Validation of Tissue Microarrays for Vascular Research
Marc K. Halushka MD; Johns Hopkins Medical Institutions; Toby Cornish MD; Johns Hopkins Medical Institutions; Jie Lu MS; Johns Hopkins Medical Institutions;
Content:
Content: While tissue microarrays (TMA) have long been the purview of oncology research, their role as research tools for other pathologies is limited. TMAs were investigated for their usefulness in the study of diabetic vascular disease.
Technology:
Technology: A global sampling of 13 vascular tissues was collected from each of 100 adult autopsies. Selected tissues include renal cortex, coronary arteries, internal mammary arteries, pulmonary arteries, carotid arteries, and dorsalis pedis arteries. Seventeen 99-1.5mm core size TMAs were used to array 1,500 tissues from these autopsies and 183 additional control tissues. Renal cortex was arrayed twice on the same TMA and again on a separate TMA.
Design:
Design: Immunohistochemistry with diamino benzidine (brown) staining was performed for 18 proteins related to diabetic vascular disease including matrix metallanoproteinases and their inhibitors (MMPs & TIMPs), advanced, glycation end products (AGEs), and receptors for AGEs. Tissue core images were marked up to delineate the region of interest (ROI) for analysis using custom macros in ImageJ (Wayne Rasband, NIH). Brown staining was then measured using color deconvolution, and a distribution of brown intensity for the ROI in each core was determined. Median staining intensity values strongly correlated with non-parametric methods of analysis (R2=0.98), and were used for subsequent measures.
Results:
Results: There was tight correlation between median staining intensity values for the glomeruli in two renal cores from the same individual summed for all MMP and TIMP immunohistochemistry staining (MMPs 1,2,3,9, TIMPs 1,2,3) both on the same TMA and across two TMAs (R2=0.92, 0.84 respectively). Intimal atherosclerosis and fibrosis was assessed as a contributor to median intensity values. Markups were created that compared tunica media alone to combined T. media and T. intima staining. In atherosclerosis prone vessels there was a reduced correlation between T. media alone and T. media and intima staining intensity, whereas atherosclerosis resistant vessels demonstrated a tight correlation (coronary artery R2 =0.54 vs. mesenteric artery R2=0.96).
Conclusion:
Conclusions: TMA is a novel research tool for investigating vascular disease. Success of this method is dependent upon large caliber (1.5 mm) punch sizes and appropriately selected tissues. Median staining values are also dependent on the layer of the vessel evaluated.
