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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 (normal).

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 sections.

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 surgery.

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)

Full access
in American Journal of Veterinary Research


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.

Full access
in American Journal of Veterinary Research