Rat permanent middle cerebral artery occlusion procedure

This study was approved by the Animal Ethics Committee of the University of Western Australia and follows guidelines outlined by the Australian Code for the Care and use of Animals for Scientific Purposes. The experimental procedure for performing the permanent middle cerebral artery occlusion (MCAO) stroke model has been described previously [20, 21]. Briefly, male Sprague–Dawley rats weighing 270–320 g were kept under controlled housing conditions with a 12 h light–dark cycle and with free access to food and water. Experimental animals were fasted overnight and subjected to filament permanent MCAO. In order to monitor blood pressure and withdraw blood samples, a cannula was inserted in the tail artery. Between 50 and 200 µL of blood was used for glucose (glucometer; MediSense Products, Abbott Laboratories, Bedford, MA, USA) and other measurements (PaO₂, PaCO₂, pH; ABL5, Radiometer, Copenhagen, Denmark). The MCAO procedure was considered successful based on a >25 % decrease from baseline in cerebral blood flow (CBF) after insertion of filament, as measured by laser Doppler flowmetry. During surgery temperature was closely monitored using a rectal probe (Physitemp Instruments, Clifton, USA) and maintained at 37.5 ± 0.5 °C, with fan heating or cooling.

Thirty minutes post-MCAO, rats were intravenously treated with the peptide (1000 nmol/kg in 600 µL over 6 min) or vehicle (0.9 % sodium chloride for injection; 600 µL over 6 min). Treatments were administered via the right internal jugular vein and infusion pump. Treatments were randomised and all procedures were performed blinded to treatment.

Twenty-fours hours post-MCAO, infarct area assessment was performed by preparing 2 mm thick cerebral coronal brain slices, and incubating in 3 % 2,3,5 triphenyltetrazolium chloride (TTC; Sigma-Aldrich, St. Louis, USA) at 37 °C for 20 min, followed by fixation in 4 % formalin at room temperature overnight. Digital images of coronal sections were acquired using a colour scanner and analysed by an operator blind to treatment status, using ImageJ software (3rd edition, NIH, Bethesda, USA). The total infarct volume was determined by measuring the areas of infarcted tissue on both sides of the 2 mm sections. These measured areas were corrected for cerebral oedema by multiplying the infarct volume for the oedema index (calculated by dividing the total volume of the stroke-affected hemisphere by the total volume of the contralateral hemisphere) [22].

A total of 42 animals were used in the trial. Five animals were excluded from the study; two animals were euthanased due to subarachnoid haemorrhage, one animal was excluded due to insufficient decrease in CBF, one animal was excluded due to pyrexia, and one died during surgical recovery for an unknown reason.