Proceedings of The Physiological Society

University College Cork (2004) J Physiol 560P, PC1



Lam,Janice ; Malpas,Simon ; Sands,Gregory ; Gerneke,Dane ; LeGrice,Ian ; Pyner,Susan ;

1. School Biological & Biomedical Sciences, University of Durham, Durham, United Kingdom. 2. Department of Physiology, University of Auckland, Auckland, New Zealand.

There is evidence that high blood pressure is associated with changes in renal function and structure of renal vasculature (Anderson et al., 2000). Thus knowledge of the architecture of the renal vasculature is likely to be important for our understanding of the relationship between renal function, structure and blood pressure. The renal vasculature is difficult to measure and reconstruct. A current method requires injection of resins and digestion of the kidney tissue to reveal casts of blood vessels that are imaged using scanning electron microscopy (Denton et al., 2000). This approach provides detail of vasculature but does not allow for easy reconstruction of a 3 dimensional view of the whole renal vessel network. Therefore, we have further developed a method first used to produce extended volume 3D images of myocardium whereby immunofluorescence labelling of blood vessel can be visualised using extended confocal microscopy (Young et al., 1998). Experimental procedures were approved by the University of Auckland Animal Ethical Committee. The animals were anaesthetised with 3% halothane in oxygen and the abdominal aorta was tied above and below the level of the renal arteries. The animals were killed humanely with sodium pentobarbitone (160 mg/kg) followed by perfusion with saline/heparin/sodium nitrite then wheat germ agglutinin conjugated with tetramethylrhodamine isothiocyanate (WGA-TRITC) and finally with 4% paraformaldehyde in phosphate buffered saline (PBS). The kidneys were removed, post fixed then stored in 0.1 M PBS until resin embedded. Confocal fluorescence laser scanning microscopy was used to obtain 3D images in a contiguous mosaic across the surface of the block. The system consisted of a confocal microscope (Leica TCS 4D) with a Kr/Ar laser and a variable speed Ultramill (Leica). Z-stack volume images were acquired for overlapping x-y areas that covered the region of interest. The scanned volume was milled and the imaging process repeated. The acquired images were combined to reconstruct the volume in 3D. The renal vasculature was revealed by the presence of fluorescing WGA-TRITC bound to the endothelium of the blood vessel wall. The resultant image reconstruction provides structural detail of the branching network of renal blood vessels as well the orientation relationships. We have successfully developed a method that allows 3D volume reconstruction of renal vessel architecture of relatively large tissue blocks. The structural information is important for our further understanding of the role of the kidney in the development of hypertension.

Where applicable, experiments conform with Society ethical requirements