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In vivo differentiation of leukocytes rolling in mesenteric postcapillary venules
G J Tangelder1, C J Janssens, D W Slaaf
1Department of Physiology, University of Limburg, Maastricht, The Netherlands.
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 (13-366 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.
Insights
This study presents a new method to identify rolling leukocytes in transparent tissues. The findings reveal that nearly all rolling leukocytes in rabbit mesenteric venules are polymorphonuclear cells (granulocytes).
Area of Science:
- Immunology
- Microcirculation Research
- In Vivo Imaging
Background:
- Leukocyte rolling in microvessels is a critical step in inflammatory responses.
- Identifying specific leukocyte subtypes involved in rolling is essential for understanding these processes.
- Current methods may lack the resolution or specificity for in vivo subtype analysis.
Purpose of the Study:
- To develop and validate a method for assessing leukocyte subtypes rolling in vivo within transparent tissues.
- To determine the predominant leukocyte subtype involved in microvessel rolling in rabbits.
- To evaluate the safety and efficacy of the staining method on physiological parameters.
Main Methods:
- Utilized intravital fluorescence video microscopy in anesthetized rabbits.
- Administered acridine yellow dye intravenously to stain leukocyte nuclei in situ.
- Classified rolling leukocytes based on nuclear morphology into polymorphonuclear (PMN) and monomorphonuclear types.
- Analyzed rolling leukocyte percentages, vessel parameters, and hemodynamic data.
Main Results:
- Acridine yellow effectively labeled all rolling leukocytes in mesenteric venules.
- Over 94% of classified rolling leukocytes were identified as PMNs (granulocytes).
- This PMN predominance was consistent across various rolling rates, vessel sizes, and flow velocities.
- The dye did not significantly alter hemodynamic parameters or systemic leukocyte counts.
Conclusions:
- The developed method accurately identifies leukocyte subtypes rolling in microvessels in vivo.
- Leukocyte rolling in rabbit postcapillary venules is almost exclusively mediated by granulocytes.
- This technique offers a valuable tool for studying inflammatory cell dynamics in real-time.