Proceedings of The Physiological Society

University College Cork (2004) J Physiol 560P, C1

Communications

DISRUPTION OF ASTROCYTIC GAP JUNCTIONS AND FAILURE OF ACTIVITY-RELATED DILATATION IN CORTICAL ARTERIOLES AFTER SIMULATED ISCHAEMIA IN BRAIN SLICES

Bate,E ; Thompson,E A; Griffiths,J L; Key,B J; Lovick,T A;

1. Depts Physiology and Pharmacology, University of Birmingham, Birmingham, United Kingdom.


Interpretation of BOLD signals from functional MRI is based on the assumption that local cerebral blood flow reflects neuronal activity. Scans from patients after recovery from a transient ischaemic attack showed abnormalities suggesting the coupling process was disrupted (1). We have investigated whether activity-related dilatation in cerebral arterioles is compromised after ischaemia and whether gap junctional communication in the neurone-astrocyte-arteriole triad could be affected. Cortical slices were prepared from urethane-anaesthetised (1.5g kg-1 i.p.) adult Wistar rats as described previously (2) and maintained at 33oC. In 10 arterioles, resting internal diameter (ID) 14.6±0.95μm (mean±SEM), addition of 75nM U46619 was used to pre-constrict vessels and induce vasomotion. Superfusion with a glutamate agonist (1μM AMPA for 10min) evoked 9.93±1.6% increase in ID and vasomotion decreased from 12.0±1.3 to 2.3±0.7min-1. 30 min after washout of AMPA, 5 slices were subjected to 5min oxygen and glucose deprivation (OGD, ACSF gassed with 95%N2/5%CO2 and glucose replaced by sucrose). OGD induced transient (<10min) dilatation (8.3±1.7% increase in ID, vasomotion decreased to 1.6±0.7min-1). 30min later, a second application of AMPA failed to evoke a significant change in ID (-0.42±1.57%, p>0.05, paired t-test) and vasomotion decreased to only 5.6±0.7min-1. In control vessels (no OGD, n=5) the response to AMPA was unchanged (7.3±1.7% increase in ID, vasomotion decreased to 5.2±1.2min-1, p>0,05 Student’s t test). Immunostaining for connexin 43, the predominant astrocytic gap junctional protein was revealed in 40μm thick sections from fixed slices using antibodies against the phosphorylated (Cx43p) and dephosphorylated (Cx43d) protein (antibodies 71-0700 and 13-8300, Zymed). In neocortex, punctate Cx43p immunoreaction product was interspersed between unstained neuronal somata. Staining for Cx43d was paler but showed a similar distribution. 30min after exposure to 5min OGD, the punctate Cx43p immunostain became paler but patches of a dark, granular immunoreaction product appeared, especially in somatosensory and auditory/visual cortices. In equivalent areas of Cx43d-stained slices, patches of unstained tissue appeared, surrounded by a border of intense punctate staining. The results indicate that activity-related dilatation in cortical arterioles is disrupted following recovery from a simulated transient ischaemic attack. The concomitant changes in Cx43 immunostaining may reflect a disruption of cellular processes in which communication through astrocytic gap junctions is compromised.

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