Vasospasm: Measurement of Diameter, Perimeter, and Wall Thickness - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 454

Take photos with microscope camera and save in tagged image fi le
format.

Alternatively, use a slide scanner that will scan the specimens
onto an optical disc. For these systems, the software program usually
allows viewing the specimen on the computer, taking pictures and
adjusts scale magnifi cation automatically.

3.5. Assessing
Vasospasm

Most image analysis software can perform this task, one of the
most common measuring softwares is Image J (available free from
the NIH).
Open Image J and drag and drop image onto the Image J tool

bar.

Adjust and measure scale bar, by drawing a line across the scale.
Subsequently, go to edit option on the toolbar 6 adjust scale bar 6
and write down the length of the scale bar (2-3 cm) corresponding
with the pixel dimensions.
Click on the free-style line icon, and draw along the lumen of
the artery under high magnifi cation. An alternative method would
be utilizing the magic tool, which automatically selects the lumen
area accurately, if enough contrast is seen in the image.
Alternatively, the perimeter is calculated initially in a similar fash-
ion to the measurement of area. The area of the artery and its radius
(area = pr 2 , where r = radius) are calculated from the perimeter value
(2 pr ) that are measured with image J. This alternative method pro-
vides an accurate approximation of the lumen areas as it would correct
for potential errors in vessel deformity. If the artery is not sectioned
perpendicular to its long axis, however, errors could occur.
Click Ctrl + M, and a new window will open up with the
measurements of the lumen area/perimeter in microns (Fig. 2 ,
one can repeat the step three times and take the average for an
accurate assessment).
To measure the thickness of the wall, select the straight line
icon and draw a line from the endothelial layer 6 tunica media and
usually not the adventitial layer (which could be irregular and not
accurately sectioned due to the fragility and variability of the con-
nective tissue). Repeat in four different regions, adjacent to each
other (90°) (Fig. 2 ).
Select all the data in the data viewer, copy and paste into a
spreadsheet. Perform analysis, by measuring the mean of the lumen
area in all samples—the standard deviation or standard error and
the average of all four measurements of the wall thickness.
Data can be expressed as separate graphs or one graph depending
on what the researcher wishes to convey. Two separate graphs can
be used to depict lumen diameter or cross-sectional area and
another as wall thickness. If there are appropriate controls, this
would demonstrate vasoconstriction/vasodilation as well as wall
remodeling. Another way to show the data would be wall thickness/
lumen diameter which increases with increasingly severe vasospasm.