Capillary blood flow around microglial somata determines dynamics of microglial processes in ischemic conditions.

Transgenic mice

All procedures follow National Institutes of Health guidelines for the humane care and use of laboratory animals and underwent yearly review by the Animal Care and Use Committee at Georgia Health Sciences University. All efforts were made to minimize animal discomfort and reduce the number of mice used. The founding mice of the B6.129P- Cx3cr1 tm1Litt /J colony [CX3CR1-EGFP] and B6.Cg-Tg(Thy1-YFPH)2Jrs/J colony [YFP-H] were purchased from Jackson Laboratories.

Mice of the CX3CR1-EGFP strain express

EGFP in microglia under control of the endogenous Cx3cr1 locus encoding the chemokine (C-X3-C) receptor 1 (CX3CR1, also known as fractalkine receptor) ( Jung et al., 2000 ). In these mice microglia cells are clearly labeled with EGFP providing high contrast, thus facilitating the imaging of microglial somata and fine processes. YFP-H mice display bright fluorescence of YFP expressed in a fraction of pyramidal neurons of the neocortex aiding dendritic imaging ( Feng et al., 2000 ). We used only heterozygous [CX3CR1-EGFP] mice in all experiments to preserve CX3CR1 signaling that affects microglia-neuron interactions ( Cardona et al., 2006 ; Ransohoff and Cardona, 2010 ). Hybrid mice, with a small proportion of both fluorescent neocortical pyramidal neurons and fluorescent microglia, were generated by crossing [YFP-H] and [CX3CR1-EGFP] strains. In total, 52 heterozygous [CX3CR1-EGFP], 12 hybrid [YFP-H]/[CX3CR1-EGFP] and 4 wild type male and female mice between 6 and 16 weeks of age were used in this study.

Preparation of mice for in vivo imaging

Microglia were imaged through a thinned skull cranial window centered at stereotaxic coordinates −1.8 mm from bregma, 2.8 mm lateral over the somatosensory cortex. Surgical procedures followed a protocol adapted from Grutzendler and Gan (2005) . Mice were anesthetized with an intraperitoneal injection of urethane (1.5 mg/g body weight). Body temperature was maintained at 37°C with a heating pad (Sunbeam). A short ~1 cm L-shaped glass capillary (1.2 mm diameter) was inserted into the trachea and secured with sutures to minimize potential breathing problems. The scalp was gently separated from the cranium. A plastic ring (13 mm diameter) was glued to the skull with dental acrylic cement (Co-Oral-lte Dental) to stabilize the head during craniotomy and imaging using a mouse headholder attached to a baseplate. A small aluminum bar with two tapped screw holes was embedded into the acrylic of the mice used in the bilateral common carotid artery occlusion (BCCAO) experiments. The heads of these mice were stabilized in the same position during consecutive imaging sessions by 2 screws tightened to a custom-made L-shaped adjustable metal arm fixed to the baseplate. A circular area of the skull (~1 mm diameter) was thinned under the Zeiss Stemi SV6 stereo zoom microscope with a high-speed dental drill (Midwest Stylus mini 540S) by ¼ bit. A final thickness of the skull (~30 μm) was achieved by gently scraping the bone with a microsurgical blade (Surgistar, #38-6900). A cortex buffer was applied to keep the thinned region moist during experiments. The baseplate containing the headholder with the mouse resting on a heating pad was affixed to the Luigs & Neumann microscope stage for imaging. Rectal temperature was monitored continuously and maintained at 37°C. A sufficient level of anesthesia was confirmed by the lack of a toe-pinch reflex and heart rate (450–650 beats/min) monitoring using DAM-60 amplifier (WPI) and maintained with only minimal supplementation if necessary (