MRI biomarkers of BME
With the aid of a custom surface coil and carefully designed MRI pulse sequence, we were able to generate images that could clearly differentiate the marrow of TNF-transgenic and WT mice on both precontrast and postcontrast scans (). Of particular interest was the hyperintense marrow of TNF-transgenic mice on precontrast scans (NBMI = 0.59) () that was markedly brighter after contrast enhancement (NMCE = 1.02) (), suggestive of highly vascular and/or permeable tissue (31
). Corresponding histologic analysis of this region () demonstrated the presence of highly cellular hematopoietic tissue throughout the marrow cavity. In contrast, the bone marrow of 5-month-old WT mice was dark in both precontrast (NBMI = 0.16) () and postcontrast (NMCE = 0.34) () sequences, and the corresponding histologic examination () showed that the tibial metaphyseal region contained a high percentage of adipocytes, indicating a predominantly yellow marrow phenotype. The hypointensity in the metaphyseal region of WT mice is partly due to fat suppression, which was incorporated into the MRI pulse sequence.
Figure 1 Differences in the bone marrow of tumor necrosis factor-transgenic (TNF-Tg) and wild-type (WT) mice. A-F, Magnetic resonance images and corresponding orange G-Alcian blue-stained histologic sections of the knee joints from representative 5-month-old TNF-Tg (more ...)
Intrareader, interreader, and inter-MRI reliability of marrow MRI biomarkers was measured using ICC analysis, and was found to be excellent for all variables. NBMI was found to have ICC values of 0.998 for intrareader, 0.997 for interreader, and 0.982 for inter-MRI reliability. NMCE quantifications had ICC values of 0.981, 0.984, and 0.944, respectively.
Conversion of red marrow to yellow marrow in aging WT mice, but not in TNF-transgenic mice
The yellow marrow pattern seen in 5-month-old WT mice led us to hypothesize that MRI could track the conversion from cellular, red marrow to fatty, yellow marrow, which is a natural consequence of aging in the bones of mammals (32
). To test this hypothesis in a natural history study, 5 WT and 5 TNF-transgenic animals received bimonthly MRI scans from 2 to 5 months of age. Quantification () showed a significant decrease in NBMI with increasing age in WT animals (slope = -0.084, P
< 0.0001) as red marrow was converted to fat (). TNF-transgenic animals, in contrast, showed a highly significant increase in this value from 2 to 5 months (slope = 0.040, P
< 0.0001). In addition, NMCE () showed a highly significant decrease in WT mice (slope = -0.201, P
< 0.0001), whereas TNF-transgenic mice had a significant increase in this value (slope = 0.065, P
< 0.05). These results indicate that the diminishing signals in WT mice are associated with red to yellow marrow conversion, while the elevated bone marrow signal persists in TNF-transgenic mice because they do not undergo marrow conversion.
Conversion of red marrow to yellow marrow in TNF-transgenic animals following anti-TNF therapy
To further validate our marrow quantifications, and to determine if yellow marrow conversion could be induced in TNF-transgenic animals, we tested the hypothesis that anti-TNF therapy administered to TNF-transgenic mice would significantly lessen both the NBMI and the NMCE. We observed a decrease in signal intensity in the marrow from baseline () to 8 weeks after the initiation of therapy () in 3-month-old TNF-transgenic mice that received anti-TNF therapy. The corresponding histologic analysis () revealed a high proportion of adipocytes in the bone marrow. In contrast, there was an increase in signal intensity in the marrow of placebo-treated TNF-transgenic mice from baseline () to 8 weeks (), characterized by highly cellular marrow throughout the bones (). Mean NBMI () and NMCE () values changed significantly over time. In mice receiving anti-TNF therapy, NBMI and NMCE values decreased significantly during the 8 weeks of treatment (slopes = -0.044, P < 0.0001 and -0.081, P < 0.0001, respectively). Mean NBMI values increased significantly in placebo-treated animals (slope = 0.011, P < 0.05), while mean NMCE remained elevated but did not change. A highly significant treatment effect (difference in slopes between placebo and anti-TNF therapy) was found for both NBMI (0.055, P < 0.0001) and NMCE (0.094, P < 0.0001). These results show that TNF is responsible for the persistence of red marrow in TNF-transgenic mice, because inhibition of this cytokine resulted in red to yellow marrow conversion.
Figure 2 Conversion of highly cellular marrow from TNF-Tg mice to yellow marrow with anti-TNF therapy. Magnetic resonance images (MRIs) of the proximal tibia were obtained at baseline (A and D) and 8 weeks after initiation of treatment (B and E) with anti-TNF (more ...)
Effect of joint synovitis on diffuse bone marrow patterns in adjacent marrow
Results of histologic examinations of 5-month-old TNF-transgenic mice and their WT littermates were compared, to better understand the origin of marrow conversion in our model. We also wanted to determine if the hypercellular marrow pattern was generated in response to signals derived from the adjacent inflamed joint, or if it resulted from endocrine effects due to elevated levels of TNF in the systemic circulation of TNF-transgenic mice. The histologic analysis showed that in WT mice, marrow of the distal lower limb at 5 months of age was entirely filled with adipose tissue (). Interestingly, the marrow in the distal tibiae of their TNF-transgenic littermates was also filled with fat, despite very severe ankle arthritis, as demonstrated by the presence of cortical breaks and synovial infiltration from the adjacent joint (). This suggests that local joint inflammation is not responsible for the induction of marrow conversion, even when it is in direct contact with yellow marrow through infiltrating pannus tissue.
Figure 3 Lack of red to yellow marrow conversion in TNF-Tg mice is caused by systemic effects rather than local joint inflammation and is reversed along a distal to proximal axis by anti-TNF therapy. A-L, Orange G-Alcian blue-stained histologic sections from representative (more ...)
We observed a different pattern of marrow cellularity in the proximal tibiae of TNF-transgenic mice, in which the histologic analysis showed hypercellular marrow irrespective of the severity of knee inflammation. As an example, shows the knee histology in a TNF-transgenic mouse with little to no joint inflammation () but predominantly red marrow () and elevated signals on MRI (NBMI = 0.587). In contrast, shows the histology in a TNF-transgenic mouse with severe arthritis (), which also exhibited hypercellular marrow (NBMI = 0.688) (). Collectively, these findings indicate that the increases in cellular marrow in TNF-transgenic animals follow a distinct pattern from central to distal joints, which is a reverse pattern from the yellow marrow conversion that takes place in growing individuals (32
Marrow conversions and their relationship to vascularity
The tibial diaphyses of 5-month-old TNF-transgenic mice show the region in which systemic marrow conversions occurred. Histologic examination of TNF-transgenic mice () revealed the marrow conversion front as a highly vascularized region that contains large numbers of erythrocytes in expanded vascular channels between the monocytes and fat. In contrast, the marrow conversion front in TNF-transgenic mice treated with anti-TNF for 8 weeks () was characterized by tissue containing few erythrocytes. To demonstrate the significance of this vascularity, we performed histomorphometry to quantify these erythrocytic regions within the red marrow of the middiaphysis in the tibiae of WT, TNF-transgenic, and anti-TNF-treated TNF-transgenic mice (). We determined that TNF-transgenic mice had a highly significant increase in these vascular areas of red marrow compared with WT animals. Furthermore, anti-TNF therapy significantly reduced these vascular areas in TNF-transgenic mice to levels observed in WT mice. Thus, our interpretation of these results is that TNF prevents conversion of red to yellow marrow along a proximal to distal axis, while loss of vascularity is associated with conversion to yellow marrow in response to effective anti-TNF therapy.
Diffuse BME signals are associated with increased myelopoiesis, while focal edema signals colocalize with invading pannus through cortical breaks
To further define the nature of the persisting red marrow in TNF-transgenic mice, we performed FACS analyses of marrow-derived cells from the tibiae of 5-month-old WT and TNF-transgenic mice (). While we failed to detect any significant differences in lymphocyte populations, FACS analysis revealed a dramatic increase in the percentage of CD11b+,Gr-1+ myeloid cells in the marrow of TNF-transgenic mice and most of these were in the CD11b+,Gr-1low
population, which consists mostly of dividing myeloid precursors, as previously described (22
). To assess a potential direct relationship between elevated MRI signals and myelopoiesis, we compared the percentage of CD11b+ cells in the tibia versus the NBMI of the tibia from MRI analysis using linear regression analysis, and found a highly significant correlation (R2
= 0.914, P
= 0.0028). This supports the conclusion that diffuse, generalized BME signals can partly be caused by TNF-induced myelopoiesis.
Figure 4 Association of generalized bone marrow edema (BME) signals with increased myelopoiesis. A-D, Bone marrow cells harvested from 5-month-old WT and TNF-Tg mice were stained with labeled antibodies specific for CD3 (A), CD45 (B), CD11b (C), or Gr-1 (D), and (more ...)
Furthermore, TNF-transgenic mice >8 months old typically have severe erosive arthritis that leads to cortical breaks and contiguous pannus that spans from the synovial lining of the knee joint into the bone marrow (27
). Interestingly, in addition to the diffuse BME signal throughout their distal femora and proximal tibiae, these mice have higher signal intensity in the marrow adjacent to the synovium (), which showed greater marrow contrast enhancement compared with the diffuse BME postcontrast (). Corresponding histologic analysis of this focal BME pattern () confirmed pannus invasion of the marrow space through a cortical break in the femur. Immunohistochemical analysis of F4/80+ cells () demonstrated that the infiltrating cells in mice with subchondral BME were predominantly monocyte/macrophages, which is consistent with the FACS data.
BME in TNF-transgenic mice is associated with increased Gd-DTPA perfusion
Although the aforementioned results suggest that the elevated marrow signals are associated with red marrow myelopoiesis, we also made several observations that clearly indicated that further abnormalities in red marrow must occur in order to present as BME on MRI. First, the MRI signal in older WT animals with incomplete marrow conversion appeared dark in both the red and the yellow marrow regions of the tibia (). Second, the vascular area was increased in TNF-transgenic mice over WT mice in these persisting red marrow areas (). Third, there was a dramatic difference in the contrast-enhancement patterns of the tibial diaphysis between WT and TNF-transgenic mice at 5 months of age (). In WT mice at this age, red marrow persisted in the diaphysis of the marrow () and was supplied with blood by the large nutrient artery (). This artery was clearly visualized on precontrast and postcontrast MRIs (). However, there was no corresponding edema signal or contrast enhancement in the red marrow surrounding this artery. In contrast, in TNF-transgenic animals, the nutrient artery was seen on histologic examination (), but could not be visualized on MRIs () due to high signal intensity throughout the marrow. These results suggest that the vascular endothelium in the marrow of TNF-transgenic mice is highly permeable, which causes increased NBMI due to excess interstitial fluid and increased NMCE due to diffusion of the Gd-DTPA throughout the marrow space.
Figure 5 Association of bone marrow edema in TNF-Tg mice with increased permeability of the blood-bone marrow barrier. A and B, Orange G-Alcian blue-stained histologic section from a 5-month-old WT mouse highlights the nutrient artery in the tibia (boxed area (more ...)
MRI thresholds of yellow marrow, normal red marrow, and BME
Given that there is a broad spectrum of signals from normal and pathologic red marrow, we performed a linear regression analysis of the NBMI versus NMCE signals from the WT and TNF-transgenic mice shown in and from the anti-TNF-treated mice shown in (). This analysis demonstrated a highly significant correlation (R2 = 0.812, P < 0.0001) between NBMI and NMCE in all of the tibiae examined. Moreover, we were able to identify apparent thresholds that defined the yellow versus normal red marrow versus pathologic red marrow that presents as BME.
Figure 6 Thresholds of yellow marrow, red marrow, and pathologic inflammatory bone marrow edema signals. Linear regression analysis of NBMI versus NMCE in the WT and TNF-Tg mice shown in and in anti-TNF-treated TNF-Tg mice after 8 weeks of therapy showed (more ...)