Animal experiments were approved by the local ethics committee. Male Wistar rats (n = 112) weighing 280-320 g (Charles River, Sulzfeld, Germany) were subjected to embolic stroke (TE) using a method modified from Busch .
In a preliminary study (n = 64 animals), the effects of different red blood clot size and its various preparation on infarct size, edema and mortality have been tested in four different groups (n = 16 each). Anaesthesia and animal preparation were performed as described for the main body of the study. No rt-PA has been given.
A whole blood sample of 0.5 ml was withdrawn into a polyethylene catheter (PE-50; NeoLab, Heidelberg, Germany) and allowed to clot for 2 hours. Then, blood clots were transferred to a 20 mM CaCl2 solution and incubated over night at 4°C or for several minutes at room temperature. Prior to cutting clots the calcified blood was washed in 0.9% NaCl to wash out the CaCl2. A blood clot of 30 mm or 24 mm length, respectively, was divided into 12 pieces on a scale paper. Clot preparation according to treatment groups has been done as follows:
Group 1: Clot incubation time 24 hours, clot size 3 mm, number of clots 12
Group 2: Clot incubation time 2 hours, clot size 3 mm, number of clots 12
Group 3: Clot incubation time 24 hours, clot size 2 mm, number of clots 12
Group 4: Clot incubation time 2 hours, clot size Group 2 mm, number of clots 12
After 24 hours, the animals have been sacrificed by an overdose of ketamine (10%) and xylazine hydrochloride (100 mg/kg body weight). After decapitation, brains were rapidly removed and dissected into 5 coronal sections of 2 mm thickness, incubated in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC) at 37°C for 15 minutes until the brains were stained sufficiently and immersion-fixed in a 4% paraformaldehyde solution. Slices were scanned into Adobe Photoshop and infarct size was measured using scion image (Scion corporation, Maryland, USA) as imaging software. Infarct size is reported as infarct volume and is corrected for edema. To compensate for the effect of brain edema, the corrected infarct volume was calculated as previous described . The following formula was used to calculate the infarct volume corrected for the cerebral edema : Corrected infarct volume = volume of the left, nonischemic hemisphere - (volume of the right, ischemic hemisphere - infarct volume). The volume of the brain edema has been calculated with the following formula: the sum of surface areas of the left, non-ischemic hemisphere divided by the sum of surface areas of the right, ischemic hemisphere. Infarct size and size of edema is expressed as % of the contralateral hemisphere.
Since mortality was the lowest in group 4 of the preliminary study and infarct size showed low standard deviations, clots for animals in the major study were prepared according to method 4.
Animals were randomly assigned to one of the following groups:
Control group (Co): 37°C body core temperature, no specific treatment (n = 12).
Thrombolysis group (T): 37°C body core temperature, rt-PA 1.5 h after TE (n = 12).
Hypothermia group (H): 34°C induced 1.5 h after TE and maintained for 4.5 h (n = 12).
Combination group (TH): 34°C induced 1.5 h after TE and maintained for 4.5 h plus rt-PA 1.5 h after TE (n = 12).
General anaesthesia and monitoring of vital signs
Animals were anesthetized using a mixture of halothane (Halocarbon laboratories), oxygen 30% and N2O (70%). Minimum alveolar concentrations were corrected for the actual body temperature. A polyethylene-catheter (PE-50; Neolab, Heidelberg, Germany) in the right femoral artery was used for continuous monitoring of blood pressure, heart frequency, and blood gases during the experiment as well as blood sampling for measuring blood protein levels. The right femoral vein was cannulated for administration of rt-PA, placebo or contrast agent (Magnevist).
Thromboembolic stroke model
For induction of TE, the right common carotid (CCA), internal carotid (ICA), and external carotid arteries (ECA) were exposed, and further dissection identified the origin of the pterygopalatine artery (PPA). The ECA and the PPA were permanently ligated, while the CCA was only temporarily clipped for embolization. A PE 50 catheter was inserted into the ECA proximal to its ligation, and 12 red blood clots (each 0.35 mm in diameter and 2 mm in length) were injected at the origin of the right middle cerebral artery (MCA).
Rectal temperature was regulated by a thermostatically controlled heating pad (Foehr Medical Instruments, Seeheim-Jugenheim, Germany). A rectal probe was inserted 4 cm into the rectum to measure the actual body core temperature. Prior experimental data indicated that the body core temperature correlates to intracranial and pericranial temperature during normothermia and therapeutic hypothermia . In normothermic rats, a target rectal temperature of 37°C was achieved by setting the heating pad to 37°C. For induction of therapeutic hypothermia, the animals were externally cooled by cooling pads attached to the abdomen of the animal in the prone position, until a temperature of 34.5°C was reached. Thereafter, cooling pads were removed from abdomen and the heating pad was set to 34°C. Within approximately 2-5 min, the animal reached 34°C which was maintained via the heating pad for the entire cooling period. The Optimal temperature of 34°C of body temperature for effective treatment of experimental stroke has been described in our laboratory and cooling of the animals was performed as described in this previous study . Rewarming was performed by setting the heating pad to the desired target temperature of 37°C. The rewarming procedure took approximately 30 min.
For thrombolysis, rt-PA (Boehringer Ingelheim, Germany) 10 mg/kg body weight was injected 1.5 hours after induction of TE as previously described .
Removal of brain tissue
Twenty-four hours after MCAO, rats were sacrificed by an overdose of ketamine (10%) and xylazine hydrochloride (100 mg/kg body weight). After decapitation, brains were rapidly removed, frozen in isopentane at -20°C, and stored until use at -80°C.