Stereotaxic frame

Induction of photothrombotic strokes was performed similar to a previous study [9 (link)]. In brief, rats were anesthetized with ketamine (75 mg/kg, i.p. Medistar, Ascheberg, Germany) and xylazine anesthesia (10 mg/kg, i.p. Bayer, Leverkusen, Germany). The head was fixed in a stereotaxic frame (Stoelting Co., Wood Dale, IL, USA). After a median skin incision, preparation and cleaning of the skull, a photothrombotic stroke was induced through the intact bone. In brief, Rose Bengal dye (10 mg/kg body weight; 7.5 mg/mL in sterile saline) was injected into the tail vein using a 24G venous line (Abbocath, Hospira, Maidenhead, UK) during the first 2 min of a 20 min illumination period using a cold light source (KL 1500, Schott AG, Mainz, Germany). To avoid calefaction damage, air-cooling was performed using a custom made ventilation system. To standardize lesion size, a 4 mm diameter stencil was placed above the forelimb area of the primary motor cortex (2 mm anterior and 2 mm lateral, relative to bregma). Blood oxygenation and heart rate were constantly monitored (MouseSTAT Pulse Oximeter for mice and rats, Kent Scientific Corporation, Torrington, CT, USA), body temperature was controlled using a heating pad (Temperature Controller TC-1000, CWE Inc., Ardmore, PA, USA). Carprofen (5 mg/kg, s.c.; Norbrook, Newry, Northern Ireland) was given after surgery for pain relief.

The mice were anesthetized with 5% isoflurane and positioned on a stereotaxic frame (Stoelting Co.). A skin incision was made along the sagittal midline to expose the skull. A burr hole was drilled 2.8 mm lateral and 0.3 mm anterior to bregma and 3.8 mm deep, and collagenase VII‐S (sterile‐filtered, 0.5 U in 0.5 μl of sterile saline, Sigma) was then injected slowly into the left basal ganglia at the abovementioned stereotactic coordinates (at a rate of 0.05 μl/min). The needle was left in place for an additional 10 min and withdrawn slowly (at a rate of 1 mm/min) to prevent reflux. The burr hole was sealed with bone wax, the skin was sutured, and the mice were allowed to recover under observation. For the mice in the control group, holes were drilled in the skull at the same location, but collagenase was not injected. Mice that died before the end of the study were excluded.

Male Wistar rats (weighting 250–300 g) were placed in a stereotaxic frame (Stoelting) connected to an anesthesia system (EZ-7000 Classic System from E-Z Anesthesia, Plexx B.V.) containing isoflurane (ISOFLO^®, Esteve Veterinaria, Spain), which maintained the animals anesthetized only during the stereotaxic procedure. Each animal received a single unilateral intrahippocampal injection (coordinates relative to Bregma: antero-posterior −3.5 mm, lateral −2.5 mm, and dorso-ventral −4.0 mm) of 1 µL at the rate of 0.2 µL/min controlled by an automatic injector system (syringe pump, model 11 plus, Harvard Apparatus), either of aCSF or of 120 nmol of QA. Within each group, some of the animals were co-administered 1 nmol of MRS2500. The osmolality of all of the injected solutions was adjusted with NaCl. The animals were monitored for convulsions for 2 h after the intrahippocampal injections and the histological evaluation was performed 24 h later.