Biomedical Engineering Reference
In-Depth Information
Although 10 % neutral-buffered formalin (NBF) is used in the
fixation of extraocular tissues of animals for toxicity studies, it is
not ideal for immersion fixation of globes and may result in artifacts
(e.g., sensory retinal separation) [
25
]. Since there is no ideal fixative
for ocular fixation, several different fixatives are used on a routine
basis (Table
1
)[
10
-
13
,
79
,
80
]. Ocular fixatives are the ones in
which the globes are entirely immersed after enucleation (e.g.,
Zenker's, Bouin's, Davidson's, or Modified Davidson's fixatives)
or a fixative, like glutaraldehyde (e.g., 4 % glutaraldehyde mixed 1:1
with 10 % NBF), that involves an intravitreal injection of the fixative
or initially submerged in the fixative for a brief (5-30 min) period of
time then having a small (5 mm) window created. With this type of
fixation, the globe is resubmerged for the set period of time.
Regardless of the fixative used, the volume of globe to fixative
should be at least 1:10 and wide mouth jars with the name of the
study, number of the animal, and what globe (right or left) should
be used. Gauze may be used to cover the globes to ensure that they
stay submerged in the fixative.
Zenker's and Bouin's fixatives have been used in the past for
fixation of globes, but require more involvement in processing of
the ocular tissue and have issues with disposal. Zenker's fixative
contains mercury and Bouin's fixative contains picric acid that is
corrosive, potentially explosive, and is difficult to completely
remove from the tissue [
25
]. These fixatives have generally been
replaced with Davidson's fixative or Modified Davidson's fixative
[
11
,
21
,
78
,
79
,
81
]. These fixatives are used only for fixation for
light microscopy and none are used for electron microscopy.
Davidson's fixative or Modified Davidson's fixative is often
used for the fixation of rodent globes generally with good results.
Fixation time is dependent upon the size of the globe [
82
,
83
]. For
globes of rodents, the time is 6-24 h, but for globes of larger
animals (i.e., rabbit, dog, monkey) the time is 24-48 h. Globes
may be then transferred to 10 % NBF (or 70 % alcohol, but excess
exposure to alcohol will harden the lens) until the time of
trimming. During this period, the cornea and sclera become firm
which helps during trimming. Excess exposure to these fixatives
may result in artifacts which may include (1) diffuse opaque gross
appearance of globes of albino rats and mice, (2) vacuolation of the
corneal epithelium, (3) oblong spaces in the corneal stroma, (4)
vacuolation of the corneal endothelium, (5) shattering of the lens,
(6) swelling of the lens with rupture of the lens capsule, (7) swelling
of lens fibers, (8) fragmentation and globule formation in the lens
of monkeys, and (9) indistinct appearance of photoreceptor inner
and outer segments. An advantage of Davidson's fixative and
Modified Davidson's fixative is the enhancement of lenticular cell
outlines.
2.7.2 Fixation for Light
Microscopy
