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Osteocyte reaction to decreased bone fluid flow

骨细胞对骨液流量减少的反应

基本信息

批准号：
6644841
负责人：
SHIJING QIU
金额：
$ 7.15万
依托单位：
HENRY FORD HEALTH SYSTEM
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-08-15 至 2005-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6644841
关键词：
apoptosis artery confocal scanning microscopy fluid flow hypoxia immunocytochemistry interstitial laboratory rat musculoskeletal circulation osteocytes pathologic bone resorption perfusion physiologic bone resorption terminal nick end labeling tibia ultrasound blood flow measurement

项目摘要

DESCRIPTION (provided by applicant): 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.

描述(申请人提供)：在骨骼中有两种类型的液体流动，循环间质流动(CIFF)和应变加速液体流动。至目前，关于骨细胞反应降低的信息很少骨液流动。我们的工作假设是：1)CIFF因以下原因而下降血液供应不足导致骨缺氧，导致大量骨细胞发生凋亡性改变和2)骨细胞凋亡率增加有助于激活骨骼重塑。在拟议的研究中， 74只成年SD大鼠右股动脉结扎减少胫骨CIFF。动物将在2，4，8，48和术后168(7天)小时。双侧皮质骨和松质骨将对胫骨进行检查。 1)测量胫前肌的血流量变化用激光多普勒血流仪(LDF)测定红细胞相对血流速度(REFV)。之后，荧光染料(异硫氰酸荧光素(FITC)-葡聚糖和埃文斯蓝色)将以一小时为间隔进行静脉灌流。所有动物都会在第二次灌流后1分钟处死。未脱钙的骨切片将使用激光扫描共聚焦显微镜进行检查。出血率骨组织中的间质液体流动可以通过定量图像来确定分析。 2)静脉注射盐酸匹莫硝唑治疗骨细胞缺氧性检查。含有阳性匹莫硝唑的骨细胞脱钙切片免疫染色暴露的加合物视为作为缺氧性骨细胞。骨细胞缺氧的发生和发展 CIFF的降低可以通过缺氧量的变化来确定骨细胞。 3)脱钙骨切片用甲苯胺蓝和末端染色脱氧核苷酸转移酶dUTP缺口末端标记法。中的变化骨细胞密度和凋亡和坏死细胞的数量将单独检查以确定骨细胞对退行性骨的反应 CIFF。 4)如果低CIFF导致的骨细胞死亡可以诱导骨重建，破骨细胞性吸收预计在动脉后约7天出现。结扎术。破骨细胞吸收与骨吸收的空间关系甲苯胺蓝切片将确定骨细胞退行性变和图奈尔污渍。