Biomedical Engineering Reference
In-Depth Information
and endothelium, (2) fewer oblong spaces in the corneal stroma,
(3) visualize lenticular cell outlines, and (4) visible outlines of
photoreceptor inner and outer segments. Potential artifacts asso-
ciated with glutaraldehyde fixation include (1) a few oblong shaped
spaces in the corneal stroma, (2) lenticular cracks, (3) distorted
shape of the cornea and lens in the globes of rodents due to
osmolarity [
77
,
86
], (4) vacuolation of the inner layers of the retina,
and (5) vacuolation in the layer of photoreceptor inner and outer
segments. There appears to be fewer artifacts associated with pro-
longed fixation in glutaraldehyde, but in general rodent globes are
fixed for 12-24 h and larger globes (i.e., rabbit, dog, monkey) for
24-48 h. After the initial fixation, globes should be transferred to
10 % NBF for a minimum of 24 h to help firm the outer tunics (i.e.,
cornea and sclera) for trimming (especially for the globes of rabbits
and monkeys).
As mentioned earlier, emersion fixation of the larger, non-
rodent globes may need to be enhanced for a fixative, such as
glutaraldehyde, but is not needed for Davidson's fixative or Mod-
ified Davidson's fixative. Importantly, no holes should be created in
a globe that has not been in fixative. Instead the globe should be
placed in fixative for 5-30 minutes and then a small window may be
created near the equator to allow better penetration of fixative into
intraocular structures.
Another method is the intravitreal injection of fixative. This
may be accomplished by use of a small (25-27 ga) needle gently
introduced into the vitreous cavity through the sclera just posterior
to the equator. The needle should be angled toward the posterior
pole to avoid hitting the lens and should be performed in an area
that is 90
away from the plane of section to avoid seeing the
injection area. For larger, nonprimate globes, this is at a nasal or
temporal location. For primate globes this is at a superior or inferior
location. With one hand holding the globe, the fixative should be
slowly injected until the globe feels firm. This usually involves a
volume of 0.15-0.3 ml of fixative. The injected globe should then
be immersed in the fixative. When done gently, artifacts, such as
retinal detachment, are not observed.
There are many references for trimming globes and the method
used will depend on the purpose of the toxicity study [
10
-
13
,
78
,
79
]. For standard systemic toxicity studies using globes from
dogs, minipigs, and rabbits, generally one section of the globe
through the optic disc and pupil that is parallel to a vertical
median (midsagittal) plane is usual. If the test animal is a primate,
the desired plane of section is one that is slightly superior to a
horizontal plane in order to have the temporal macula and the
optic disc in the same section. Although it would be ideal to have
2.7.3 Trimming for
Paraffin Embedding
