The pharmacotherapy of focal cortical ischaemia in the mouse

Abstract

The measurement of corticalω₃ (peripheral-type benzodiazepine binding) site densities provides an accurate index for the detection and quantification of ischaemic brain lesions following middle cerebral artery occlusion (MCAO) in mice. Here, we have used this marker to assess the neuroprotective activity of potential anti-ischaemic drugs belonging to several chemical classes. In untreated mice, the mean infarcted volume measured 96 h after unilateral coagulation of the middle cerebral artery was27.9 ± 4.3mm³ (17.5% of the hemisphere volume) andω₃ site densities (measured by incubation with³H-PK 11195) were increased by107.3 ± 4.8% (cortical homogenates) or by 81% (coronal brain sections). The administration of the anti-ischaemic agent SL 82.0715 (10 mg/kg i.p.), 5 min, 6 h and 18 h after the occlusion and then twice daily until sacrifice evoked a decrease of similar magnitude (ca. 60–70%) in the volume of the infarction and in the proliferation ofω₃ sites. The constant tissue sparing effect of SL 82.0715 allowed the examination of the window of therapeutic opportunity. A significant diminution of corticalω₃ sites was still noted when the first administration was delayed until 3 h post-occlusion. Moreover, the protective effect of SL 82.0715 was enhanced by repeated treatment for the first 36 h but not thereafter. Based on the histological, autoradiographic and homogenate binding results obtained with SL 82.0715, we studied the protective effects of several competitive and non-competitive NMDA receptor antagonists. When administered according to the above-described standard protocol, these drugs reducedω₃ site levels in cortical homogenates from MCAO mice in a dose-dependent manner. The dose preventing by 50% the increase in ω₃ site levels (in mg/kg i.p.) and the maximal inhibition were respectively: MK-801 (0.2, 93%); TCP (1.6, 66%); kynurenate (260, 58%); ifenprodil (7.0, 58%); SL 82.0715 (1.1, 72%); CGS 19755 (46% at 10 mg/kg); dextromethorphan (46% at 30 mg/kg). In contrast, agents acting preferentially upon sigma (σ) opiate receptors ((+)-3PPP, 1–10 mg/kg i.p. and haloperidol, 0.3–3 mg/kg i.p.) did not provide a significant protection. In general, calcium channel blockers (nimodipine, flunarizine, verapamil, perhexiline, diltiazem) were devoid of a clear neuroprotective potential when administered at non-toxic doses after the coagulation of the middle cerebral artery. Diltiazem (3 and 10 mg/kg i.p.) provided a significant protection when the first administration was performed 10 min prior to the occlusion. Limited protection was observed with adenosine A₁ receptor agonists (N⁶-cyclohexyladenosine and 2-chloro-adenosine). No protection was afforded by the platelet activating factor (PAF) receptor antagonists, brotizolam and alprazolam at the dose of 10 mg/kg or by aGingko biloba extract at 30 mg/kg. The anti-inflammatory agent, dexamethasone, and the minor tranquillizer, clonazepam, also failed to alterω₃ site densities when administered at the dose of 10 mg/kg. Some putative cerebroprotective compounds also prevented the increases in ω₃ site densities in MCAO mice with 50% effective doses (in mg/kg i.p.) and maximal effects as follows: vincamine (0.2, 74%); cyclandelate (15, 63%) and piribedil (4, 66%) although many others were inactive (eg. naftidrofuryl, pyrithioxine, papaverine). In conclusion, the results with both NMDA receptor-channel blockers (MK-801, TCP) and atypical non-competitive NMDA receptor antagonists (ifenprodil, SL 82.0715) suggest that NMDA antagonists constitute the first coherent pharmacological class for the treatment of focal cerebral ischaemia. The mouse model in which tissue loss and sparing are indirectly assessed by the quantification ofω₃ sites further allows not only the rapid evaluation of putative neuroprotective agents but possibly also the determination of an optimal treatment regimen for active compounds.