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The nuclei of mouse connective tissue fibroblasts contain chromocenters which are well-defined zones of heterochromatin that can be used as positional landmarks to examine nuclear remodeling in response to a mechanical perturbation. This study used component tree analysis, an image segmentation algorithm that detects high intensity voxels that are topologically connected, to quantify the spatial organization of chromocenters in fibroblasts within whole mouse connective tissue fixed and stained with 4',6-diamidino-2-phenylindole (DAPI). The component tree analysis method was applied to confocal microscopy images of whole mouse areolar connective tissue incubated for 30 min ex vivo with or without static stretch. In stretched tissue, the mean distance between chromocenters within fibroblast nuclei was significantly greater (vs. non-stretched, P < 0.001), corresponding to an average of a 500-nm increase in chromocenter separation (~10% strain). There was no significant difference in chromocenter number or average size between stretch and no stretch. Average chromocenter distance was positively correlated with nuclear cross-sectional area (r = 0.78, P < 0.0001), and nuclear volume (r = 0.42, P < 0.0001), and negatively correlated with nuclear aspect ratio (r = -0.65, P < 0.0001) and nuclear concavity index (r = -0.44, P < 0.0001). These results demonstrate that component trees can be successfully applied to the morphometric analysis of nuclear chromocenters in fibroblasts within whole connective tissue. Static stretching of mouse areolar connective tissue for 30 min resulted in substantially increased separation of nuclear chromocenters in connective tissue fibroblasts. This interior remodeling of the nucleus induced by tissue stretch may impact transcriptionally active euchromatin within the inter-chromocenter space., (© 2013 Anatomical Society.)

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 fibroblast's 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., (Copyright © 2010 Wiley-Liss, Inc.)

Studies in cultured cells have shown that nuclear shape is an important factor influencing nuclear function, and that mechanical forces applied to the cell can directly affect nuclear shape. In a previous study, we demonstrated that stretching of whole mouse subcutaneous tissue causes dynamic cytoskeletal remodeling with perinuclear redistribution of alpha-actin in fibroblasts within the tissue. We have further shown that the nuclei of these fibroblasts have deep invaginations containing alpha-actin. In the current study, we hypothesized that tissue stretch would cause nuclear remodeling with a reduced amount of nuclear invagination, measurable as a change in nuclear concavity. Subcutaneous areolar connective tissue samples were excised from 28 mice and randomized to either tissue stretch or no stretch for 30 min, then examined with histochemistry and confocal microscopy. In stretched tissue (vs. non-stretched), fibroblast nuclei had a larger cross-sectional area (P < 0.001), smaller thickness (P < 0.03) in the plane of the tissue, and smaller relative concavity (P < 0.005) indicating an increase in nuclear convexity. The stretch-induced loss of invaginations may have important influences on gene expression, RNA trafficking and/or cell differentiation.

Alpha smooth muscle actin (alpha-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 alpha-SMA in relation to the nucleus. Furthermore, we explored colocalization analysis as a method for quantifying the amount of alpha-SMA in close approximation to the nucleic acid marker, 4',6-diamidino-2-phenyl-indole, dihydrochloride. Our findings indicate the presence of alpha-SMA within nuclear invaginations in close proximity to the nuclear membrane, but not in the nucleoplasm. Although the function of these alpha-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.

Background: Although acupuncture-needle manipulation is an important component of acupuncture therapy, little information is currently available on whether or not specific types of needle manipulation produce different effects on the body. Bidirectional (back-and-forth) rotation is one of the most common forms of needle manipulation used in acupuncture practice., Objectives: In this study, we hypothesized that bidirectional acupuncture needle rotation causes dose-dependent active cytoskeletal remodeling in connective tissue fibroblasts similar to that previously demonstrated with unidirectional rotation., Interventions: Subcutaneous tissue explants from 18 mice were randomized to varying amounts of bidirectional rotation cycles (8-64) and rotation-cycle amplitude (180 degrees -720 degrees ) ex vivo for 30 minutes, followed by tissue fixation, confocal microscopy, and measurement of fibroblast cell body cross-sectional area., Results: As with unidirectional rotation, fibroblasts responded to bidirectional rotation with extensive cell spreading and lamellipodia formation. Bidirectional needle rotation had a significant overall effect on fibroblast cell body cross sectional area (analysis of variance, p < 0.001). The cellular response to bidirectional rotation was nonmonotonic with maximal responses occurring within specific stimulus windows with regard to cycle amplitude and cycle number., Conclusions: These findings demonstrate that subtle differences in acupuncture-needle manipulation techniques can affect cellular responses in mouse subcutaneous connective tissue. Further studies will be needed to determine whether these connective-tissue responses are related to therapeutic effects.

Cytoskeleton-dependent changes in cell shape are well-established factors regulating a wide range of cellular functions including signal transduction, gene expression, and matrix adhesion. Although the importance of mechanical forces on cell shape and function is well established in cultured cells, very little is known about these effects in whole tissues or in vivo. In this study we used ex vivo and in vivo models to investigate the effect of tissue stretch on mouse subcutaneous tissue fibroblast morphology. Tissue stretch ex vivo (average 25% tissue elongation from 10 min to 2 h) caused a significant time-dependent increase in fibroblast cell body perimeter and cross-sectional area (ANOVA, P < 0.01). At 2 h, mean fibroblast cell body cross-sectional area was 201% greater in stretched than in unstretched tissue. Fibroblasts in stretched tissue had larger, "sheetlike" cell bodies with shorter processes. In contrast, fibroblasts in unstretched tissue had a "dendritic" morphology with smaller, more globular cell bodies and longer processes. Tissue stretch in vivo for 30 min had effects that paralleled those ex vivo. Stretch-induced cell body expansion ex vivo was inhibited by colchicine and cytochalasin D. The dynamic, cytoskeleton-dependent responses of fibroblasts to changes in tissue length demonstrated in this study have important implications for our understanding of normal movement and posture, as well as therapies using mechanical stimulation of connective tissue including physical therapy, massage, and acupuncture.