Fig. 7.

Five-day neurovascular co-culture. (A, B) Fluorescent images of tyrosine hydroxylase sympathetic axons growing towards femoral (A) or carotid (B) artery segments for five days in co-culture. In femoral–SCG co-cultures axons grew out to, but did not pass the vessel in 6/7 co-cultures. In addition, the neurons appeared to make contact with and wrap around the vessel, forming a network parallel and perpendicular to the axis of the vessel similar to that seen in ex-vivo whole mount preparations (data not shown). Scale bar=200 μm. (C, D) Confocal images of tyrosine hydroxylase immunofluorescence of SCG explants cultured with femoral (C) or carotid (D) artery segments for five days in 2.5 mg/ml collagen. Neurons comprising the femoral network were arranged both parallel and perpendicular to the vessel axis, investing the vessel, while neurons comprising the carotid network were arranged mainly perpendicular to the vessel axis with occasional axons migrating past the vessel segment (arrows) in 5/7 co-cultures. Scale bar=200 μm. (E, F) Merged images of a five-day co-culture of a femoral artery segment with a SCG (E) and a segment of femoral artery cultured alone (F), illustrating synaptophysin staining (green fluorescence) and tyrosine hydroxylase staining (red fluorescence) of presynaptic varicosities, revealing co-localization of a portion of the varicosities suggestive of sympathetic re-innervation (orange fluorescence) in (E) and no detectable tyrosine hydroxylase staining in the mono-cultured femoral artery segment (F). Scale bar=100 μm. (G, H) High power magnification insets of (E) and (F) illustrating specific synaptophysin labeled varicosities (green fluorescence denoted by black arrows) and synaptophysin/tyrosine hydroxylase co-labeled varicosities (orange fluorescence denoted by white arrows) in (G) and specific synaptophysin labeled varicosities (green fluorescence denoted by black arrows) in the monoculture in(H). Scale bar=25 μm.