Biomedical Engineering Reference
In-Depth Information
several efflux transporters located in the retina, similar to those in
the BBB, that are largely responsible for removal of compounds
from the retina. It is this combination of selective permeability and
highly active efflux transporters [
7
,
8
], that has led to a lag in the
development of orally active compounds which can be used to treat
and prevent major ocular diseases including glaucoma and macular
degeneration. ADME analysis for ocular drug delivery will require
examining various transporters and metabolites in the retina, the
optic nerve, and optic tract targets in the brain. The following
method covers the removal of these tissues from experimental
rodents after perfusion with phosphate-buffered saline. Non-fixed
tissues (absent blood products) are necessary for downstream anal-
ysis by LC-MS/MS or HPLC.
2 Materials and Methods
1. Peristaltic pump with tubing and needle appropriate for PBS
delivery (25-18G).
2. Straight scissors for trimming fascia, fat, muscle.
3. Hemostat for grasping xiphoid process.
4. Bone cutting shears for head removal.
5. Curved toothed forceps for eye and optic nerve removal.
6. Straight smooth or 45
smooth forceps for lens and retina
removal.
7. Curved scissors for eye removal [alternative].
8. Small surgical spring scissors [Vannas, Moria, or Castroviejo]
for corneal removal.
9. Rongeurs for brain dissection.
2.1 Materials
Figure
1
shows representation of main steps to obtain tissue.
2.2 Methods:
Dissection
1. Make 0.1 M phosphate-buffered saline (PBS; pH 7.4).
2. Purge the peristaltic pump then fill its tubing with PBS. Affix a
needle (25G is sufficient for mouse, 18G for rat) to the end of
the pump tubing. This needle will be placed into the exposed
heart to deliver the PBS throughout the body, eliminating all
blood.
3. Subject the rodent to an overdose of injectable or inhalant
anesthetic.
4. Once the diaphragm has stopped contracting, but prior to cessa-
tion of the heartbeat, use straight scissors to make a horizontal
incision through the skin and then the fascia directly posterior to
the rib cage.
