Connexin hemichannel blockade improves survival of striatal GABA-ergic neurons after global cerebral ischaemia in term-equivalent fetal sheep

Abstract

Basal ganglia injury at term remains a major cause of disability, such as cerebral palsy. In this study we tested the hypotheses that blockade of astrocytic connexin hemichannels with a mimetic peptide would improve survival of striatal phenotypic neurons after global cerebral ischaemia in term-equivalent fetal sheep, and that neuronal survival would be associated with electrophysiological recovery. Fetal sheep (0.85 gestation) were randomly assigned to receive a short or long (1 or 25 h) intracerebroventricular infusion of a mimetic peptide or vehicle, starting 90 minutes after 30 minutes of cerebral ischaemia. Sheep were killed 7 days after ischaemia. Cerebral ischaemia was associated with reduced numbers of calbindin-28k, calretinin, parvalbumin and GAD positive striatal neurons (P < 0.05 ischaemia + vehicle, n = 6 vs. sham ischaemia, n = 6) but not ChAT or nNOS positive neurons. Short infusion of peptide (n = 6) did not significantly improve survival of any striatal phenotype. Long infusion of peptide (n = 6) was associated with increased survival of calbindin-28k, calretinin, parvalbumin and GAD positive neurons (P < 0.05 vs. ischaemia + vehicle). Neurophysiological recovery was associated with improved survival of calbindin-28k, calretinin and parvalbumin positive striatal neurons (P < 0.05 for all). In conclusion, connexin hemichannel blockade after cerebral ischaemia in term-equivalent fetal sheep improves survival of striatal GABA-ergic neurons.

Figure 1

Representative continuous EEG and sleep state cycling data (1 min averages) from a fetus in the ischaemia + vehicle group (A,B). Note the continued suppression of EEG power after asphyxia (0 h) followed by a large prolonged rise in EEG activity between 10 and 168 h, reflecting electrographic seizure activity, and no sleep state cycling by 168 h (7 d). In the examples from a fetus in the ischaemia + short infusion (C,D) and ischaemia + long infusion groups (E,F), the pattern of seizure activity was much more discrete than after ischaemia + vehicle and sleep state cycling returned by 96 and 72 h (days 4 and 3), respectively.

Figure 2

Phenotypic striatal neuronal density in the striatum (including the caudate nucleus and putamen). Data are mean ± SD from n = 6/group. ^#p < 0.05 vs. sham ischaemia; *P < 0.05 vs. ischaemia + vehicle.

Figure 3

Representative photomicrographs of striatal phenotypic neurons in the caudate nucleus from sham ischaemia, ischaemia + vehicle, ischaemia + short infusion and ischaemia + long infusion groups at 40x magnification. Arrowheads with tail show examples of neurons that were counted, arrowheads show examples of neurons that were not counted. Scale bar is 200 µm.

Figure 4

Representative photomicrographs of striatal phenotypic neurons in the putamen from sham ischaemia, ischaemia + vehicle, ischaemia + short infusion and ischaemia + long infusion groups at 40x magnification. Arrowheads with tail show examples of neurons that were counted, arrowheads show examples of neurons that were not counted. Scale bar is 200 µm.

Figure 5

Correlations between neurophysiological outcomes and histopathology in ischaemia + vehicle, n = 6; ischaemia + short infusion, n = 6 and ischaemia + long infusion, n = 5 (neurophysiological data were not available from 1 subject in the ischaemia + long infusion group). Panels (A–C) Correlations between total seizure burden and survival of striatal calbindin-28k, calretinin and parvalbumin positive neurons. (D–F) Correlations between the day that sleep state cycling (SSC) resumed after ischaemia and survival of striatal calbindin-28k, calretinin and parvalbumin positive neurons. (G–I) Correlations between EEG power on day 7 of recovery and survival of striatal calbindin-28k, calretinin and parvalbumin positive neurons.

Figure 6

Photomicrograph showing the striatal area that was outlined at 2x and randomly sampled at 20x magnification, using a sampling grid set at 2200 × 2200. Striatal phenotypic neuronal populations were quantified from each hemisphere across 2 sections.