Arteriogenesis depends on circulating monocytes and macrophage accumulation and is severely depressed in op/op mice.

It has been suggested that monocytes/macrophages represent the pivotal cell type during early adaptive growth of pre-existent arterial anastomoses toward functional collateral arteries (arteriogenesis) upon arterial occlusion. This hypothesis was supported by previous studies providing evidence that elevation of the peripheral monocyte count, increased monocyte survival (e.g., granulocyte macrophage-colony stimulating factor), as well as enhanced attraction or adhesion (e.g., monocyte chemoattractant protein 1; intercellular adhesion molecule 1) of the latter cells correlates directly with the arteriogenic response to restore tissue perfusion. However, the experimental proof of the essential role of monocytes/macrophages remains to be given. We therefore hypothesized that arteriogenesis is reduced in a genuine, nonpharmocologically induced monocyte/macrophage-deficient model of femoral artery occlusion in osteopetrotic (op/op) mice. Total leukocyte count did not differ between op/op mice and control (B6C3Fe a/a-Csf1(+/+)) mice. op/op mice show a significant monocytopenia (0.67%+/-0.38% vs. 1.53%+/-0.32%), granulocytosis (33.66%+/-6.67% vs. 22.83+/-7.47%), and a concomitant, relative lymphopenia (65.67%+/-6.58% vs. 75.65%+/-7.31%). Microsphere-based perfusion measurement 7 days after femoral artery occlusion demonstrated a significantly reduced perfusion restoration upon femoral artery occlusion in op/op mice as compared with controls (28.19%+/-0.91% vs. 47.88%+/-2.49%). The application of a novel method of high resolution (microfocus X-ray system) angiography revealed a reduction of proliferation and diameter of collateral arteries. Quantitative immunohistology showed significantly lower numbers of macrophages in the surrounding tissue of proliferating arteries. This study provides additional evidence for the preeminent role of monocytes/macrophages during arteriogenesis in a genuine model of monocyte deficiency, i.e., without pharmacological intervention.

Quantification of collateral artery growth by automated fluorescent microsphere perfusion.

BACKGROUND: Since their introduction, genetically modified mice have become more and more important to examine molecular mechanisms involved in vascular growth. Today the gold standard for measuring vessel conductivity is to directly assess in vivo perfusion. However, this usually becomes more complicated the smaller the animal, especially due to the need for extensive instrumentation and requirement of maximal vasodilation. METHODS: We developed an automated system that allows pressure-controlled in vivo perfusion of small animals with differently labeled fluorescent microspheres. RESULTS: Besides precise operation of the system (mean pressures divergence 0.08%), automation of small animal microsphere perfusion is reliable and highly accurate in mice with and without femoral artery occlusion. In sham-operated control mice, which did not undergo femoral occlusion, highly reproducible measurements of hind limb perfusion (right vs. left=1.03 ± 0.037) could be assessed. In mice after unilateral femoral artery occlusion, mean perfusion ratios of the automated method (ratio occluded vs. non-occluded hind limb=0.598 ± 0.046) were comparable to the manual approach (0.561 ± 0.062). However, inter-individual variances were significantly smaller with the automated system. CONCLUSION: We describe here a novel and innovative technical approach for pressure-controlled fluid handling specifically designed for microsphere perfusion measurements in small animals.