Biomedical Engineering Reference
In-Depth Information
When these preparations are on the microscope stage, various conditions can be investi-
gated to determine the effects on flow properties or the blood cells. For instance, various
agonists can be added to the tissue perfusate, which is used to keep the tissue moist dur-
ing the experiments, to determine how the addition of a compound to the entire vascular
network affects that vascular bed (e.g., flow through it, communication along it, among
others). For clarification, this perfusate bathes the entire dissected tissue so that it does not
dry out. Additionally, micropipette techniques can be used to localize the injection of ago-
nists to determine the endothelial cell communication throughout the vascular network
and how that communication affects flow conditions. Changes in the vascular network,
such as vessel diameter, flow rate, and cell volume, can all be investigated with intravital
microscopy. Therefore, there are many possible outcomes for this type of research.
In recent years, the information that has been collected from intravital microscopy has
improved significantly with better optics, better cameras, and better markers (targeting,
longevity, and optical properties). Better optics/cameras have allowed the researchers to
target smaller capillary bed segments more accurately, as well as small blood vessels. The
use of fluorescent markers has allowed researchers to target specific compounds, specific
cell receptors, and other indicators to understand the changes that occur within the vascu-
lar bed down to the molecular level. As an example, many cellular processes are regulated
by the movement of calcium through a single endothelial cell and by the calcium move-
ment through gap junctions connecting neighboring endothelial cells. By using fluorescent
markers that can target intracellular calcium compartments and/or intracellular calcium
signaling molecules, intravital microscopy can investigate physiological and pathological
events instead of just physical parameters of the vascular bed. This includes changes in
blood cell flux, hematocrit, intracellular ion concentrations, and the expression of cell
membrane-bound receptors. Also, the adhesion/rolling of white blood cells onto the endo-
thelial cells membrane can be investigated with intravital microscopy. Therefore, many
physiological and pathological conditions such as ischemia/reperfusion, inflammatory
responses, endothelial cell barrier integrity, graft success, and nutrient delivery can all be
investigated with this technique.
A standard intravital microscopy apparatus (
Figure 15.1
) consists of an epi-fluorescence
upright (or inverted) microscope, which is connected to a camera, and then feeds directly
into a recording device and a live video monitor. A perfusion system is needed to keep
the tissue moist during the experiment. An anesthesia controlling system is needed to
make sure that the animal is kept under anesthetic during the entire experiment, which
can last 6 to 8 hours. It is critical to choose anesthetics that do not alter vascular properties.
A micropipette system can be used to locally inject antagonists within the vascular bed or
they can be injected via the perfusate. Data collected from this apparatus are typically fed
into an image analysis system which can be used to accurately quantify the particular data
of interest from the experiment.
Intravital microscopy is a very powerful research tool to investigate changes to vascular
networks under multiple conditions. The importance of this method is that the vascular
network that is under investigation remains connected to the animal and is under physio-
logical regulation from the animal. Many groups use this technique to understand the
development of diseases and fundamental vascular physiology because as described
above, various conditions can be applied to the entire tissue or locally to individual cells.
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