Biology Reference
In-Depth Information
At microscopic level, a highly vascularized tissue is recognizable
among the trabeculae of the sponges treated with rhuEpo. The tissue
consists of newly formed blood vessels growing perpendicularly to
the plane of the CAM, mainly capillaries within an abundant network
of collagen fibers. In contrast, no blood vessels are present among
trabeculae of the implants treated with vehicle alone (Fig.
1
).
4
Notes
1. Chick embryos from different vendors can vary significantly in
their degree of vascularization and developmental status. Thus,
consistent use of the same vender can decrease experimental
variability.
2. Cleaning the egg shell before incubation will remove any debris
associated with the outer surface and decrease the risk of infec-
tion. Furthermore, specific incubation conditions, including
constant temperature and humidity, are of critical importance
for proper vascularization and embryo survival.
3. Make sure the needle opening is pointing away from the
embryo during albumen aspiration. In addition, regular
changes of needle and syringe limit the carryover of infection
from egg to egg.
4. If large pieces of shell fall onto the CAM it may possible to
remove these using fine forceps. It is probably best to discard
eggs where pieces of shell have fallen onto the CAM and have
not been removed very easily, as these eggs may develop false
positive response due to inflammation. Reject eggs with an
excessively humid CAM; otherwise, sponges may float off dur-
ing the incubation period.
5. The CAM is an expanding membrane with vessels developing
over its entire surface. It is preferable not place the sponges on
its outer edges.
6. Two operators, preferably blinded to the sample identity
should grade the angiogenic response. Different test substances
can produce a range of different types of angiogenic response,
such as a mixed response of microvascular growth and large
vessel deformation/growth towards the point of application.
Samples may also induce local bleeding and the presence and
severity of these reactions should be noted, as the response
may be secondary to the bleeding or inflammation.
Acknowledgement
Supported by European Seventh Framework Programme
(FTP/2007-2013) under grant agreement No 278570 to DR.
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