Thank you for your excellent questions. The animals were approximately 9 wks of age. The vessels were fixed as we have previously described for our model (Unthank, et al. 1996, Circ. Res.). In brief, the segment of interest was placed within an observation chamber. The vasculature was maximally dilated (topical application of 0.1mM adenosine, 0.01mM sodium nitroprusside). The suffusion solution was then replaced with formalin warmed to 37 degrees and also containing 0.1mM adenosine, 0.01mM sodium nitroprusside. This allowed for fixation of the vessels at their in vivo pressure. After a few minutes, the animals' arterial pressure begins to drop as the fixative diffuses into the vasculature and circulates systemically. At this point, the terminal ileum with its vasculature was ligated and placed in formalin overnight. The next day vessels were isolated for processing and embedding in plastic.
I would also like to comment that our results are not really that much different from what you and others have reported. You can see from Figure 4 that medial area tends to be increased in the SHR vessels. Medial thickness is increased in SHR for both control and collateral vessels. The primary purpose of our study to determine if shear-mediated luminal expansion occurred in collaterals of the SHR. Because our data indicated statistical significance in luminal expansion in WKY relative to SHR and the enlargement of the WKY was similar to what we observed in Wistar rats previously, we limited our study to 5 WKY animals. Had we done 10 WKY as we did SHR, I expect that statistical significance would have been achieved between the control vessels of the WKY and SHR as many other studies have shown.