2.6. In vivo leukocyte adhesion and rolling in live capillaries

Murine Dorsal Window Chamber Preparation: Eight week-old C57/BL6 WT male mice were fitted with a dorsal window chamber. The mice window chamber model is widely used for microvascular studies in the unanesthetized state, and the complete surgical technique is described in detail elsewhere [26], [27]. Briefly, the animal was prepared for chamber implantation by an IP injection of pentobarbital for anesthesia. Sutures were used to lift the dorsal skin away from the animal, and one frame of the chamber was positioned on the animal's back. A chamber consisted of two identical titanium frames with a 12 mm circular window. With the aid of a stereomicroscope, one side of the skin fold was removed following the outline of the window until only a thin layer of retractor muscle and the intact subcutaneous skin of the opposing side remained. The intact skin of the other side was exposed to the ambient environment. Animals were allowed 2 days for recovery. Finally, catheters (PE-50) were implanted in the carotid artery and jugular vein. Catheters were tunneled under the skin, exteriorized at the dorsal side of the neck, and securely attached to the window frame. Three to four days after the initial surgery, the microvasculature was examined, and only animals passing an established systemic and microcirculatory inclusion criteria entered the study [28], [29]. For inclusion criteria, mice were considered suitable for the experiments if: 1) systemic parameters were within normal range. Namely, heart rate (HR) > 400 beat/min, mean arterial blood pressure (MAP) > 80 mmHg, systemic hematocrit (Hct) > 45%, and arterial PO2 pressure > 60 mmHg; and 2) microscopic examination of the tissue in the chamber observed under x650 magnification did not reveal signs of edema or bleeding. For systemic parameters, MAP and heart rate (HR) were recorded continuously (MP 150, Biopac System).

Tourniquet ischemia reperfusion (IR): Ischemia was induced for one-hour tourniquet clamp occlusion of the tissue in the window chamber model. Briefly, the periphery of the window was occluded by pressing a thin flat rubber ring (circular clamp). Mice restrained in a Plexiglas tube during ischemia [30]. Flow obstruction was induced by slowly tightening a precision threaded screw fixed to the side of the intact skin of the window chamber. The rubber ring was pressed against the intact skin and towards the cover-glass. Microvascular flow was continuously monitored under trans-illumination, until it ceased in all feeding and draining microvessels leading in and out of the clamped area without compression injury. The chamber was checked during the clamping period to ensure (no flow) that ischemia was maintained.

Experimental Setup: The animals were restrained in a tube and the protruding window chamber was fixed to the microscopic stage for trans-illumination (BX51WI, Olympus). Measurements were carried out using a 40X (LUMPFL-WIR, numerical aperture 0.8, Olympus) water immersion objective. Detailed mappings were made of the chamber vasculature so that the same vessels studied at baseline could be followed throughout the experiment. Six to eight arterioles and venules were selected in each preparation. Fields of observation and vessels were chosen for study at locations in the tissue where the vessels were in sharp focus. Leukocyte-endothelium interaction was studied in all the vessels included in the study.

Microhemodynamics: A video image-shearing method was used to measure vessel diameter (D) [31]. Changes in arteriolar and venular diameter from baseline were used as indicators of a change in vascular tone. Arteriolar and venular centerline velocities were measured on-line using the photodiode cross-correlation method (Photo Diode/Velocity Tracker Model 102B, Vista Electronics). The measured centerline velocity (V) was corrected according to vessel size to obtain the mean RBC velocity [32]. Blood flow (Q) was calculated from the measured values as Q = π × V (D/2)2.

Functional capillary density (FCD): Capillaries were considered functional if red blood cells (RBCs) transit through the capillary segments during a 45 s period. FCD was tabulated from the capillary lengths with RBC transit in an area comprised of 10 successive microscopic fields (420 × 320 μm²). FCD (cm^-1) is the total length of RBC-perfused capillaries divided by the area of the field of view.

Leukocyte-endothelium interaction: Leukocytes were labeled by intravenous injection of Texas Red anti-CD45 antibodies (10 μg, CalTag, catalog# MCD4517). Fluorescently-labeled leukocytes were excited and images were captured with a Vivid Standard (XF42 filter, Omega Filters) using a low light camera (ORCA 9247, Hamamatsu). Briefly, a straight portion of blood vessels was illuminated for 60 s and video was recorded (10 frames/s). Leukocytes were counted during video playback in a 100 μm length segment and categorized according to their flow behavior as “free-flowing”, “rolling” on the endothelium, and “immobilized” cells.

Tissue viability: Equal volumes of Annexin V (Alexafluor 488 conjugate; Molecular Probes) and propidium iodide (PI, 0.2 mg/mL, Molecular Probes) were mixed, and injected 30 min before visualization by intravital microscopy (8 h after the exchange transfusion). Microscopic images were obtained with a low light video camera (ORCA 9247, Hamamatsu). Labeled cells were counted in the skin fold window and data are given as the average number of fluorescent cells counted in 40 selected visual fields (210 × 160 μm). Sebaceous glands and hair follicles excluded from the cell counts due to their consistently high necrosis and apoptosis rate.