Objective: Induce focal cortical cerebral ischemia through electrocoagulation of the distal middle cerebral artery to create an animal stroke model
Gather these items before starting the experiment. Check off items as you prepare.
National Institutes of Health
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House mice in temperature-controlled environment with controlled light-dark cycle and humidity
Note: 12 h light-dark cycle with food and water available ad libitum; room humidity controlled at 55 ± 5%
“All mice were housed in a temperature-controlled environment (22 ± 2 °C) on a 12 h light–dark cycle with food and water available ad libitum. Room humidity was controlled at 55 ± 5%”
Anesthetize male mice weighing 23-26g with isoflurane in oxygen and nitrous oxide mixture
Note: Specific anesthesia induction time not mentioned
“Male mice weighing 23–26 g were anesthetized with 1–1.5% isoflurane in 30% oxygen and 70% nitrous oxide”
Make a 2 cm curved skin incision between the right eye and the right ear using surgical scissors
Note: Curved incision for optimal surgical access
“After making a 2 cm curved skin incision between the right eye and the right ear using surgical scissors”
Retract the temporal muscle laterally to expose the surgical field
“the temporal muscle was retracted laterally”
Under operating microscope, use high-speed micro drill to open a 1.5 mm-diameter window in the skull just rostral to the foramen ovale
Note: Window location is critical - just rostral to foramen ovale
“Under an operating microscope, a 1.5 mm-diameter window was opened using a high-speed micro drill (Stoelting, CellPoint Scientific, Maryland) just rostral to the foramen ovale”
Carefully open the dura mater to expose the distal middle cerebral artery
Note: Careful handling required to avoid damage to underlying tissue
“The dura mater was carefully opened and the distal MCA was exposed and isolated”
Expose, isolate, and electrocauterize the distal MCA using bipolar electrocoagulation forceps, then disconnect it just distal to crossing the olfactory tract
Note: Disconnection location is critical - just distal to olfactory tract crossing
“the distal MCA was exposed and isolated, electrocauterized using bipolar electrocoagulation forceps, and disconnected just distal to crossing the olfactory tract”
Immediately following MCA occlusion, occlude the right common carotid artery with a microvascular clip
Note: Must be done immediately after MCA occlusion; maintained for 15 minutes
“Immediately following the occlusion, the right common carotid artery (CCA) was occluded with a microvascular clip (Fine Science Tools, Foster City, CA) for 15 min”
Close surgical wounds and allow mice to recover from anesthesia
Note: Recovery conditions not specified
“After closing of the surgical wounds, mice were allowed to recover from the anesthesia”
For sham-operated control animals, expose the MCA and CCA but do not perform occlusion
Note: Sham controls undergo identical surgical procedure except for the occlusion step
“Sham-operated animals only had the MCA and the CCA exposed”
Prepare six coronal sections of 20 μm thickness from brain tissue
Note: Specific timing for tissue collection not mentioned
“For cerebral infarction, six coronal sections (20 μm) were stained with hematoxylin and eosin”
Stain coronal brain sections with hematoxylin and eosin
“six coronal sections (20 μm) were stained with hematoxylin and eosin”
Measure infarct volume using NIH ImageJ 1.46r software and present as percentage of contralateral hemisphere
Note: Infarct volume normalized to contralateral hemisphere for comparison
“Infarct volume was measured using the NIH ImageJ 1.46r software and was presented as percentage of the contralateral hemisphere”
PAD4 -/- mice purchased from The Jackson Laboratory; WT controls from SLAC Laboratory Animal Co. Ltd, Shanghai, China