Joanne O. Davidson

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.

Astrocytes and microglia in acute cerebral injury underlying cerebral palsy associated with preterm birth

Cerebral palsy is one of the most devastating consequences of brain injury around the time of birth, and nearly a third of cases are now associated with premature birth. Compared with term babies, preterm babies have an increased incidence of complications that may increase the risk of disability, such as intraventricular hemorrhage, periventricular leukomalacia, sepsis, and necrotizing enterocolitis. The response to injury is highly dependent on brain maturity, and although cellular vulnerability is well documented, there is now evidence that premyelinating axons are also particularly sensitive to ischemic injury. In this review, we will explore recent evidence highlighting a central role for glia in mediating increased risk of disability in premature infants, including excessive activation of microglia and opening of astrocytic gap junction hemichannels in spreading injury after brain ischemia, in part likely involving release of adenosine triphosphate (ATP) and overactivation of purinergic receptors, particularly in white matter. We propose the hypothesis that inflammation-induced opening of connexin hemichannels is a key regulating event that initiates a vicious circle of excessive ATP release, which in turn propagates activation of purinergic receptors on microglia and astrocytes. This suggests that developing effective neuroprotective strategies for preterm infants requires a detailed understanding of glial responses.

Connexin Hemichannel Blockade Is Neuroprotective after Asphyxia in Preterm Fetal Sheep

Asphyxia around the time of preterm birth is associated with neurodevelopmental disability. In this study, we tested the hypothesis that blockade of connexin hemichannels would improve recovery of brain activity and reduce cell loss after asphyxia in preterm fetal sheep. Asphyxia was induced by 25 min of complete umbilical cord occlusion in preterm fetal sheep (103–104 d gestational age). Connexin hemichannels were blocked by intracerebroventricular infusion of mimetic peptide starting 90 min after asphyxia at a concentration of 50 µM/h for one hour followed by 50 µM/24 hour for 24 hours (occlusion-peptide group, n = 6) or vehicle infusion for controls (occlusion-vehicle group, n = 7). Peptide infusion was associated with earlier recovery of electroencephalographic power after asphyxia compared to occlusion-vehicle (p<0.05), with reduced neuronal loss in the caudate and putamen (p<0.05), but not in the hippocampus. In the intragyral and periventricular white matter, peptide administration was associated with an increase in total oligodendrocyte numbers (p<0.05) and immature/mature oligodendrocytes compared to occlusion-vehicle (p<0.05), with a significant increase in proliferation (p<0.05). Connexin hemichannel blockade was neuroprotective and reduced oligodendrocyte death and improved recovery of oligodendrocyte maturation in preterm fetuses after asphyxia.

Therapeutic hypothermia for neonatal hypoxic-ischemic encephalopathy - where to from here?

Hypoxia–ischemia before or around the time of birth occurs in approximately 2/1000 live births and is associated with a high risk of death or lifelong disability. Therapeutic hypothermia is now well established as standard treatment for infants with moderate to severe hypoxic–ischemic encephalopathy but is only partially effective. There is compelling preclinical and clinical evidence that hypothermia is most protective when it is started as early as possible after hypoxia–ischemia. Further improvements in outcome from therapeutic hypothermia are very likely to arise from strategies to reduce the delay before starting treatment of affected infants. In this review, we examine evidence that current protocols are reasonably close to the optimal depth and duration of cooling, but that the optimal rate of rewarming after hypothermia is unclear. The potential for combination treatments to augment hypothermic neuroprotection has considerable promise, particularly with endogenous targets such as melatonin and erythropoietin, and noble gases such as xenon. We dissect the critical importance of preclinical studies using realistic delays in treatment and clinically relevant cooling protocols when examining combination treatment, and that for many strategies overlapping mechanisms of action can substantially attenuate any effects.

Connexin hemichannel blockade is neuroprotective after, but not during, global cerebral ischemia in near-term fetal sheep.

There is increasing evidence that connexin hemichannels, the half gap junctions that sit unopposed in the cell membrane, can open during ischemia and that blockade of connexin43 hemichannels after cerebral ischemia can improve neural outcomes. However, it is unclear whether connexin blockade during ischemia is protective. In the present study global cerebral ischemia was induced by 30 min of bilateral carotid artery occlusion in near-term (128 ± 1 day gestation age) fetal sheep. A specific mimetic peptide that blocks connexin43 hemichannels was infused into the lateral ventricle for either 1h before and during ischemia (intra-ischemia group, n=6) or for 25 h starting 90 min after the end of ischemia (post-ischemia group, n=7). The vehicle was infused in the ischemia-vehicle group (n=6) and sham-controls received sham occlusion plus vehicle (n=10). The post-ischemia group showed enhanced recovery of EEG power from day five until the end of the experiment (-5 ± 1.6 dB) compared to ischemia-vehicle (-13 ± 1.9 dB, p<0.05) and intra-ischemia infusion (-14.4 ± 3.6 dB, p<0.05). Post-ischemic infusion was associated with higher neuronal counts compared to ischemia-vehicle and intra-ischemia in the cortex (p<0.05) but not the CA1 and CA3 regions of the hippocampus. Oligodendrocyte cell counts in the intragyral and periventricular white matter were significantly higher in the post-ischemia group compared to ischemia-vehicle and intra-ischemia infusion (p<0.05). These large animal data support the hypothesis that connexin hemichannel opening after, but not during, ischemia contributes to the spread of white and gray matter injury of the developing brain.

Maternal dexamethasone and EEG hyperactivity in preterm fetal sheep.

Non‐technical summary Long‐acting glucocorticoids such as dexamethasone are commonly given to women at risk of preterm labour. While they significantly improve survival of the prematurely born infant, their effects on preterm brain activity is surprisingly unclear. We found that, in sheep, a conventional clinical course of maternal dexamethasone treatment was associated with dramatic, evolving low‐frequency hyperactivity of the fetal EEG. This activity reflected a striking shift to less‐frequent but higher amplitude EEG waveforms, and unexpectedly, EEG waveforms whose duration and pattern were highly consistent with seizure activity. After resolution of hyperactivity, the EEG showed changes consistent with maturation of sleep architecture, and reassuringly there was no histological evidence of brain injury 5 days after first exposure. The long‐term implications are uncertain, but these effects may contribute to improved neonatal outcomes.

The Use of Connexin-Based Therapeutic Approaches to Target Inflammatory Diseases

Alterations in Connexin43 (Cx43) expression levels have been shown to play a role in inflammatory processes including skin wounding and neuroinflammation. Cx43 protein levels increase following a skin wound and can inhibit wound healing. Increased Cx43 has been observed following stroke, epilepsy, ischemia, optic nerve damage, and spinal cord injury with gap junctional communication and hemichannel opening leading to increased secondary damage via the inflammatory response. Connexin43 modulation has been identified as a potential target for protection and repair in neuroinflammation and skin wound repair. This review describes the use of a Cx43 specific antisense oligonucleotide (Cx43 AsODN) and peptide mimetics of the connexin extracellular loop domain to modulate Cx43 expression and/or function in inflammatory disorders of the skin and central nervous system. An overview of the role of connexin43 in inflammatory conditions, how antisense and peptide have allowed us to elucidate the role of Cx43 in these diseases, create models of diseases to test interventions and their potential for use clinically or in current clinical trials is presented. Antisense oligonucleotides are applied topically and have been used to improve wound healing following skin injury. They have also been used to develop ex vivo models of neuroinflammatory diseases that will allow testing of intervention strategies. The connexin mimetic peptides have shown potential in a number of neuroinflammatory disorders in ex vivo models as well as in vivo when delivered directly to the injury site or when delivered systemically.

Role of Hemichannels in CNS Inflammation and the Inflammasome Pathway

Neurodegenerative, cardiovascular, and metabolic disorders, once triggered, share a number of common features, including sustained inflammatory cell activation and vascular disruption. These shared pathways are induced independently of any genetic predisposition to the disease or the precise external stimulus. Glial cells respond to injury with an innate immune response that includes release of proinflammatory cytokines and chemokines. Vascular endothelial cells may also be affected, leading to opening of the blood-brain barrier that facilitates invasion by circulating inflammatory cells. Inflammation can trigger acute neural injury followed by chronic inflammation that plays a key role in neurodegenerative conditions. Gap junction channels normally allow direct cell-to-cell communication. They are formed by the docking of two hemichannels, one contributed by each of the neighboring cells. While the opening probability of these channels is tightly controlled under resting conditions, hemichannels can open in response to injury or inflammatory factors, forming a large, relatively nonselective membrane pore. In this review, we consider the CNS immune system from the perspective that modulating connexin hemichannel opening can prevent tissue damage arising from excessive and uncontrolled inflammation. We discuss connexin channel roles in microglia, astrocytes, and endothelial cells in both acute and chronic inflammatory conditions, and in particular describe the role of connexin hemichannels in the inflammasome pathway where they contribute to both its activation and its spread to neighboring cells. Finally, we describe the benefits of hemichannel block in animal models of brain injury.

Battle of the hemichannels--Connexins and Pannexins in ischemic brain injury.

Perinatal ischemic brain injury can occur as a result of a global ischemic insult or focal ischemic stroke in the preterm or full‐term neonate. One of the most striking features of HI injury is that, after initial recovery of cellular oxidative metabolism, there is a delayed, ‘secondary’ mitochondrial failure that spreads over time from the most severely damaged areas outwards, into previously undamaged regions. This secondary failure is accompanied by transient seizure activity and cytotoxic edema.

How long is too long for cerebral cooling after ischemia in fetal sheep

Therapeutic hypothermia can partially reduce long-term death and disability in neonates after hypoxic-ischemic encephalopathy. The aim of this study was to determine whether prolonging the duration of cooling from 3 days to 5 days could further improve outcomes of cerebral ischemia in near-term fetal sheep. Fetal sheep (0.85 gestation) received 30 minutes bilateral carotid artery occlusion followed by 3 days of normothermia (n = 8), 3 days of hypothermia (n = 8), or 5 days of hypothermia (n = 8) started 3 hours after ischemia. Sham controls received sham ischemia followed by normothermia (n = 8). Cerebral ischemia was associated with profound loss of electroencephalography power and spectral edge, with greater and more rapid recovery in both hypothermia groups (P < 0.05). Ischemia was associated with severe loss of neurons in the cortex, hippocampus and thalamus (P < 0.05), with a significant improvement in both hypothermia groups. However, the ischemia-3-day hypothermia group showed greater neuronal survival in the cortex and dentate gyrus compared with ischemia-5-day hypothermia (P < 0.05). Ischemia was associated with induction of iba1-positive microglia, which was attenuated in both hypothermia groups (P < 0.05). Extending the duration of delayed therapeutic hypothermia from 3 to 5 days did not improve outcomes after severe ischemia, and was associated with reduced neuronal survival in some regions.