The animal experiments were performed on male Wistar rats, weighing 300-350 g (Møllegaard’s Breeding Center, Copenhagen, Denmark), which were fasted overnight with free access to tap water. The experiments were approved by the Malmö/Lund Ethical Committee on Animal Experiments and were in accordance with the guidelines of the National Institute of Health (NIH). All efforts were made to minimize animal suffering and to reduce the number of animals used.
The two-vessel occlusion (2-VO) model of global cerebral ischemia was used . In brief, the animals were initially anaesthetized with 3.5-4% isoflurane in N2O/O2 (70:30) and then intubated and connected to a small animal respirator. The isoflurane concentration was reduced to 1.5% for the remainder of the surgery. The tail artery and vein were cannulated for blood pressure recordings, blood sampling and drug infusions. The right jugular vein was used to insert a soft silastic catheter into the superior caval vein for rapid withdrawal of blood and reduction of the blood pressure to 45–50 mmHg during ischemia. Both common carotid arteries were dissected and encircled with loose ligatures. In addition, EEG electrodes were placed into the temporal muscles and EEG activity recorded. Thereafter, both common carotid arteries were reversible clamped for 10 minutes. At the end of surgery, isoflurane concentration was reduced to 0.5-1% and the ventilation and O2 supply was adjusted to an arterial PCO2 of 35–40 mmHg and a PO2 of close to 100 mmHg. Heparin (90 IU.kg-1) was given prior to the first blood gas measurement. During the experiment, animals were paralyzed with vecuronium bromide (2 mg.h-1). The animals’ head and body temperature were kept close to 37°C using a heating lamp and a thermostat-regulated pad.
Immediately after ischemia, 0.5 ml of a 0.6 mol/L NaHCO3 solution was given intravenously to neutralize systemic acidosis, and isoflurane supply was discontinued. When the animals regained spontaneous breathing they were extubated and disconnected from the respirator and were then housed in cages with free access to tap water and pellet food. Sham-operated animals were treated in the same way as those in the ischemia group except for the occlusion of the carotid arteries and the reduction of blood pressure. In total 23 rats were operated, thereof 2 rats were excluded immediately after surgery due to surgery problems, one animal died on day 3 after surgery.
Randomization, EPO administration and EPO analysis
Every other animal was randomized into the treatment groups and received either an intravenous bolus injection of EPO (Neorecormon, Roche, Switzerland) immediately after surgery calculated based on the following equation: 80 IU multiplied with the volume of distribution (VD; 0.057 mL/g BW). Thereafter, maintenance intravenous infusion was performed at 160 IU per hour via an osmotic minipump (mini-osmotic alzet pump2001 D) for 72 h or an intravenous saline injection immediately after surgery and saline infusion via an osmotic minipump for additional 72 h (mini-osmotic alzet pump 1003D). Serum was analyzed for EPO 10 to 15 minutes after the bolus injection and 5 and 72 h after the surgery using an automated ELISA system (Quantikine® IVD® ELISA, R&D Systems, UK).
For histopathological analyses, animals were re-anaesthetized with 3.5% isoflurane, tracheotomized, artificially ventilated, and perfusion-fixed with phosphate-buffered 4% formaldehyde after 7 days of reperfusion. The brains were allowed to fix in situ for one hour and were then removed, dehydrated, and embedded in paraffin. The paraffin embedded brains were sectioned at 5 μm, and stained with celestine blue and acid fuchsin. Sections were deparaffinized, incubated in celestin blue solution (0,5%) for 1 minute and after washing with acetic acid incubated in acid fuchsin solution (1%) for 1 minute. After dehydration and mounting, brain damage was quantified by visual counting of intact violet-stained neurons in a blinded manner according to Nellgard and Wieloch . Undamaged neurons, which had an intact nucleus and stained violet, were counted in three defined fields, each 400 μm in length, of the CA1 subsector of the dorsal hippocampus (3.8 to 4 mm caudal to bregma): the medial, hipp (1), the middle, hipp (2), and the lateral, hipp (3) CA1 (at the border to CA3). Cortical (Cx) damage was assessed in a circular area of 400 μm in diameter of the temporal cortex which is most sensitive area in the model of global ischemia. Striatal (Str) damage was assessed as neuronal score: score 0 - no damage, score 1 - damage to dorsal striatum or ventral striatum, 2- both ventral and dorsal rim, 3- entire striatum affected.
The assay was performed according to the manufacturers instructions using the protocol for paraffin-embedded tissue sections (Roche Diagnostics GmbH, Germany). TUNEL positive cells were counted from the entire CA1 region of the hippocampus (approximately 3.8 to 4 mm caudal to bregma) and presented as absolute numbers.
This test combines a sensori-motor test and a memory test of the T-maze type . The rotating pole test measures coordination and integration of movements . Rats traverse an elevated wooden pole (elevation 700 mm, diameter 40 mm, length 1500 mm) rotating at a speed of 10 rpm to the left or right. Prior to ischemia the animals are trained several days before ischemia until they can cross the pole onto a platform and enter the home cage. The home cage, made of transparent plexi-glas with an entrance opening at the left edge, is placed in front of the platform at the end of the pole. A cardboard wall is placed perpendicular to the cage wall, immediately to the right of the rotating pole with the entrance accessible, but not seen by the rat. This part of the test requires retention of memory. At 7 days after ischemia, each animal was tested twice, while recorded by a video camera. A score (0 to 6) was subsequently assigned to each performance. 0 means falling off the pole immediately; 1 means unable to traverse the pole; 2 means falling off the pole while crossing; 3 denotes crossing the pole while slipping and jumping with the hind limbs; 4 means crossing the pole with at least 50% slips; 5 means crossing with few slips; 6 indicates crossing with no foot slips. Score 5–6 is considered as normal performance. Correct or failed entry into the homecage were registered.
Differences in the number of cells in the hippocampus and cortex in saline and EPO treated rats were analyzed by the Students t-test. Effects of EPO treatment on neuronal survival in the striatum were analyzed by the Mann–Whitney test. Individual physiological parameters between saline and EPO treated animals at the same time point were analyzed by the Students t-test. Physiological parameters within a treatment group were analyzed by ANOVA for repeated measurements and Dunnetts T3 posthoc test. Behavioral tests were anlyzed by the Mann–Whitney test. Statistical analyses were performed using IBM SPSS Statistics 20.0 (IBM Svenska AB, Sweden).