A nonocclusive silicone cuff placed around the rabbit carotid artery results in a diffuse intimal thickening. The early stages of this phenomenon were studied by light microscopy, immunohistochemistry, and electron microscopy. Neointimal formation appeared to be triphasic. The first phase started 2 hours after cuff placement, with vascular infiltration by polymorphonuclear leukocytes (PMNs). In the second phase, starting within 12 hours, 1.90±0J6% of the medial smooth muscle cells (SMCs) were

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... plicating, as demonstrated by their immunoreactivity for proliferating cell nuclear antigen (PCNA). The third phase was characterized by the appearance, from day 3 onward, of subendothelial SMCs that were immunoreactive for or-SMC actin and vimentin. A few cells showed immunoreactivity for PCNA. During this phase all the PMNs disappeared, but SMC replication in the media was still present, as indicated by the presence of mitoses and the persisting immunoreactivity for PCNA (0.76±0.22% at day 7). In the third phase the number of subendothelial cells increased (104±15 SMC nuclei per section at day 7, of which 8.89±2.26% were PCNA-positive) and was associated with deposition of collagen type IV and flbronectin. At 14 days a complete, circular neointima was present and contained 2.13 ±0.28% replicating SMCs. The media showed 0.44±0.08% cell-cycling SMCs, which was still four times higher than normal. During the first week there was also a significantly higher PCNA activity in the media of sham-operated carotid arteries (no cuff present) than in nonsurgical ones. However, this did not lead to the formation of a neointima. We conclude that in the cuff system SMC replication in the media precedes the neointimal formation. The system can be used to study SMC replication, migration, and neointimal formation with minimal medial SMC damage. (Arteriosclerosis and Thrombosis 1992;12:1447-1457) KEY WORDS • intima • smooth muscle cells • experimental atherosclerosis • proliferating cell nuclear antigen Methods Experimental Design Male New England White rabbits (2.5-3 kg) were anesthetized with sodium pentobarbital (30 mg/kg body wt i.v.), and both carotid arteries were exposed and dissected from the surrounding tissues. A nonocclusive, biologically inert, soft and flexible silicone cuff (20 mm long, 4.7-mm inner diameter; Silastic 732, Dow Corn-by guest on