Permanent middle cerebral artery occlusion surgical procedure

This study was approved by the Animal Ethics Committee of the University of Western Australia (Approval number: RA/3/100/1362) and follows the guidelines outlined by the Australian Code for the Care and Use of Animals for Scientific Purposes. The surgical procedure for performing the permanent middle cerebral artery occlusion (MCAO) stroke model in rats, induced using an intraluminal monofilament, has been described previously [5,6]. Briefly, healthy male Wistar rats weighing 280–320 g were housed under controlled conditions; 12 hour light/dark cycle, free access to food (rat chow) and water and environmental enrichment (tissue/paper towels, gnawing blocks and red plastic cylinder in cage). Rats were purchased from the Animal Resources Centre, Murdoch, Western Australia.

Experimental animals, that had been fasted overnight, were anesthetised with the use of a facemask with 4% isoflurane (mix 30% oxygen/70% nitrous oxide) and anesthetic maintenance with 2–3% isoflurane. A laser Doppler probe was attached 1 mm posterior to the bregma and 4 mm lateral from the midline of the skull to measure cerebral blood flow. The tail artery was cannulated to allow blood pressure monitoring and withdrawal of blood samples for measurement of arterial blood gases (PaO₂, PaCO₂), pH (ABL5, Radiometer, Copenhagen, Denmark) and plasma glucose levels (MediSense Optium, Abbott Laboratories, USA). The MCAO procedure was considered successful if there was at least 25% decrease from baseline in cerebral blood flow (CBF) after insertion of the filament to occlude the right middle cerebral artery, as measured by laser Doppler flowmetry. In addition, using a rectal probe (Physitemp Instruments, USA), the animal body temperature was closely monitored during surgery and maintained between 37–37.8°C, with fan heating or cooling, as required. Treatments were administered intravenously through the right internal jugular vein, thirty minutes after MCAO, using an infusion pump (600 μl over 6 minutes).

Treatment groups consisted of R18 and R18D at 300 nmol/kg, and vehicle (0.9% sodium chloride). The 300 nmol/kg dose was selected because in previous studies examining the effectiveness of R18 in permanent and transient MCAO stroke, this dose reduced infarct volume to a lesser extent than a 1000 nmol/kg dose, thus if R18D was more neuroprotective than R18, the 300 nmol/kg dose would increase the likelihood of being able to demonstrate a difference in the efficacy of the L- and D-isoforms. Treatments were randomised and all procedures were performed while being blinded to treatments.