Biomedical Engineering Reference
In-Depth Information
6. Iris/Ciliary Body/Retina/Choroid
The pigment melanin is found in several tissues of the eye
including those of the uveal tract (iris, ciliary body, and cho-
roid) and the retina. Melanin may play a critical role in drug
binding and has been associated with long residence times,
drug efficacy, and possible ocular toxicity [
57
]. Melanin bind-
ing, however, may not be predictive of ocular toxicity [
58
].
Melanin is a polymer composed of dihydroxyindolyl and dihy-
droxyindole-2-carboxyl units and is ultimately derived from
tyrosine [
59
,
60
]. As such, melanin has multiple sites contain-
ing hydroxyl and carboxylic acid groups, which can interact
with drugs, especially basic drugs [
58
,
59
]. Examples of drugs
known to bind to melanin in the eye include ephedrine, atro-
pine, chlorpromazine, timolol, chloroquine, and levofloxacin
[
57
,
59
,
61
]. When developing bioanalytical assays for iris,
ciliary body, choroid, or retina, the drug binding characteristics
of melanin must be taken into consideration. Drug binding to
melanin can sequester the drug, making it unavailable for
analysis. This may not be of consequence when melanin-
containing tissues are used to generate calibration curves as
long as drug binding is comparable across the concentration
range of the assay and when sensitivity is sufficient. However,
when using a surrogate matrix for the calibration curve (includ-
ing tissues from non-pigmented animals such as New Zealand
White rabbits), recovery from the pigmented tissues and surro-
gate matrix may be different and this must be accounted for
when developing the assay. The use of high concentrations of
salts (such as NaCl or MgCl
2
) or other agents to disrupt the
melanin-drug binding may be required for the development of
an appropriate assay [
60
].
Proper sample collection is critical for ocular bioanalytical studies.
Improper sample collection procedures can result in cross contami-
nation of tissues or fluids which will compromise the value of even
the best bioanalytical techniques. The main challenges encountered
during sample collection are the following:
2.2 Eye Dissection
and Ocular Tissue
Collection
1. The biological ocular barriers that stop foreign substances
including drugs from penetrating eyes or traveling across vari-
ous tissues within the eye are destroyed immediately upon
euthanasia of the animal, allowing drugs to equilibrate across
tissues within the eye. As mentioned earlier, to prevent this
equilibration, eyes need to be enucleated immediately upon
euthanasia and flash frozen in liquid nitrogen or in a dry ice/
alcohol bath as soon as possible. The eyes must be maintained
in a frozen state during dissection to prevent or minimize drug
diffusion to adjacent tissues.
2. As mentioned previously, ocular tissues contain several
drug-metabolizing enzymes. These may provide a challenge
