Biology Reference
In-Depth Information
such as a laminar flow hood to minimize the risk of infection
(see Note 4).
5. On day 8 of incubation, open the window under sterile condi-
tions within a laminar-flow hood and implant a 1-mm
3
sterilized
gelatin sponge onto the CAM. For this purpose, the sponge is
cut by hand with a blade and gently placed on top of the grow-
ing CAM. Use sterile gloves to minimize the risk of infection
(see Note 5).
6. Pipette rhEpo (dissolved in 1-3 ml of sterile routine tissue
culture medium at doses ranging between 10 and 50 U/
implant) onto the sponge. Sponge containing vehicle alone is
used as negative control.
7. On incubation day 12, macroscopic observation shows that
the gelatin sponge treated with rhuEpo is surrounded by allan-
toic vessels that develop radially towards the implant in a
“spoked-wheel” pattern. Macroscopic evaluation of the vaso-
proliferative response can be performed as follows: for each
egg, the total number of vessels converging towards the graft
can be counted under the stereomicroscope at 10× magni fication
at different time points after implantation from day 8 to day
12. Then, for each experimental group (
n
³10) data are
expressed as mean ± SD and kinetics curves can be obtained for
proangiogenic stimulus compared to controls (see Note 6).
8. Fix the embryos and their membranes in ovo by pipetting 10 ml
of Bouin's fluid solution on the CAM surface and fix for 3 h at
room temperature.
9. Cut the sponges, the underlying and the immediately adjacent
CAM portions with curved tip-forceps and process the speci-
men for paraffin embedding.
10. Deydrate tissue samples in ethanol, cleared in toluene and
immerse in the embedding paraffin for 2 h, according to stan-
dard procedure.
11. Cut eight-micrometer serial sections of paraffin-embedded
CAMs in parallel to the surface of the membrane, stain sections
with a 0.5% aqueous solution of toluidine blue for 1 min at
room temperature and observe under a light microscope.
12. Microscopic evaluation of the angiogenic response. Quantitative
evaluation of the angiogenic response, expressed as microvessel
density, can be obtained by applying a morphometric method
of “point counting” on histological CAM sections. Briefly, two
investigators simultaneously identify transversally cut microve-
ssels (diameter ranging from 3 to 10 mm) among the gelatin
sponge trabeculae with a double headed photomicroscope at
250× magnification. A square mesh consisting of 12 lines per
side, giving 144 intersection points, is inserted in the eyepiece.
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