In vivo differentiation of leukocytes rolling in mesenteric postcapillary venules

Abstract

A method is presented to assess in vivo in transparent tissues the leukocyte subtypes that roll in microvessels. In nine rabbits anesthetized with ketamine-xylazine, leukocyte nuclei were stained in situ with acridine yellow (3 mg/kg i.v. for 5 min). Intravital fluorescence video microscopy in 24 mesenteric venules (17–29 microns, median 21) indicated labeling of all rolling leukocytes. On the basis of the shape of their nucleus, 67–100% (median 89) could be classified unequivocally (13366 cells analyzed, median 77) as polymorphonuclear (PMN, i.e., granulocytes) or monomorphonuclear (lymphocytes and monocytes). Of these classified cells, 94–100% were PMNs (median 100, including 1 stray value of 69%). This PMN percentage was independent of the level of leukocyte rolling (2–36/min, median 14), vessel diameter, flow velocity (0.5–2.5 mm/s), or duration of the experiment (< 6 h). The dye had no significant influence on hemodynamic parameters, systemic leukocyte counts (1.5–7.8 x 10(9)/l), or in vitro differentiation pattern (27–38% granulocytes, 0–2% monocytes, 61–71% lymphocytes). In conclusion, our method demonstrated that the leukocytes that roll in postcapillary venules of the exteriorized rabbit mesentery are almost exclusively granulocytes.