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Gap junctions between astrocytes support a functional syncytium that is thought to play an important role in neural homeostasis. In order to investigate regulation of this syncytium and of connexin43 (Cx43), a principal astrocytic gap junction protein, we determined the sequelae of gap junction and Cx43 disposition in a rat cerebral focal ischaemia model with various ischaemia/reperfusion times using sequence-specific anti-Cx43 antibodies (designated 13-8300, 18A, 16A and 71-0700) that exhibit differential recognition of Cx43, perhaps reflecting functional aspects of gap junctions. Antibody 13-8300 specifically detects only an unphosphorylated form of Cx43 in both Western blots and tissue sections. In hypothalamus after brief (15 min) ischaemic injury, Cx43 at intact gap junctions undergoes dephosphorylation, accompanied by reduced epitope recognition by antibodies 16A and 71-0700. Tissue examined 24 h after reperfusion showed that these effects were reversible. Astrocytic gap junction internalization occurring 1 h after ischaemia was accompanied by decreased immunodetection with 13-8300. At this time, gap junctions were absent in the ischaemic core, coinciding with a loss of Cx43 recognition with 18A and 13-8300, but elevated labelling of internalized Cx43 with 16A and 71-0700. Unphosphorylated Cx43 persisted at intact gap junctions confined to a thin corridor at the ischaemic penumbra which contained presumptive apoptotic cell profiles. Similar results were obtained in ischaemic striatum and cerebral cortex, though with a delayed time course that depended on the severity of the ischaemic insult. These results demonstrate that astrocytic Cx43 epitope masking, dephosphorylation and cellular redistribution occur after ischaemic brain injury, proceed as a temporally and spatially ordered sequence of events and culminate in differential patterns of Cx43 modification and sequestration at the lesion centre and periphery. These observations suggest an attempt by astrocytes in the vicinity of injury to remodel the junctional syncytium according to altered tissue homeostatic requirements.

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We previously reported that kainic acid (KA) lesion sites in rat brain exhibit an absence of astrocytic gap junctions at 1 week post-lesion. Loss of immunocytochemical reactivity with a sequence-specific antibody against the astrocytic gap junctional protein connexin43 (Cx43) suggested epitope masking since persistence of Cx43 was observed on Western blots. Here, we determined the fate of Cx43 at various times after thalamic KA and striatal NMDA lesions. In normal tissue and at 6 hr post-KA lesion, Cx43 immunoreactivity predominated at typical astrocytic gap junctions. Immunolabelled junctions were still seen at 3 days, with epitope masking already present, and were virtually absent by 6 days post-lesion. Gap junction remodeling was indicated by the appearance of intracellular immunostained annular profiles and uncharacteristically extensive gap junctions between symmetrically immunolabelled membranes and between labelled astrocytic and unlabelled oligodendrocytic membranes. Labelled multivesicular clusters emerged at 2 days, were numerous at 3 days and constituted the sole Cx43 sequestration site by post-lesion day 6. Ultrastructural disruption and gap junction disassembly progressed more slowly in NMDA-injected tissue where immunoreactivity persisted, albeit at markedly decreasing levels until the final survival time examined (16 days). Intense Cx43 immunolabelling was seen in filopodia of putative reactive astrocytes at the lesion periphery at 6-8 days and was associated at 16 days with an increased number of gap junctions primarily between fine astrocytic processes. These results demonstrate that massive neuronal loss alone or in conjunction with direct actions of excitotoxins on astrocytes precipitates an astrocytic reaction accompanied initially by removal of their gap junctions followed by redistribution of Cx43, and suggest that the astrocytic syncytium may undergo reorganization in a manner leading to isolation of the lesion site.