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In vivo microscopy of rat skeletal muscle after ischemia using labelled neutrophils (PMN)
M Richardson1, A Berker, A Roberts
1UCLA Medical Center.
Abstract:
Neutrophils (PMN) have been implicated as mediators of the "no-reflow" phenomenon seen in skeletal muscle during reperfusion after ischemia. In order to evaluate the PMN contribution to the changes seen in the microcirculation of skeletal muscle after ischemia, we evaluated PMN velocity using in vivo microscopy in a rat model. After the induction of anesthesia, the right iliac and femoral arteries were isolated. The right anterior tibialis muscle was exposed in situ, covered with a plexiglass disc, and perfused with Kreb's solution. Fluorescein-labeled bovine albumin was given intravenously, which identified the capillaries under microscopic magnification as viewed on the video screen. Acridine orange was then administered intravenously, which selectively fluoresced the PMN. The right iliac and femoral arteries were clamped for ischemia intervals of 5, 10, 15, 20, 25, and 30 min. Acridine orange was given immediately after the arteries were unclamped, after 30 min of reperfusion and after 60 min of reperfusion. PMN velocity was determined by the distance traveled by the PMN over time using the videotape and frame-by-frame review. Results demonstrated no change in PMN velocity (mm/sec) after 5 min of ischemia. After 10, 15, and 20 min of ischemia, PMN velocity initially slowed and then recovered, which was not statistically significant. After 25 min of ischemia, PMN velocity decreased significantly, which persisted (P < 0.05 compared to 5-min ischemia by ANOVA). No flow was seen after 30 min of ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Neutrophil (PMN) velocity in skeletal muscle slows significantly after 25 minutes of ischemia, contributing to the no-reflow phenomenon during reperfusion. This study quantifies PMN behavior in a rat model.
Area of Science:
- Physiology
- Microcirculation Research
- Ischemia-Reperfusion Injury
Background:
- Neutrophils (PMN) are implicated in the no-reflow phenomenon in skeletal muscle post-ischemia.
- Understanding PMN dynamics is crucial for addressing microcirculatory dysfunction.
Purpose of the Study:
- To evaluate the contribution of neutrophils to microcirculatory changes in skeletal muscle after ischemia.
- To quantify neutrophil velocity in vivo during reperfusion following varying ischemia durations.
Main Methods:
- Utilized a rat model with in vivo microscopy of the anterior tibialis muscle.
- Ischemia was induced via iliac and femoral artery clamping for 5-30 minutes.
- Neutrophils were fluorescently labeled with acridine orange for velocity measurement via videotape analysis.
Main Results:
- No significant change in neutrophil velocity was observed after 5 minutes of ischemia.
- Transient, non-significant slowing of neutrophil velocity occurred after 10-20 minutes of ischemia.
- A significant decrease in neutrophil velocity was noted after 25 minutes of ischemia, persisting through reperfusion. No flow was observed after 30 minutes.
Conclusions:
- Neutrophil velocity reduction is a significant factor in skeletal muscle microcirculation impairment after prolonged ischemia.
- The findings support the role of neutrophils in the no-reflow phenomenon, particularly after 25 minutes of ischemia.
- Targeting neutrophil activity may be a therapeutic strategy for ischemia-reperfusion injury.