Excessive release of glutamate has traditionally been accepted to be the first detrimental event leading to brain damage at the onset of stroke. However, within 2 min of stroke onset, neurons and glia in the ischemic core undergo a profound loss of membrane potential caused by failure of the Na+/K+ ATPase pump. This ‘ischemic’ or ‘anoxic’ depolarisation (AD) results in cellular swelling and acute neuronal injury which is not inhibited by NMDA receptor antagonists or ion channel blockers although a ‘soup’ of both may block it in slices1, 2, 3. While this result complements current thinking that a cocktail of drugs may be needed to treat stroke4, Anderson and colleagues5 found that sigma-1-receptor ligands display a remarkable ability to inhibit AD suggesting that currently unknown mechanisms are involved in AD and the ensuing brain damage. The present study compared the effects of the novel compound AM-36, an antioxidant, Na+ channel blocker and a sigma-1-receptor ligand, carbetapentane (CP, a sigma -1-receptor ligand) and dibucaine (a Na+ channel blocker) upon AD onset. AD was imaged using intrinsic optical signalling5 (IOS) in submerged neocortical slices as a focal increase in light transmittance that then propagated at 2 mm/min across gray matter. Slices (400 μm) from Sprague Dawley rats (21–30 d) were equilibrated in aCSF bubbled with 95%O2/5%CO2 and superfused with aCSF at 3 ml/min. Slices were incubated with or without drugs at 31°C for 45 min prior to imaging and exposure to oxygen-glucose deprivation (1 mM glucose aCSF bubbled with 95% N2/5%CO2) at 35°C. AD onset was blocked or delayed vs vehicle controls (6.2±0.2 min) by 50 μM AM-36 (8.2±0.5 min), 10 μM dibucaine (7.8±0.3 min) or 50μM CP (7.4±0.0 min). In separate experiments, neocortical field potentials were evoked from slices incubated with 10 μM dibucaine or 50 μM AM-36. Dibucaine reduced field potentials by 88% while AM-36 and CP did not alter the response. Thus AD suppression appears mediated by sigma-receptor activation and not Na+ channel blockade. Competition binding studies in progress confirm μM affinity of AM-36 and dibucaine for sigma-1-receptors. In vivo, both AM-36 (6 mg/kg ip), and the sigma-1-receptor ligand 4-PPBP (10 μmol/kg ip) potently reduced neuronal damage following endothelin-1-induced focal ischemia in conscious rats6. These effects were mediated at least in part via inhibition of neuronal and inducible nitric oxide synthase (NOS) upregulation after stroke. In brain slices, preliminary studies showed no effect of pre-incubation with the specific neuronal NOS inhibitor 7-nitroindazole on AD (control: 6.6±0.7 vs 7-NI: 7.2±0.5 min, P>0.05). The present studies have found potent inhibition of neuronal damage in vivo and in brain slices by drugs acting on sigma-1-receptors. The mechanism of action of sigma ligands is yet to be determined, but may involve inhibition of NOS in vivo.
