There are two types of fluid flow in bone, a circulatory interstitial flow (CIFF) and a strain accelerated fluid flow. To date, there is little information about the osteocyte reaction to decreased bone fluid flow. Our working hypotheses are: 1) Declined CIFF arising from insufficient blood supply causes bone hypoxia, leading to a large number of osteocytes undergoing apoptotic changes and 2) increased ostesocyte apoptosis contributes to the activation of bone remodeling. In the proposed study, the right femoral artery in 74 adult Sprague-Dawley rats will be ligated to decrease CIFF in the tibia. The animals will be sacrificed at 2, 4, 8, 48, and 168 (7days) hours after surgery. The cortical and cancellous bone in bilateral tibias will be examined. 1) The changes in blood flow in tibialis anterior muscle will be measured by relative erythrocyte flow velocity (REFV) using laser-Doppler flowmetry (LDF). After that, fluorochromes (fluorescein isothiocyanate (FITC)-dextran and Evans blue) will be perfused intravenously with one hour interval. All animals will be sacrificed 1 minute after second perfusion. The undecalcified bone sections will be examined using laser-scanning confocal microscopy. The rate of blood and interstitial fluid flow in bone can be determined by quantitative image analysis. 2) Intravenous injection of pimonidazole hydrochloride will be applied for the examination of osteocyte hypoxia. Osteocytes containing positive pimonidazole adduct that is exposed by immunostain of the decalcified sections are regarded as hypoxic osteocytes. The onset and development of osteocyte hypoxia following decreased CIFF can be determined by the alterations in the amount of hypoxia osteocytes. 3) Decalcified bone sections will be stained with toluidine blue and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). The changes in osteocyte density and the amount of apoptotic and necrotic cells will be examined separately for determining the reaction of osteocytes to declined CIFF. 4) If osteocyte death resulting from lower CIFF could induce bone remodeling, osteoclastic resorption is expected to appear about 7 days after artery ligation. The spatial relationship between osteoclastic resorption and osteocyte degeneration will be determined in the sections with toluidine blue and TUNEL stain.
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