CORRELATION OF XE-133 CLEARANCE, BLOOD-FLOW AND HISTOLOGY IN THE RAT SPONGE MODEL FOR ANGIOGENESIS - FURTHER-STUDIES WITH ANGIOGENIC MODIFIERS

BACKGROUND: We have previously described a method of quantitating angi ogenesis by using a simple Xe-133 clearance technique for repeated mea surement of relative blood flow changes through s.c. sponge implants o ver a period of 14 days. The quantitative requirement of this bioassay is that the measurements of 6-minute Xe-133 clearance should provide a fast and reliable means to detect relative blood flow changes in the neovasculature, so a more vigorous validation of the use of the Xe-13 3 clearance technique as an indicator of angiogenesis is needed. EXPER IMENTAL DESIGN: Four different techniques were used: (a) to measure ab solute blood flow in the sponges using Sn-113 microspheres; (b) to qua ntitate the levels of hemoglobin and total protein in the implants; (c ) to determine the amount of neovasculature in the sponges by the carm ine dye method; and (d) to carry out histologic and morphometric analy sis of sponge implants. To confirm parallel changes in Xe-133 clearanc e and in the other techniques, the effects of selected angiogenic prom oters and inhibitors were also investigated. RESULTS: There was a good correlation between Xe-133 clearance from the sponges and absolute bl ood flow (r = 0.952, p < 0.01); the levels of hemoglobin (r = 0.982, p < 0.01) and total protein (r = 0.962, p < 0.01); the amount of carmin e dye (r = 0.974, p < 0.01); the fibrovascular growth areas (r = 0.992 , p < 0.01); and the vascular density (r = 0.997, p < 0.01) in the imp lants. Daily administration of 3 pmol of IL-1 alpha or IL-8 caused int ense neovascularization. When given alone, lower doses of IL-1 alpha ( 0.3 pmol) or bradykinin (10 pmol) produced no apparent effect. However , co-administration of these doses to a single sponge together caused an increase in the rate of angiogenesis similar to that seen with a hi gher dose of IL-1 alpha (3 pmol) acting alone. In contrast, daily co-a dministration of a potent and selective protein kinase C inhibitor, ca lphostin C (4 mu g), inhibited the neovascular response elicited by 3 pmol of IL-1 alpha. Furthermore, daily doses of 5 pg of dexamethasone for 14 days inhibited sponge-induced angiogenesis. CONCLUSIONS: The re sults clearly show that the Xe-133 clearance technique not only gives an indication of the rate of perfusion of the sponges with blood but a lso gives a good estimate of its functional vascularity. Thus, the mea surement of Xe-133 clearance in the sponge implant provides a simple a nd objective method for routine studies of modifiers of angiogenesis.