There were no 1000�C2000 ��m thenthereby vascularised macrometastases present. The delta�Cdelta cycle threshold (����Ct) method was used to calculate the real-time PCR results, which measures gene expression between the model and a housekeeping gene ( cross threshold), then between the model and control groups ( cross threshold). First, calculated the ��Ct values that are the difference between the VEGF Ct value and the ��-actin Ct value from three zones. Then calculated the ����Ct values that are the difference between the ��Cts from three zones. Statistical analysis was done with Microsoft Excel 2008 (Redmond, Washington, USA). A one-way ANOVA was used to determine statistical significance of differences in serum VEGF levels over time.

A correlation coefficient analysis was used to determine the correlation of VEGF serum levels to the location and number of hepatic micrometastases. The level of significance was set at a p value <0.05. Results Serum VEGF levels rose after inoculation of C57Bl/6 mouse eyes with the B16LS9 melanoma cells (figure 2). On day 14 there was a statistically significant (p<0.05) increase in VEGF levels in the serum of mice inoculated with the cells, with an average peak level of 37.985 pg/ml at day 21. Mice injected with PBS did not exhibit the same rise in serum VEGF expression and VEGF expression remained relatively stable over the time course studied. Figure 2 Averaged serum levels of vascular endothelial growth factor (VEGF) over time. Mice were inoculated with either B16LS9 murine melanoma cells or phosphate buffered saline (PBS) and serum VEGF levels were measured by ELISA at the indicated post-inoculation .

.. A representative photomicrograph of a mouse hepatic lobules is shown in figure 1B. Zones 1�C3 micrometastastic locations are depicted in figures 1B (asterisks). VEGF protein expression was confirmed with immunohistochemistry for VEGF in identified hepatic micrometastases. A representative micrometastasis with corresponding VEGF staining is shown in figure 3. Figure 3 Uveal melanoma hepatic micrometastases express vascular endothelial growth factor (VEGF) protein. (A) Micrometastastic melanoma in liver (arrow) (haematoxylin and eosin staining, magnification, ��100). (B) Hepatic micrometastasis expresses VEGF … The number and location of micrometastases was determined for each mouse and correlated with their averaged and peak VEGF serum levels (figure 4).

Peak serum VEGF levels weakly correlated with the total number of hepatic micrometastases (Pearson R=0.444, p<0.05). There was moderate correlation of peak VEGF serum levels with zone 2 (Pearson R=0.644, p<0.05) and zone 3 micrometastases Drug_discovery (Pearson R=0.572, p<0.05), although this is a preliminary study in a limited number of mice. R2 values are also reported, which indicate the percentage of variability of serum VEGF levels as it relates to the number and location of hepatic micrometastases.