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Principal Investigator:
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Susan A. Bloomfield, Ph.D.
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Organization:
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Texas A&M University
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Project Ended:
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2000
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Blood flow to bone has been shown to affect bone mass and presumably bone strength. Preliminary data indicate that blood flow to the rat femur decreases after 14 days of simulated microgravity, using hindlimb unloading (HU). If adult rats subjected to HU are given dobutamine, a synthetic catecholamine which can cause peripheral vasodilation and increased blood flow, the loss of cortical bone area usually observed is prevented. The primary aim of this project is to characterize changes in 1) bone blood flow, 2) indices of bone mass, geometry, and strength, and 3) changes in gene expression for candidate genes for mechanotransduction in bone after 3, 7, 14, 21, and 28 days of HU in the adult rat. Using a rat of at least 5 months of age avoids inadvertently studying effects of simulated microgravity on growing, rather than adult, bone.
The key contributions of these studies lie in the quantification of blood flow to various compartments of bone and marrow with the acute adjustment to the tail suspension posture (after 10 minutes) and after 7 and 28 days of tail suspension. Ten minutes of HU significantly decreases flow to the proximal femur, the femoral shaft and to femoral shaft marrow. By 7 days of HU, blood flow to all portions of the femur, including distal metaphysis, is sigificantly less than flow to these regions in cage-activity control rats. These decreases in flow are due to an increase in vascular resistance; this increased resistance is maintained through 28 days of HU. Conversely, blood flow to bone of the forelimb and head (skull, mandible) is increased during HU with the acute shift to the tail suspension posture. However, blood flow returns to normal with 7 days HU due to compensatory increases in vascular resistance. These alterations in blood flow were accompanied by changes in bone mass, with those bones experiencing lower flows during HU decreasing in mass (femur, tibia), whereas those bones experiencing that acute increase in flow with HU increasing in mass (mandible, clavicle, humerus).
Because radiolabeled microspheres are used to measure blood flow, rendering all tissues radio-active, bones from these animals could not be further processed. Separate experiments were performed to document the time course of alterations in bone mineral content, bone geometry, mechanical properties and gene expression with HU. Histomorphometric analyses of fluorochrome-labeled bone reveal large decrements in mineral apposition rate and bone formation rate at the tibio-fibular junction (60 percent and 90 percent declines, respectively) in these mature adult rats. Our data confirm that of earlier investigators (Dehority et al., Amer. J. Physiol., 1999) in that decreases in bone formation are slower to develop in the mature rat skeleton as opposed to the better characterized response of young growing rats, but more prolonged.
Few changes were noted in bone mineral density (BMD) of mid-shaft cortical bone. Alterations in tibial BMD and cross-sectional area appear to parallel growth-related changes in the humerus. Not surprisingly, mechanical properties at this site were not affected either. Analyses using pQCT of the proximal tibial metaphysis reveal a compartment-specific alterations in BMD, where cancellous (trabecular) bone is decreasing in BMD even as the cortical shell gains in BMD during 28d HU. Novel analyses of cancellous bone mechanical properties reveal a decline in both elastic modulus and ultimate stress in this core of the proximal tibial metaphysis after 28d HU. Clearly, mechanical strength of bone is compromised at sites rich in cancellous bone in the unloaded limb, even in the mature adult skeleton in which growth processes are minimal.
Gene expression is altered in proximal femur samples from these unloaded rats. Early changes at 3 days reveal an increased expression of cytokines favoring bone resorption (interleukin-6 and interferon-γ) and a decrease in expression of TGF-β1, which normally stimulates bone formation activity. These alterations remain to be confirmed in longer duration studies. Microarray data indicate upregulation of a number of genes potentially important in regulating bone cell activity: integrin abv3 and abv5, nitric oxide synthase, prostaglandin synthase 1 and 2. A down-regulation of several oncogenes (fos, abl) was noted, as well as for BMP receptors types I and II. A very recent finding with micro-array analysis points to an upregulation of a membrane kinase previously unidentified in bone in proximal femurs from rats subjected to 28d HU.
These results have important implications for the development of countermeasures to ameliorate bone loss with prolonged exposure to weightlessness. We have established a time course for declines in blood flow to bone, using state-of-the-art microsphere studies, which precede reductions in bone formation (at mid-shaft tibia) and BMD and mechanical propertiesof cancellous bone in the proximal tibia. If, indeed, continuing experiments can demonstrate a causal link between altered blood flow and shifts in bone remodeling activity, a whole new category of countermeasures might be considered. Some pharmacological agents may effect a general vasodilation and therefore increase in blood flow, but relatively benign physical measures heating, exercise, lower body negative pressure (with or without exercise) can be tested for effectiveness in earth-based studies in increasing blood flow to bone. These data on mature adult rats, a better model for adult human bone than the more widely used growing rat, also suggest that sites rich in cancellous bone (proximal femur, proximal tibia, distal femur) in unloaded limbs are at the highest risk for fracture with prolonged exposure to microgravity. We may have over-estimated the rate of change in cortical bone BMD and cortical bone geometry in the past having relied on information from rapidly growing young rats. Our gene expression data provide some mechanistic data on which to build future countermeasure strategies, given early changes in regulatory peptides important in regulating bone cell activity.
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