.

Perinatal brain injury secondary to hypoxia-ischemia and/or infection/inflammation remains a major cause of disability. Therapeutic hypothermia significantly improves outcomes, but in randomized controlled trials nearly half of infants still died or survived with disability, showing that additional interventions are needed. There is growing evidence that brain injury spreads over time from injured to previously uninjured regions of the brain. At least in part, this spread is related to opening of connexin hemichannels and pannexin channels, both of which are large conductance membrane channels found in many brain cells. Opening of these membrane channels releases adenosine triphosphate (ATP), and other neuroactive molecules, into the extracellular space. ATP has an important role in normal signaling, but pathologically can trigger the assembly of the multi-protein inflammasome complex. The inflammasome complex promotes activation of inflammatory caspases, and release of inflammatory cytokines. Overall, the connexin hemichannel appears to play a primary role in propagation of injury and chronic disease, and connexin hemichannel blockade has been shown to be neuroprotective in multiple animal models. Thus, there is potential for some blockers of connexin or pannexin channels to be developed into targeted interventions that could be used in conjunction with or separate to therapeutic hypothermia.

Recombinant erythropoietin does not augment hypothermic white matter protection after global cerebral ischaemia in near-term fetal sheep.

Therapeutic hypothermia for hypoxic-ischaemic encephalopathy provides partial white matter protection. Recombinant erythropoietin reduces demyelination after hypoxia-ischaemia, but it is unclear whether adjunct erythropoietin treatment can further improve outcomes after therapeutic hypothermia. Term-equivalent fetal sheep received sham-ischaemia (n = 9) or cerebral ischaemia for 30 min (ischaemia-vehicle, n = 8), followed by intravenous infusion of recombinant erythropoietin (ischaemia-Epo, n = 8; 5000 IU/kg bolus dose, then 833.3 IU/kg/h), cerebral hypothermia (ischaemia-hypothermia, n = 8), or recombinant erythropoietin plus hypothermia (ischaemia-Epo-hypothermia, n = 8), from 3 to 72 h post-ischaemia. Foetal brains were harvested at 7 days after cerebral ischaemia. Ischaemia was associated with marked loss of total Olig2-positive oligodendrocytes with reduced density of myelin and linearity of the white matter tracts (P < 0.01), and microglial induction and increased caspase-3-positive apoptosis. Cerebral hypothermia improved the total number of oligodendrocytes and restored myelin basic protein (P < 0.01), whereas recombinant erythropoietin partially improved myelin basic protein density and tract linearity. Both interventions suppressed microgliosis and caspase-3 (P < 0.05). Co-treatment improved 2',3'-cyclic-nucleotide 3'-phosphodiesterase-myelin density compared to hypothermia, but had no other additive effect. These findings suggest that although hypothermia and recombinant erythropoietin independently protect white matter after severe hypoxia-ischaemia, they have partially overlapping anti-inflammatory and anti-apoptotic effects, with little additive benefit of combination therapy.

The Role of Connexin and Pannexin Channels in Perinatal Brain Injury and Inflammation.

Perinatal brain injury remains a major cause of death and life-long disability. Perinatal brain injury is typically associated with hypoxia-ischemia and/or infection/inflammation. Both hypoxia-ischemia and infection trigger an inflammatory response in the brain. The inflammatory response can contribute to brain cell loss and chronic neuroinflammation leading to neurological impairments. It is now well-established that brain injury evolves over time, and shows a striking spread from injured to previously uninjured regions of the brain. There is increasing evidence that this spread is related to opening of connexin hemichannels and pannexin channels, both of which are large conductance membrane channels found in almost all cell types in the brain. Blocking connexin hemichannels within the first 3 h after hypoxia-ischemia has been shown to improve outcomes in term equivalent fetal sheep but it is important to also understand the downstream pathways linking membrane channel opening with the development of injury in order to identify new therapeutic targets. Open membrane channels release adenosine triphosphate (ATP), and other neuroactive molecules, into the extracellular space. ATP has an important physiological role, but has also been reported to act as a damage-associated molecular pattern (DAMP) signal mediated through specific purinergic receptors and so act as a primary signal 1 in the innate immune system inflammasome pathway. More crucially, extracellular ATP is a key inflammasome signal 2 activator, with purinergic receptor binding triggering the assembly of the multi-protein inflammasome complex. The inflammasome pathway and complex formation contribute to activation of inflammatory caspases, and the release of inflammatory cytokines, including interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-18, and vascular endothelial growth factor (VEGF). We propose that the NOD-like receptor protein-3 (NLRP3) inflammasome, which has been linked to inflammatory responses in models of ischemic stroke and various inflammatory diseases, may be one mechanism by which connexin hemichannel opening especially mediates perinatal brain injury.