Role of gap junctional coupling in astrocytic networks in the determination of global ischaemia-induced oxidative stress and hippocampal damage.

While there is evidence that gap junctions play important roles in the determination of cell injuries, there is not much known about mechanisms by which gap junctional communication may exert these functions. Using a global model of transient ischaemia in rats, we found that pretreatment with the gap junctional blockers carbenoxolone, 18alpha-glycyrrhetinic acid and endothelin, applied via cannulae implanted into the hippocampus in one hemisphere, resulted in decreased numbers of TUNEL-positive neurons, as compared with the contralateral hippocampus that received saline injection. Post-treatment with carbenoxolone for up to 30 min after the stroke injury still resulted in decreased cell death, but post-treatment at 90 min after the ischaemic insult did not result in differences in cell death. However, quinine, an inhibitor of Cx36-mediated gap junctional coupling, did not result in appreciable neuroprotection. Searching for a possible mechanism for the observed protective effects, possible actions of the gap junctional blockers in the electrical activity of the hippocampus during the ischaemic insult were assessed using intracerebral recordings, with no differences observed between the saline-injected and the contralateral drug-injected hippocampus. However, a significant reduction in lipid peroxides, a measure of free radical formation, in the hippocampus treated with carbenoxolone, revealed that the actions of gap junctional coupling during injuries may be causally related to oxidative stress. These observations suggest that coupling in glial networks may be functionally important in determining neuronal vulnerability to oxidative injuries.

Functional contribution of specific brain areas to absence seizures: role of thalamic gap-junctional coupling.

The synchronized discharges typical of seizures have a multifactorial origin at molecular, cellular and network levels. During recent years, the functional role of gap-junctional coupling has received increased attention as a mechanism that may participate in seizure generation. We have investigated the possible functional roles of thalamic and hippocampal gap-junctional communication (GJC) in the generation of spike-and-wave discharges in a rodent model of atypical absence seizures. Seizures in this model spread throughout limbic, thalamic and neocortical areas. Rats were chronically implanted with cannulae to deliver drugs or saline, and local field potentials recordings were performed using intracerebral electrodes positioned in distinct brain areas. Initially, the effects on synaptic transmission of the gap-junctional blockers used in this study were determined. Neither carbenoxolone (CBX) nor 18-alpha-glycyrrhetinic acid altered chemical synaptic transmission at the concentrations tested. These two compounds, when injected via cannulae into the reticular nucleus of the thalamus (NRT), decreased significantly the duration of seizures as compared with saline injections or injections of the CBX inactive derivative glycyrrhizic acid. CBX injections into the hippocampus resulted in diminished seizure activity as well. NRT injections of trimethylamine, which presumably causes intracellular alkalinization (thereby promoting gap-junctional opening), enhanced seizures and spindle activity. These observations suggest that, in this rodent model, thalamic and limbic areas are involved in the synchronous paroxysmal activity and that GJC contributes to the spike-and-wave discharges.

Anti-porin antibodies prevent excitotoxic and ischemic damage to brain tissue.

The mitochondrial permeability transition (MPT) is a converging event for different molecular routes leading to cellular death after excitotoxic/oxidative stress, and is considered to represent the opening of a pore in the mitochondrial membrane. There is evidence that the outer mitochondrial membrane protein porin is involved in the MPT and apoptosis. We present here a proof-of-principle study to address the hypothesis that anti-porin antibodies can prevent excitotoxic/ischemia-induced cell death. We generated anti-porin antibodies and show that the F(ab)(2) fragments penetrate living cells, reduce Ca(2+)-induced mitochondrial swelling as other MPT blockers do, and decrease neuronal death in dissociated and organotypic brain slice cultures exposed to excitotoxic and ischemic episodes. These observations present direct evidence that anti-porin antibody fragments prevent cell damage in brain tissue, that porin is a crucial protein involved in mitochondrial and cell dysfunction, and that it is conceivable that antibodies can be used as therapeutic agents.

Ischemia-induced brain damage depends on specific gap-junctional coupling.

Ischemic brain injury results in neuronal loss and associated neurologic deficits. Although there is some evidence that intercellular communication via gap junctions can spread oxidative cell injury, the possible role of gap-junctional communication in ischemia-induced cell death is the object of debate. Because gap junctions directly connect the cytoplasms of coupled cells, they offer a way to propagate stress signals from cell to cell. The authors investigated the contribution of gap-junctional communication to cell death using an in vitro ischemia model, which was reproduced by submersion of organotypic hippocampal slices into glucose-free deoxygenated medium. The gap-junctional blocker carbenoxolone significantly decreased the spread of cell death, as measured by propidium iodide staining, over a 48-hour period after the ischemic episode. Carbenoxolone ameliorated the hypoxia-induced impairment of the intrinsic neuronal electrophysiologic characteristics, as measured by whole-cell patch clamp recordings. To determine whether specific connexins were involved in the spread of postischemic cell death, the authors partially reduced the synthesis of specific connexins using antisense oligodeoxynucleotides. Simultaneous knockdown of two connexins localized mostly in neurons, connexins 32 and 26, resulted in significant neuroprotection 48 hours after the hypoxic-hypoglycemic episode. Similarly, partial reduction of the predominant glial connexin 43 significantly decreased cell death. These results indicate that gap-junctional communication contributes to the propagation of hypoxic injury and that specific gap junctions could be a novel target to reduce brain damage.

Specific gap junctions enhance the neuronal vulnerability to brain traumatic injury.

Traumatic brain injury results in neuronal loss and associated neurological deficits. Although most research on the factors leading to trauma-induced damage focuses on synaptic or ionic mechanisms, the possible role of direct intercellular communication via gap junctions has remained unexplored. Gap junctions connect directly the cytoplasms of coupled cells; hence, they offer a way to propagate stress signals from cell to cell. We investigated the contribution of gap junctional communication (GJC) to cell death using an in vitro trauma model. The impact injury, induced by a weight dropped on the distal CA1 area of organotypic hippocampal slices, results in glutamate-dependent cell loss. The gap junctional blockers carbenoxolone and octanol decreased significantly post-traumatic cell death, measured by propidium iodide staining over a 72 hr period after the impact. Dye coupling in the pyramidal layers was enhanced immediately after the injury and decreased over the following 24 hr. To determine whether specific connexins were involved in the spread of trauma-induced cell death, we used organotypic slices from connexin43 (Cx43) knock-out mice, as well as acute knock-outs by incubation with antisense oligodeoxynucleotides. Simultaneous knockdown of two neuronal connexins resulted in significant neuroprotection. Slices from the null-mutant Cx43 mice, as well as the acute Cx43 knockdown, also showed decreased cell death after the impact. The gap junctional blockers alleviated the trauma-induced impairment of synaptic function as measured by electrophysiological field potential recordings. These results indicate that GJC enhances the cellular vulnerability to traumatic injury. Hence, specific gap junctions could be a novel target to reduce injury and secondary damage to the brain and maximize recovery from trauma.