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1.  Validation of power Doppler Versus Contrast Enhanced Magnetic Resonance Imaging Quantification of Joint Inflammation in Murine Inflammatory Arthritis 
Contrast enhancement magnetic resonance imaging (CE-MRI) of synovial volume is the radiographic gold standard to quantify joint inflammation but cost limits use. Therefore, we examined if power Doppler-ultrasound (PD-US) outcomes of synovitis in tumor necrosis factor transgenic (TNF-Tg) mice correlate with CE-MRI. TNF-Tg mice underwent PD-US of their knees to measure the joint space volume (JSV) and PD volume (PDV), and the results were correlated with synovial volume determined by CE-MRI. Immunohistochemistry for CD31 was performed to corroborate the PD signal. Synovial volume strongly correlated with both JSV and PDV (p<0.01). CD31+ blood vessels were observed in inflamed synovium proximal to the joint surface, which corresponded to areas of intense PD signals. JSV and PDV are valid measures of joint inflammation that correlate with synovial volume determined by CE-MRI and are associated with vascularity. Given the emergence of PD-US as a non-quantitative outcome of joint inflammation, we find JSV and PDV to be feasible and highly cost-effective for longitudinal studies in animal models. Furthermore, given the increasing utilization of PD-US in standard clinical practice, JSV and PDV could be translated to better quantify joint flare and response to therapy in RA patients.
PMCID: PMC4376603  PMID: 25359523
Bone ultrasound; Rheumatoid Arthritis (RA); Magnetic Resonance Imaging (MRI)
2.  Efficacy of B cell Depletion Therapy on Joint Flare is Associated with Increased Lymphatic Flow 
Arthritis and rheumatism  2013;65(1):130-138.
B cell depletion therapy (BCDT) ameliorates rheumatoid arthritis by mechanisms that are incompletely understood. Arthritic flare in tumor necrosis factor transgenic (TNF-Tg) mice is associated with efferent lymph node (LN) “collapse,” triggered by B cell translocation into lymphatic spaces and decreased lymphatic drainage. We examined whether BCDT efficacy is associated with restoration of lymphatic drainage due to removal of obstructing nodal B cells.
We developed contrast-enhancement (CE) MRI imaging, near-infrared indocyanine green (NIR-ICG) imaging, and intravital immunofluorescent imaging to longitudinally assess synovitis, lymphatic flow, and cell migration in lymphatic vessels in TNF-Tg mice. We tested to see if BCDT efficacy is associated with restoration of lymphatic draining and cell egress from arthritic joints.
Unlike active lymphatics to normal and pre-arthritic knees, afferent lymphatic vessels to collapsed LNs in inflamed knees do not pulse. Intravital immunofluorescent imaging demonstrated that CD11b+ monocytes/macrophages in lymphatic vessels afferent to expanding LN travel at high velocity (186 ± 37 micrometer/sec), while these cells are stationary in lymphatic vessels afferent to collapsed PLN. BCDT of flaring TNF-Tg mice significantly decreased knee synovial volume by 50% from the baseline level, and significantly increased lymphatic clearance versus placebo (p<0.05). This increased lymphatic drainage restored macrophages egress from inflamed joints without recovery of the lymphatic pulse.
These results support a novel mechanism in which BCDT of flaring joints lessens inflammation by increasing lymphatic drainage and subsequent migration of cells and cytokines from the synovial space.
PMCID: PMC3535508  PMID: 23002006
Rheumatoid Arthritis (RA); Flare; Tumor Necrosis Factor (TNF); B cells in Inflamed Lymph Nodes (B-in); Lymphatic Pulse
3.  Power Doppler Ultrasound Phenotyping of Expanding versus Collapsed Popliteal Lymph Nodes in Murine Inflammatory Arthritis 
PLoS ONE  2013;8(9):e73766.
Rheumatoid arthritis is a chronic inflammatory disease manifested by episodic flares in affected joints that are challenging to predict and treat. Longitudinal contrast enhanced-MRI (CE-MRI) of inflammatory arthritis in tumor necrosis factor-transgenic (TNF-Tg) mice has demonstrated that popliteal lymph nodes (PLN) increase in volume and contrast enhancement during the pre-arthritic “expanding” phase of the disease, and then suddenly “collapse” during knee flare. Given the potential of this biomarker of arthritic flare, we aimed to develop a more cost-effective means of phenotyping PLN using ultrasound (US) imaging. Initially we attempted to recapitulate CE-MRI of PLN with subcutaneous footpad injection of US microbubbles (DEFINITY®). While this approach allowed for phenotyping via quantification of lymphatic sinuses in PLN, which showed a dramatic decrease in collapsed PLN versus expanding or wild-type (WT) PLN, electron microscopy demonstrated that DEFINITY® injection also resulted in destruction of the lymphatic vessels afferent to the PLN. In contrast, Power Doppler (PD) US is innocuous to and efficiently quantifies blood flow within PLN of WT and TNF-Tg mice. PD-US demonstrated that expanding PLN have a significantly higher normalized PD volume (NPDV) versus collapsed PLN (0.553±0.007 vs. 0.008±0.003; p<0.05). Moreover, we define the upper (>0.030) and lower (<0.016) quartile NPDVs in this cohort of mice, which serve as conservative thresholds to phenotype PLN as expanding and collapsed, respectively. Interestingly, of the 12 PLN phenotyped by the two methods, there was disagreement in 4 cases in which they were determined to be expanding by CE-MRI and collapsed by PD-US. Since the adjacent knee had evidence of synovitis in all 4 cases, we concluded that the PD-US phenotyping was correct, and that this approach is currently the safest and most cost-effective in vivo approach to phenotype murine PLN as a biomarker of arthritic flare.
PMCID: PMC3767819  PMID: 24040061
4.  Measuring intranodal pressure and lymph viscosity to elucidate mechanisms of arthritic flare and therapeutic outcomes 
Rheumatoid arthritis (RA) is a chronic autoimmune disease with episodic flares in affected joints, whose etiology is largely unknown. Recent studies in mice demonstrated alterations in lymphatics from affected joints precede flares. Thus, we aimed to develop novel methods for measuring lymph node pressure and lymph viscosity in limbs of mice. Pressure measurements were performed by inserting a glass micropipette connected to a pressure transducer into popliteal lymph nodes (PLN) or axillary lymph nodes (ALN) of mice and determined that the lymphatic pressures were 9 and 12 cm of water, respectively. We are also developing methods for measuring lymph viscosity in lymphatic vessels afferent to PLN, which can be measured by multi-photon fluorescence recovery after photobleaching (MP-FRAP) of FITC-BSA injected into the hind footpad. These results demonstrate the potential of lymph node pressure and lymph viscosity measurements, and warrant future studies to test these outcomes as biomarkers of arthritic flare.
PMCID: PMC3334848  PMID: 22172039
Rheumatoid Arthritis; Lymph Node; Flare; Lymphatic Pressure; Lymph Viscosity
5.  Biomaterial Guides for Lymphatic Endothelial Cell Alignment and Migration 
Acta biomaterialia  2010;7(3):1104-1113.
Axillary dissection during breast cancer surgery produces extensive lymphatic vessel damage that often leads to life-long secondary lymphedema of the arm. We have developed a biodegradable material conduit for lymphatic vessel reconstruction where fibers electrospun along the conduit lumen promote endothelial cell alignment and migration, in vitro. The diameter and density of the electrospun fibers were optimized for cell migration and direction on 2-dimensional substrates by seeding human lymphatic endothelial cells (LECs) onto aligned fibers of varying diameters and densities, randomly oriented fibers, and film substrates with no fibers. We found that LECs became aligned in the fiber direction, with cells seeded on the randomly oriented fibers becoming oriented in random directions, whereas cells seeded on the highly aligned fibers became highly aligned. Cell migration was dependent upon fiber alignment and density, with optimal migration found on 1300 nm diameter aligned fibers of low density. Blood endothelial cells seeded on the fibers exhibited similar behavior as the LECs. Fiber alignment was preserved upon rolling the 2-dimensional substrate into the tubular geometry of a lymphatic vessel. The data suggest that aligned electrospun fibers may promote endothelial migration across the conduit in a manner that is independent of lymphatic growth factors.
PMCID: PMC3031728  PMID: 20974299
lymphatic regeneration; endothelial cell; aligned fibers; biodegradable materials

Results 1-5 (5)