![]() The dicom files were opened in OsiriX version 3.3.2 (OsiriX Foundation, Geneva, Switzerland), and the regions of interest were manually delineated. The T2-weighted (T2W) imaging protocol parameters were as follows: a two-dimensional fast spin echo sequence with echo time (TE) = 82.5 ms, repetition time (TR) = 4000 ms, echo train length = 8, axial slice thickness = 0.6 mm with no spacing, field of view = 3 cm, matrix = 128 × 128, number of excitations (NEX) = 10. An in-house 2-cm diameter surface coil was placed on the mouse skull to obtain the images. The mice were anesthetized with 2% isoflurane in 2 L/minute of medical grade oxygen while the respiratory rates were monitored, and the surface body temperature was also monitored and kept at 34☌ throughout the imaging. Magnetic resonance imaging (MRI) was performed at Stanford Small Animal Imaging Facility by using the GE Healthcare (Waukesha, WI) Micro-Signa software environment version 12M5 with a Varian 7 Tesla magnet, Research Resonance Instruments BFG-150 to 90 gradient insert. Arterial blood gas and lactate levels were measured by iSTAT CG4+ cartridges and a handheld blood analyzer (Abbott Laboratories, Abbott Park, IL). A noninvasive CODA Monitor system (Kent Scientific, Torrington, CT) was used to measure blood pressures and pulse rates. No differences were found in the presurgical weights of mice in any experiment. The probe was placed onto the skull 2 mm posterior and 5 to 6 mm lateral to the bregma on the left side. We measured cerebral blood flow by using a laser Doppler flow meter. The surgical wound was closed, and the mice were returned to their cages with free access to water and food. ![]() Thirty minutes after its insertion, the filament was removed to permit reperfusion. The core body temperature was measured by a rectal probe and maintained at 37☌ throughout the surgery. A hole was made in the common carotid artery, and a 7-0, silicon rubber-coated, reusable monofilament (70SPRe2045 Doccol Inc., Sharon, MA) was inserted and advanced toward the internal carotid artery 9 to 10 mm after the carotid bifurcation to occlude the left middle cerebral artery. The left external and common carotid arteries were permanently ligated. Briefly, mice were anesthetized by 1.5% to 2% isoflurane in a mixture of 1 L/minute of air and 0.2 L/minute of oxygen. The mice were habituated in the surgery room overnight, and all of the surgeries were initiated early in the morning. These findings indicate a novel role for mast cells in the meninges, the membranes that envelop the brain, as potential gatekeepers for modulating brain inflammation and pathology after stroke. We also obtained evidence that two mast cell-derived products, interleukin-6 and, to a lesser extent, chemokine (C-C motif) ligand 7, can contribute to stroke pathology. With the use of genetic and cell transfer approaches in mice, we identified evidence that meningeal mast cells can importantly contribute to the key features of stroke pathology, including infiltration of granulocytes and activated macrophages, brain swelling, and infarct size. Although different immune cells traffic through meningeal vessels en route to the brain, mature mast cells do not circulate but are resident in the meninges. An understudied but important factor is the role of meningeal-located immune cells in modulating brain pathology. Inflammation is thought to play an important role in stroke pathology, but the factors that promote inflammation in this setting remain to be fully defined. Stroke is the leading cause of adult disability and the fourth most common cause of death in the United States.
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