An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
Homozygous eNOS-KO mice and C57BL/6 wild-type were 12 to 14 week-old. FAL was by ligation proximal to the popliteal artery and distal to the lateral caudal femoral artery (LCFA) (Figure 1A, left, green arrows, less severe model)2 (link),18 (link) or proximal to the LCFA for more severe ischemia (Figure 1A, left, red arrow). The superior epigastric artery was ligated in both models (Figure 1A, left, blue arrow). Analyses were conducted blindly. Hindlimb perfusion was obtained using a perfusion imager modified for high resolution and depth of penetration.18 (link),19 (link) “Appearance” and “use” scores were obtained.2 (link) Number of native pial collaterals interconnecting the middle and anterior cerebral artery trees was determined by imaging of yellow MicrofilP casting after heparinization, vasodilation and fixation,2 (link),18 (link) and in embryonic day (E)18.5 embryos postnatal day (P)1 pups by whole-mount immunohistochemistry with anti-NG2 antibody. Twenty-one days after FAL or after acute FAL in naïve mice, the abdominal aorta was cannulated, followed by maximal dilation, heparinization, fixation, and MicrofilP casting. Collaterals in the abductor/adductor were imaged either by high resolution x-ray arteriography,2 (link) directly by successive removal of overlying muscle fibers after alcohol-methyl salicylate clearing, or by cross-section histomorphometry (see below). Intact collaterals were identified according to the Longland criteria.20 (link) Histomorphometry for collateral diameter, capillary density and immunohistochemical staining was as detailed previously.2 (link) Proliferation was measured by 5-bromodeoxyuridine (BrdUrd) incorporation. LCFA diameter was measured by stereomicroscope and flow velocity was measured with a Doppler microprobe. Microarray analysis of gene expression was performed on microdissected anterior and posterior gracilis collaterals 24 hour after unilateral femoral ligation and after acute contralateral ligation (control) (Figure 1A, left, black arrows). For each RNA replicate, collaterals from 15 mice (30 ligated for 24 hour and 30 acutely ligated) were pooled. Three replicates for C57BL/6 and eNOS-KO each were hybridized. Real time quantitative RT-PCR was performed for representative genes in each functional gene category identified in the array studies. All data were obtained while blinded to mouse strain.