Nicole A. Bouffard

Subcutaneous tissue fibroblast cytoskeletal remodeling induced by acupuncture: Evidence for a mechanotransduction‐based mechanism

Acupuncture needle rotation has been previously shown to cause specific mechanical stimulation of subcutaneous connective tissue. This study uses acupuncture to investigate the role of mechanotransduction‐based mechanisms in mechanically‐induced cytoskeletal remodeling. The effect of acupuncture needle rotation was quantified by morphometric analysis of mouse tissue explants imaged with confocal microscopy. Needle rotation induced extensive fibroblast spreading and lamellipodia formation within 30 min, measurable as an increased in cell body cross sectional area. The effect of rotation peaked with two needle revolutions and decreased with further increases in rotation. Significant effects of rotation were present throughout the tissue, indicating the presence of a response extending laterally over several centimeters. The effect of rotation with two needle revolutions was prevented by pharmacological inhibitors of actomyosin contractility (blebbistatin), Rho kinase (Y‐27632 and H‐1152), and Rac signaling. The active cytoskeletal response of fibroblasts demonstrated in this study constitutes an important step in understanding cellular mechanotransduction responses to externally applied mechanical stimuli in whole tissue, and supports a previously proposed model for the mechanism of acupuncture involving connective tissue mechanotransduction. J. Cell. Physiol.

Reduced thoracolumbar fascia shear strain in human chronic low back pain

BackgroundThe role played by the thoracolumbar fascia in chronic low back pain (LBP) is poorly understood. The thoracolumbar fascia is composed of dense connective tissue layers separated by layers of loose connective tissue that normally allow the dense layers to glide past one another during trunk motion. The goal of this study was to quantify shear plane motion within the thoracolumbar fascia using ultrasound elasticity imaging in human subjects with and without chronic low back pain (LBP).MethodsWe tested 121 human subjects, 50 without LBP and 71 with LBP of greater than 12 months duration. In each subject, an ultrasound cine-recording was acquired on the right and left sides of the back during passive trunk flexion using a motorized articulated table with the hinge point of the table at L4-5 and the ultrasound probe located longitudinally 2 cm lateral to the midline at the level of the L2-3 interspace. Tissue displacement within the thoracolumbar fascia was calculated using cross correlation techniques and shear strain was derived from this displacement data. Additional measures included standard range of motion and physical performance evaluations as well as ultrasound measurement of perimuscular connective tissue thickness and echogenicity.ResultsThoracolumbar fascia shear strain was reduced in the LBP group compared with the No-LBP group (56.4% ± 3.1% vs. 70.2% ± 3.6% respectively, p < .01). There was no evidence that this difference was sex-specific (group by sex interaction p = .09), although overall, males had significantly lower shear strain than females (p = .02). Significant correlations were found in male subjects between thoracolumbar fascia shear strain and the following variables: perimuscular connective tissue thickness (r = -0.45, p <.001), echogenicity (r = -0.28, p < .05), trunk flexion range of motion (r = 0.36, p < .01), trunk extension range of motion (r = 0.41, p < .01), repeated forward bend task duration (r = -0.54, p < .0001) and repeated sit-to-stand task duration (r = -0.45, p < .001).ConclusionThoracolumbar fascia shear strain was ~20% lower in human subjects with chronic low back pain. This reduction of shear plane motion may be due to abnormal trunk movement patterns and/or intrinsic connective tissue pathology. There appears to be some sex-related differences in thoracolumbar fascia shear strain that may also play a role in altered connective tissue function.

Ultrasound evidence of altered lumbar connective tissue structure in human subjects with chronic low back pain.

BackgroundAlthough the connective tissues forming the fascial planes of the back have been hypothesized to play a role in the pathogenesis of chronic low back pain (LBP), there have been no previous studies quantitatively evaluating connective tissue structure in this condition. The goal of this study was to perform an ultrasound-based comparison of perimuscular connective tissue structure in the lumbar region in a group of human subjects with chronic or recurrent LBP for more than 12 months, compared with a group of subjects without LBP.MethodsIn each of 107 human subjects (60 with LBP and 47 without LBP), parasagittal ultrasound images were acquired bilaterally centered on a point 2 cm lateral to the midpoint of the L2-3 interspinous ligament. The outcome measures based on these images were subcutaneous and perimuscular connective tissue thickness and echogenicity measured by ultrasound.ResultsThere were no significant differences in age, sex, body mass index (BMI) or activity levels between LBP and No-LBP groups. Perimuscular thickness and echogenicity were not correlated with age but were positively correlated with BMI. The LBP group had ~25% greater perimuscular thickness and echogenicity compared with the No-LBP group (ANCOVA adjusted for BMI, p < 0.01 and p < 0.001 respectively).ConclusionThis is the first report of abnormal connective tissue structure in the lumbar region in a group of subjects with chronic or recurrent LBP. This finding was not attributable to differences in age, sex, BMI or activity level between groups. Possible causes include genetic factors, abnormal movement patterns and chronic inflammation.

Tissue Stretch Decreases Soluble TGF-β1 and Type-1 Procollagen in Mouse Subcutaneous Connective Tissue: Evidence From Ex Vivo and In Vivo Models

Transforming growth factor beta 1 (TGF‐β1) plays a key role in connective tissue remodeling, scarring, and fibrosis. The effects of mechanical forces on TGF‐β1 and collagen deposition are not well understood. We tested the hypothesis that brief (10 min) static tissue stretch attenuates TGF‐β1‐mediated new collagen deposition in response to injury. We used two different models: (1) an ex vivo model in which excised mouse subcutaneous tissue (N = 44 animals) was kept in organ culture for 4 days and either stretched (20% strain for 10 min 1 day after excision) or not stretched; culture media was assayed by ELISA for TGF‐β1; (2) an in vivo model in which mice (N = 22 animals) underwent unilateral subcutaneous microsurgical injury on the back, then were randomized to stretch (20–30% strain for 10 min twice a day for 7 days) or no stretch; subcutaneous tissues of the back were immunohistochemically stained for Type‐1 procollagen. In the ex vivo model, TGF‐β1 protein was lower in stretched versus non‐stretched tissue (repeated measures ANOVA, P < 0.01). In the in vivo model, microinjury resulted in a significant increase in Type‐1 procollagen in the absence of stretch (P < 0.001), but not in the presence of stretch (P = 0.21). Thus, brief tissue stretch attenuated the increase in both soluble TGF‐β1 (ex vivo) and Type‐1 procollagen (in vivo) following tissue injury. These results have potential relevance to the mechanisms of treatments applying brief mechanical stretch to tissues (e.g., physical therapy, respiratory therapy, mechanical ventilation, massage, yoga, acupuncture). J. Cell. Physiol. 214: 389–395, 2008.

FIBROBLAST CYTOSKELETAL REMODELING CONTRIBUTES TO CONNECTIVE TISSUE TENSION

The visco‐elastic behavior of connective tissue is generally attributed to the material properties of the extracellular matrix rather than cellular activity. We have previously shown that fibroblasts within areolar connective tissue exhibit dynamic cytoskeletal remodeling within minutes in response to tissue stretch ex vivo and in vivo. Here, we tested the hypothesis that fibroblasts, through this cytoskeletal remodeling, actively contribute to the visco‐elastic behavior of the whole tissue. We measured significantly increased tissue tension when cellular function was broadly inhibited by sodium azide and when cytoskeletal dynamics were compromised by disrupting microtubules (with colchicine) or actomyosin contractility (via Rho kinase inhibition). These treatments led to a decrease in cell body cross‐sectional area and cell field perimeter (obtained by joining the end of all of a fibroblasts processes). Suppressing lamellipodia formation by inhibiting Rac‐1 decreased cell body cross‐sectional area but did not affect cell field perimeter or tissue tension. Thus, by changing shape, fibroblasts can dynamically modulate the visco‐elastic behavior of areolar connective tissue through Rho‐dependent cytoskeletal mechanisms. These results have broad implications for our understanding of the dynamic interplay of forces between fibroblasts and their surrounding matrix, as well as for the neural, vascular, and immune cell populations residing within connective tissue. J. Cell. Physiol. 226: 1166–1175, 2011.

Alpha smooth muscle actin distribution in cytoplasm and nuclear invaginations of connective tissue fibroblasts

Alpha smooth muscle actin (α-SMA) was recently shown to be present in mouse subcutaneous tissue fibroblasts in the absence of tissue injury. In this study, we used a combination of immunohistochemistry and correlative confocal scanning laser and electron microscopy to investigate the structural organization of α-SMA in relation to the nucleus. Furthermore, we explored colocalization analysis as a method for quantifying the amount of α-SMA in close approximation to the nucleic acid marker, 4′,6-diamidino-2-phenyl-indole, dihydrochloride. Our findings indicate the presence of α-SMA within nuclear invaginations in close proximity to the nuclear membrane, but not in the nucleoplasm. Although the function of these α-SMA-rich nuclear invaginations is at present unknown, the morphology of these structures suggests their possible involvement in cellular and nuclear mechanotransduction as well as nuclear transport.

Stretching of the Back Improves Gait, Mechanical Sensitivity and Connective Tissue Inflammation in a Rodent Model

The role played by nonspecialized connective tissues in chronic non-specific low back pain is not well understood. In a recent ultrasound study, human subjects with chronic low back pain had altered connective tissue structure compared to human subjects without low back pain, suggesting the presence of inflammation and/or fibrosis in the low back pain subjects. Mechanical input in the form of static tissue stretch has been shown in vitro and in vivo to have anti-inflammatory and anti-fibrotic effects. To better understand the pathophysiology of lumbar nonspecialized connective tissue as well as potential mechanisms underlying therapeutic effects of tissue stretch, we developed a carrageenan-induced inflammation model in the low back of a rodent. Induction of inflammation in the lumbar connective tissues resulted in altered gait, increased mechanical sensitivity of the tissues of the low back, and local macrophage infiltration. Mechanical input was then applied to this model as in vivo tissue stretch for 10 minutes twice a day for 12 days. In vivo tissue stretch mitigated the inflammation-induced changes leading to restored stride length and intrastep distance, decreased mechanical sensitivity of the back and reduced macrophage expression in the nonspecialized connective tissues of the low back. This study highlights the need for further investigation into the contribution of connective tissue to low back pain and the need for a better understanding of how interventions involving mechanical stretch could provide maximal therapeutic benefit. This tissue stretch research is relevant to body-based treatments such as yoga or massage, and to some stretch techniques used with physical therapy.

Dynamic morphometric characterization of local connective tissue network structure in humans using ultrasound

BackgroundIn humans, connective tissue forms a complex, interconnected network throughout the body that may have mechanosensory, regulatory and signaling functions. Understanding these potentially important phenomena requires non-invasive measurements of collagen network structure that can be performed in live animals or humans. The goal of this study was to show that ultrasound can be used to quantify dynamic changes in local connective tissue structure in vivo. We first performed combined ultrasound and histology examinations of the same tissue in two subjects undergoing surgery: in one subject, we examined the relationship of ultrasound to histological images in three dimensions; in the other, we examined the effect of a localized tissue perturbation using a previously developed robotic acupuncture needling technique. In ten additional non-surgical subjects, we quantified changes in tissue spatial organization over time during needle rotation vs. no rotation using ultrasound and semi-variogram analyses.Results3-D renditions of ultrasound images showed longitudinal echogenic sheets that matched with collagenous sheets seen in histological preparations. Rank correlations between serial 2-D ultrasound and corresponding histology images resulted in high positive correlations for semi-variogram ranges computed parallel (r = 0.79, p < 0.001) and perpendicular (r = 0.63, p < 0.001) to the surface of the skin, indicating concordance in spatial structure between the two data sets. Needle rotation caused tissue displacement in the area surrounding the needle that was mapped spatially with ultrasound elastography and corresponded to collagen bundles winding around the needle on histological sections. In semi-variograms computed for each ultrasound frame, there was a greater change in the area under the semi-variogram curve across successive frames during needle rotation compared with no rotation. The direction of this change was heterogeneous across subjects. The frame-to-frame variability was 10-fold (p < 0.001) greater with rotation than with no rotation indicating changes in tissue structure during rotation.ConclusionThe combination of ultrasound and semi-variogram analyses allows quantitative assessment of dynamic changes in the structure of human connective tissue in vivo.

Fibroblast cytoskeletal remodeling induced by tissue stretch involves ATP signaling

Fibroblasts in whole areolar connective tissue respond to static stretching of the tissue by expanding and remodeling their cytoskeleton within minutes both ex vivo and in vivo. This study tested the hypothesis that the mechanism of fibroblast expansion in response to tissue stretch involves extracellular ATP signaling. In response to tissue stretch ex vivo, ATP levels in the bath solution increased significantly, and this increase was sustained for 20 min, returning to baseline at 60 min. No increase in ATP was observed in tissue incubated without stretch or tissue stretched in the presence of the Rho kinase inhibitor Y27632. The increase in fibroblast cross sectional area in response to tissue stretch was blocked by both suramin (a purinergic receptor blocker) and apyrase (an enzyme that selectively degrades extracellular ATP). Furthermore, connexin channel blockers (octanol and carbenoxolone), but not VRAC (fluoxetine) or pannexin (probenecid) channel blockers, inhibited fibroblast expansion. Together, these results support a mechanism in which extracellular ATP signaling via connexin hemichannels mediate the active change in fibroblast shape that occurs in response to a static increase in tissue length. J. Cell. Physiol. 228: 1922–1926, 2013.

Visualization of macro-immune complexes in the antiphospholipid syndrome by multi-modal microscopy imaging

The antiphospholipid syndrome (APS) is an autoimmune thrombotic condition that is marked by autoantibodies against phospholipid-binding proteins. The mechanism(s) of thrombogenesis has (have) resisted elucidation since its description over thirty years ago. Nevertheless, a defining aspect of the disorder is positivity for clinical laboratory tests that confirm antibody binding to anionic phospholipids. It is remarkable that, to our knowledge, the binding of proteins from plasmas of APS patients to phospholipid has not been previously imaged. We therefore investigated this with high resolution microscopy-based imaging techniques that have not been previously used to address this question, namely atomic force microscopy and scanning electron microscopy. Atomic force microscopy imaging of APS plasmas incubated on an anionic planar phospholipid layer revealed the formation of distinct complex three-dimensional structures, which were morphologically dissimilar to structures formed from control plasmas from healthy patients. Likewise, scanning electron microscopy analysis of phospholipid vesicles incubated with APS plasmas in suspension showed formation of layered macro-immune complexes demonstrated by the significant agglomeration of a complex proteinaceous matrix from soluble plasma and aggregation of particles. In contrast, plasmas from healthy control samples bound to phospholipid vesicles in suspension generally displayed a more flattened, mat-like appearance by scanning electron microscopy. Scanning electron microscopy of plasma samples incubated on planar phospholipid layers and previously imaged by atomic force microscopy, corroborated the results obtained by mixing the plasmas with phospholipids in solution. Analysis of the incorporated proteins by silver stained SDS-polyacrylamide gel electrophoresis indicated considerable heterogeneity in the composition of the phospholipid vesicle-adsorbed proteins among APS patients. To our knowledge, these results provide the first images of plasma-derived APS immune complexes at high resolution, and show their consistent presence and heterogeneous compositions in APS patients. These findings demonstrate how high resolution microscopic techniques can contribute to advancing the understanding of an enigmatic disorder and may lay additional groundwork for furthering mechanistic understanding of APS.