Buprenorphine Does Not Aggrevate Ischemic Neuronal Injury in Experimental Focal Cerebral Ischemia

Animals were anesthesized with 4 vol% isoflurane (30% O ₂, remainder N ₂ O). Rectal temperature was maintained between 36.5 and 37.0°C using a feedback-controlled heating system. During the experiments, cerebral blood flow was measured by Laser Doppler Flowmetry (LDF) using a flexible fiber optic probe (Perimed, Stockholm), which was attached to the intact skull overlying the middle cerebral artery territory (1 mm posterior/5 mm lateral from bregma). Laser doppler flowmetry changes were monitored up to 90 minutes after the onset of ischemia. Focal cerebral ischemia was induced using an intraluminal filament technique. 9 A midline neck incision was made, and the common carotid artery (CCA) was temporarily closed with two microvascular clips (FE691, Aesculap, Tuttlingen, Germany). A 4–0 nylon monofilament (Ethilon; Ethicon, Norderstedt, Germany) coated with silicon resin (Xantopren, Bayer Dental, Osaka, Japan) was introduced through a small incision into the common carotid artery and advanced distal to the carotid bifurcation for transient occlusion of the middle cerebral artery. The filament was removed after 90 minutes,and the CCA incision was closed by single interrupted sutures,using a 10–0 silk suture. We discontinued anesthesia and placed animals into their home cages. Arterial blood gases, pH, hemoglobin, mean arterial blood pressure, and heart rate were measured during ischemia and reperfusion.

The animals were deeply anesthetized with isoflurane 24 hours after middle cerebral artery occlusion, and then decapitated. Brains were immersion-fixed in 3.8% buffered formaldehyde for 5 days and automatically processed for embedding in paraffin (Bavimed 2050Z2; Haska AG, Bern, Switzerland) through a series of ethanol baths (70–100%) into xylol and paraffin for over 21 hours. Serial coronal sections (10 μm thick, three brains per block) were cut at 1-mm intervals through the forebrain and stained with toluidine blue ( Figure 1a ). The boundaries of the infarcted cerebral cortex and neostriatum were marked by an unbiased observer and traced from a monitor (at 21x magnification) onto a digitizing tablet interfaced to an image analysis system (DiaSys Datalab; H. Meyer, Thöringen, Switzerland). The infarct volumes were calculated by numeric integration of the infarct areas on sequential slices without correction for edema.

All values are given as means (SD). We compared differences between groups by using independent sample t tests. We considered p values <0.05 to indicate statistical significance.

Laser Doppler flow values decreased to approximately 40% of preischemic levels immediately after thread insertion. After thread retraction, laser Doppler flowmetry values slightly increased above preischemic control levels. Again, no differences were detected between different animal groups ( Figure 2 ). Arterial blood gas parameters and pH were kept within the physiological range ( Table 1 ). The mean arterial blood pressure and the heart rate moved in a physiological range prior to the occlusion and 90 minutes after reperfusion, and there were no significant differences between animal groups.

We obtained reproducible brain infarcts in the 2 mg/kg of buprenorphine or 0.2 ml of vehicle treated groups ( Figure 1 ). Intraperitoneal administration of 2 mg/kg buprenorphine had no effect on the infarct volume compared with control animals ( Figure 1 ).