Deleterious effects of high dose connexin 43 mimetic peptide infusion after cerebral ischaemia in near-term fetal sheep

Abstract

Hypoxic-ischaemic brain injury at birth is associated with 1-3/1000 cases of moderate to severe encephalopathy. Previously, we have shown that connexin 43 hemichannel blockade, with a specific mimetic peptide, reduced the occurrence of seizures, improved recovery of EEG power and sleep state cycling, and improved cell survival following global cerebral ischaemia. In the present study, we examined the dose response for intracerebroventricular mimetic peptide infusion (50 μmol/kg/h for 1 h, followed by 50 μmol/kg/24 h (low dose) or 50 μmol/kg/h for 25 h (high dose) or vehicle only (control group), starting 90 min after the end of ischaemia), following global cerebral ischaemia, induced by 30 min bilateral carotid artery occlusion, in near-term fetal sheep (128 ± 1 days gestation). Both peptide infusion groups were associated with a transient significant increase in EEG power between 2-12 h after ischaemia. The ischaemia-low dose group showed a significant recovery of EEG power from day five compared to the ischaemia-vehicle and -high dose groups. In contrast, the high dose infusion was associated with greater secondary increase in impedance (brain cell swelling), as well as a trend towards a greater increase in lactate concentration and mortality. These data suggest that higher doses of connexin mimetic peptide are not beneficial and may be associated with adverse outcomes, most likely attributable to uncoupling of connexin 43 gap junctions leading to dysfunction of the astrocytic syncytium.

Keywords: connexins; fetus; gap junctions; hemichannels; ischaemia; mimetic peptide.

Figure 1

EEG power shown as hourly averages from 12 h before until seven days after ischaemia (A), and five minute averages from two hours before until 12 h after ischaemia (B), in the ischaemia-vehicle, -low dose and -high dose groups, where peptide infusion was started 90 min after the end of ischaemia (the start of infusions is shown by the red arrow). Following the onset of occlusion, EEG power is rapidly suppressed in all three groups. A secondary rise in EEG power was seen in all groups, although this was significantly greater between 2–12 h in the ischaemia-low and -high dose groups. This was followed by a reduction in EEG power to below baseline levels, which persisted at day seven. Data are mean ± SEM. * p < 0.05.

Figure 2

Examples of one minute averaged EEG data from individual fetal sheep showing (A) the typical pattern of alternating periods of low and high amplitude EEG activity characteristic of sleep state cycling during the baseline period. (B–D) One minute averaged EEG data on day seven in the ischaemia-vehicle (B), ischaemia-low dose (C) and ischaemia-high dose (D) groups, with clear sleep state cycling only being seen in the ischaemia-low dose group.

Figure 3

(A) Spectral edge was suppressed at the onset of occlusion and remained below baseline in all groups. (B) Impedance increased during occlusion in all groups. After release of occlusion, impedance returned towards baseline, followed by a secondary increase that was significantly greater in the ischaemia-high dose group. Data are mean ± SEM, p < 0.05.

Figure 4

There were no significant differences in nuchal EMG, mean arterial pressure, fetal heart rate, carotid artery blood flow or brain temperature between the ischaemia-vehicle, -low dose and-high dose groups. Data are mean ± SEM.