Objective—To develop a computer-assisted image
analysis procedure for quantitation of neovascularization
in formalin-fixed paraffin-embedded specimens
of thyroid gland tissue from dogs with and
without thyroid gland neoplasia.
Sample Population—47 thyroid gland carcinomas,
8 thyroid gland adenomas, and 8 specimens of thyroid
tissue from dogs without thyroid gland abnormalities
Procedure—Serial tissue sections were prepared
and stained with antibodies against human CD31 or
factor VIII-related antigen (factor VIII-rag). The areas
of highest vascularity were identified in CD31-
stained sections, and corresponding areas were
then identified in factor VIII-rag-stained sections.
Image analysis was used to calculate the total vascular
density in each section, and neovascularization,
expressed as a percentage, was determined
as the absolute value of the total vascular density
derived from factor VIII-rag-stained sections minus
the vascular density derived from CD31-stained
Results—Mean vascular density of thyroid gland
carcinomas derived from CD31-stained sections
was significantly greater than density derived from
factor VIII-rag-stained sections. This incremental difference
was presumed to represent degree of neovascularization.
However, significant differences
were not detected between vascular densities
derived from CD31 and factor VIII-rag-stained sections
for either normal thyroid gland tissue or thyroid
gland adenomas. No significant correlations
were found between vascular density in thyroid
gland carcinomas and survival time following
Conclusion and Clinical Relevance—A computerassisted
image analysis method was developed for
quantifying neovascularization in thyroid gland
tumors of dogs. This method may allow identification
of dogs with tumors that are most likely to
respond to treatment with novel antiangiogenesis
agents. (Am J Vet Res 2002;63:363–369)
Objective—To determine expression of cyclooxygenase (COX) genes 1 and 2 (also called prostaglandin-endoperoxide synthases 1 and 2) and stability of housekeeping gene expression during low-flow ischemia and reperfusion in the jejunum of horses.
Animals—5 healthy adult horses.
Procedures—Horses were anesthetized, and two 30-cm segments of jejunum were surgically exteriorized. Blood flow was maintained at baseline (untreated) values in 1 (control) segment and was decreased to 20% of baseline (low-flow ischemia) for 75 minutes, followed by 75 minutes of reperfusion, in the other (experimental) segment. Biopsy samples were collected from experimental segments at baseline (T0), after 75 minutes of ischemia (T1), and after 75 minutes of reperfusion (T2); samples were collected from control segments at T0 and T2. Horses were euthanized 24 hours after induction of ischemia (T3), and additional samples were collected. Samples were evaluated histologically. Total RNA was extracted; expression of COX genes and stability of 8 housekeeping genes were determined via quantitative real-time PCR assays.
Results—COX-1 and COX-2 genes were constitutively expressed in baseline samples. Low-flow ischemia resulted in significant upregulation of COX-2 gene expression at each subsequent time point, compared with baseline values. The most stably expressed reference genes were β-actin and hypoxanthine phosphoribosyltransferase, whereas glyceraldehyde 3-phosphate dehydrogenase and β-2 microglobulin were the least stably expressed.
Conclusions and Clinical Relevance—Low-flow ischemia resulted in upregulation of COX-2 gene expression in the jejunum of horses. Housekeeping genes traditionally used as internal standards may not be stable in this tissue during arterial low-flow ischemia and reperfusion.