Correlation of 133Xe 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 angiogenesis by using a simple 133Xe clearance technique for repeated measurement of relative blood flow changes through s.c. sponge implants over a period of 14 days. The quantitative requirement of this bioassay is that the measurements of 6-minute 133Xe 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 133Xe clearance technique as an indicator of angiogenesis is needed.


METHODS
Four different techniques were used: (a) to measure absolute blood flow in the sponges using 113Sn microspheres; (b) to quantitate the levels of hemoglobin and total protein in the implants; (c) to determine the amount of neovasculature in the sponges by the carmine dye method; and (d) to carry out histologic and morphometric analysis of sponge implants. To confirm parallel changes in 133Xe clearance and in the other techniques, the effects of selected angiogenic promoters and inhibitors were also investigated.


RESULTS
There was a good correlation between 133Xe clearance from the sponges and absolute blood 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 carmine 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 implants. Daily administration of 3 pmol of IL-1 alpha or IL-8 caused intense 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 higher dose of IL-1 alpha (3 pmol) acting alone. In contrast, daily co-administration of a potent and selective protein kinase C inhibitor, calphostin C (4 micrograms), inhibited the neovascular response elicited by 3 pmol of IL-1 alpha. Furthermore, daily doses of 5 micrograms of dexamethasone for 14 days inhibited sponge-induced angiogenesis.


CONCLUSIONS
The results clearly show that the 133Xe clearance technique not only gives an indication of the rate of perfusion of the sponges with blood but also gives a good estimate of its functional vascularity. Thus, the measurement of 133Xe clearance in the sponge implant provides a simple and objective method for routine studies of modifiers of angiogenesis.
Hu
D E
DE
Department of Pharmacology, University of Cambridge, United Kingdom.
Hiley
C R
CR
Smither
R L
RL
Gresham
G A
GA
Fan
T P
TP
D000818:Animals; D003907:Dexamethasone / Q000494:pharmacology; D007375:Interleukin-1 / Q000502:physiology; D016209:Interleukin-8 / Q000502:physiology; D009281:Naphthalenes; D009389:Neovascularization, Pathologic / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D011083:Polycyclic Compounds / Q000494:pharmacology; D019736:Prostheses and Implants; D051381:Rats; D012039:Regional Blood Flow / Q000502:physiology; D015503:Surgical Sponges; D014980:Xenon Radioisotopes
D007375:Interleukin-1; D016209:Interleukin-8; D009281:Naphthalenes; D011083:Polycyclic Compounds; D014980:Xenon Radioisotopes; D003907:Dexamethasone; C058819:calphostin C