Deficiency of von Willebrand factor protects mice from ischemic stroke
Blood Kleinschnitz et al.
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Supplemental materials for: Kleinschnitz et al
Induction of cerebral ischemia
Focal cerebral ischemia was induced in 6–8-week-old mice by 60 min transient middle cerebral artery occlusion (tMCAO) as described.1–3 Mice were anesthetized with 2.5% isoflurane (Abbott, Wiesbaden, Germany) in a 70% N2O/30% O2 mixture. Following a midline skin incision in the neck, the proximal common carotid artery, and the external carotid artery were ligated, and a standardized silicon rubber-coated 6.0 nylon monofilament (6021; Doccol Corp., Redlands, CA, USA) was inserted and advanced via the right internal carotid artery to occlude the origin of the right MCA. Operators (MA, CK, and TS) were blinded to the genotype and operation time per animal did not exceed 15 minutes. The intraluminal suture was left in situ for 60 minutes. Then animals were re-anesthetized, and the occluding monofilament was withdrawn to allow reperfusion. Neurological function was assessed by two independent and blinded investigators 24 h after tMCAO.
Assessment of functional outcome
24 hours after tMCAO the modified Bederson score4 was used to determine global neurological function according to the following scoring system: 0, no deficit; 1, forelimb flexion; 2, decreased resistance to lateral push; 3, unidirectional circling; 4, longitudinal spinning; 5, no movement. Motor function and coordination were evaluated by the grip test.5 For this test, the mouse was placed midway on a string between two supports and rated as follows: 0, falls off; 1, hangs onto string by one or both forepaws; 2, as for 1, and attempts to climb onto string; 3, hangs onto string by one or both forepaws plus one or both hindpaws; 4, hangs onto string by fore- and hindpaws plus tail wrapped around string; 5, escape (to the supports).
Laser-Doppler flowmetry
Laser-Doppler flowmetry (Moor Instruments, Axminster, U.K.) was used in some animals (n=3/group) to monitor regional cerebral blood flow (rCBF) in the MCA territory (6 mm lateral and 2 mm posterior from bregma).6 After advancing the thread the decrease in rCBF was similar between VWF−∕− and wild-type (WT) mice indicating sufficient occlusion of the MCA origin (12.1 ± 2.4% of baseline level in WT mice versus 10.2 ± 6.2% of baseline level in VWF−∕− mice; p>0.05) (Fig. S1A). Ten minutes after reperfusion rCBF was reconstituted to >60% of baseline levels and again did not significantly differ between WT and VWF−∕− animals (74.8 ± 8.3 % of baseline level in WT mice and 77.3 ± 5.4% of baseline level in vWF−∕− mice; p>0.05).
Assessment of the cerebral vasculature
For assessment of the cerebral vasculature WT and VWF−∕− mice (n=3/group) were deeply anesthetized with CO2 and transcardially perfused with 4% paraformaldehyde (PFA), followed by 3 ml black ink diluted in 4% PFA (1:5 v/v). Brains were carefully removed, fixed in 4% PFA overnight at 4°C and the Circle of Willis and major arteries were examined under a microscope (Fig. S1B). A complete Circle of Willis was identified in all animals studied, and the distribution of the MCA trunk and branch appeared to be anatomically identical among the genotypes. To further quantitatively examine the vascular structures, the development of the posterior communicating arteries (PComAs) which can affect brain sensitivity to ischemia7 was examined. The mean score of PComAs in both hemispheres showed no significant differences between the groups (3.2 ± 0.6 in WT mice versus 3.6 ± 0.4 in VWF−∕− mice; p>0.05).
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