Microvasculature and loose connective tissue in freeze-fracture preparations

Summary Tissues of the young chick and chick embryo were prepared in a relatively unaltered condition by the freeze-fracture technique. The ultrastructure of the microvasculature and surrounding interstitial region is compared with that seen in conventional thin-sectioned material. In the undifferentiated vessels of the 3-day chick embryo, no distinct basement lamina can be distinguished in either type of preparation. In the 3-week chick, a continuous basement lamina is present beneath the endothelium only in chemically fixed and sectioned tissue; it cannot be distinguished from the remaining interstitial substance in freeze-fracture preparations. Blood-tissue exchange may depend on permeability characteristics of the entire interstitial region rather than on the basement lamina alone.Work supported by the U.S. Atomic Energy Commission.We acknowledge the services of George T. Chubb and Paul W. Ellwanger in maintaining the electron microscope center.     

Ultrastructural changes in loose connective tissue histiocytes in dehydration and starvation]

Changes in the parameters of the volumetric fractions of the main cytoplasmic organellae were studied in the subcutaneous connective of albino rats subjected to experimental dehydration and starvation. There were some common features in the histiocytes reaction under these conditions, i. e. hypertrophy of the lysosomal apparatus and cell "infiltration" with lipids. At the same time the following differences are emphasized: dehydration caused an increase of the volumetric mitochondrial fractions and of the rough endoplasmic reticulum, and starvation of the phagosomal fraction.     

Tissue stretch induces nuclear remodeling in connective tissue fibroblasts

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 α-actin in fibroblasts within the tissue. We have further shown that the nuclei of these fibroblasts have deep invaginations containing α-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.     

Lysozyme concentration in the loose connective tissue of animals with different species sensitivity to brucellosis]

Lysozyme content was determined in the subcutaneous cellular tissue of guinea pigs and albino rats differing by species resistance to brucella infection. Experiments were conducted on intact animals and those inoculated subcutaneously with live Br. abortus 19-BA vaccine. Connective tissue was taken from the site of the vaccine administration and from the contralateral side (control). Observations showed connective tissue of the animals highly sensitive to brucella infection to be exceedingly poor in this enzyme; as to connective tissue of albino rats with low sensitivity--it contained high amount of this enzyme. Lysozyme content increased considerably in the animals belonging to both species in the inflammatory focus developing at the site of the vaccine inoculation.     

结缔组织铺片脂肪组织油红染色在组织学实验教学中的应用

目的在传统结缔组织铺片基础上开展脂肪组织油红染色方法在医学本科生组织学实验教学中的应用。方法学生先进行疏松结缔组织铺片,并施行脂肪组织油红o-甲苯胺兰-伊红三重染色,然后镜下观察。结果油红o染色把结缔组织中的脂肪细胞内脂滴保存下来并染上红色。脂肪组织中央的细胞脂滴均匀红染,充满胞浆,周边的脂肪细胞胞浆中油红染色很少,细胞呈空泡状,显示出脂肪细胞亚群存在。甲苯胺兰染色使得疏松结缔组织中肥大细胞染成紫红色,胞核染色浅,细胞数量多、成群分布。伊红可使得结缔组织内除脂肪细胞、肥大细胞意外的其他细胞的胞浆和胶原纤维染成淡红色。结论传统的组织学平铺片技术基础上引入脂肪油红o-甲苯胺兰-伊红三重染色,可增强学生动手能力,并能很好地了解输送结缔组织中细胞的不同表型和分布,丰富组织学内容,把教学、科研连接一起,达到提高实验教学质量的目的。     

Rapid and efficient preparation of free amino acids from strong acid salts on columns of crosslinked poly-4-vinylpyridine

Acidic and neutral amino acids were recovered from their strong acid salts or from large excesses of strong acids by passage through columns of crosslinked poly-4-vinylpyridine. Basic amino acids eluted as the monoacid salts. High recoveries were possible with submicromolar samples.     

Organization of connective tissue patterns by dermal fibroblasts in the regenerating axolotl limb

A set of tendons, aponeurotic sheets and retinaculae, which transduce muscle action from proximal limb levels to flexion and extension of the digits, is found in limbs of many vertebrates. This set of structures, here termed the digit tendon complex, is described for the axolotl forelimb. We show that the complex forms autonomously in muscleless axolotl limb regenerates produced from a cuff of unirradiated dermis surrounding an irradiated limb stump, and persists for up to a year after amputation. The pattern of other connective tissue structures, including the skeleton, is also normal. Fibroblast condensations that may represent sets of these cells normally associated with muscles in the extensor and flexor compartments of the carpal region also form in muscleless limbs. The results are discussed in terms of the importance of the dermis in pattern regulation, selforganization of connective tissues in general and autonomous development of the digit tendon complex in particular.     

Structural investigation of loose connective tissue by using a series of dextran fractions as non-interacting macromolecular probes.

The ability of the uncharged open-coil dextran molecules to penetrate tissue space, without coil-shape change, was utilized to probe (by partitioning experiments) the structural arrangement of the collagen-fibre network and the proteoglycan system. Hyaluronidase digests most of the proteoglycans away and enables the respective contributions to the exclusion volume to be evaluated by using a series of different-molecular-weight dextrans. It appears that the major part of the exclusion volume is due to the collagen-fibril as a rod and the dextran coil as an impenetrable sphere. The additional exclusion due to the proteoglycans could be accounted for by a set of points (regions of high proteoglycan-segment density) over which the dextran coild cannot pass. These points are an average of 50 nm apart and are indicative of local extensive entanglement of high-molecular-weight proteoglycans with each other. Reasons are given why these entanglements could not act as cross-links in long-term elastic loading of the tissue.     

Macromolecular basis of globular protein exclusion and of swelling pressure in loose connective tissue (Umbilical cord)

The macromolecular basis of tissue swelling pressure and of the ability of tissue to exclude globular proteins, according to size, have been investigated using human umbilical cord. Exclusion data of tissue, and tissue from which the polysaccharides had been removed by hyaluronidase were compared. Exclusion of globular proteins by the polysaccharides, obtained by difference from the two sets of data, was similar to that reported for isolated polysaccharides in solution. It can be described by a sphere/cylinder geometric exclusion model. The exclusion behavior of the polysaccharide-free tissue was accounted for in terms of the component collagen fibrils, glycoprotein microfibrils and cells. Average pore diameters of 18 and 110 nm, respectively, for the intact tissue and for the polysaccharide-free tissue were estimated. Swelling pressure measurements were performed on intact, on hyaluronidase-treated and on hyaluronidase and then Pronase-treated tissues to obtain the contributions of the polysaccharides, of collagen and of microfibrils. Close to the in vivo volume of tissue, the swelling pressure is given almost entirely by the polysaccharides and is consistent with the osmotic pressure expected from the relative amounts of hyaluronic acid and proteoglycan present and their distribution in the extrafibrillar, extracellular space. Upon swelling or deswelling a small net contribution of the fibrillar system to the swelling pressure is evident.     

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.     

Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration

Muscle regeneration requires the coordinated interaction of multiple cell types. Satellite cells have been implicated as the primary stem cell responsible for regenerating muscle, yet the necessity of these cells for regeneration has not been tested. Connective tissue fibroblasts also are likely to play a role in regeneration, as connective tissue fibrosis is a hallmark of regenerating muscle. However, the lack of molecular markers for these fibroblasts has precluded an investigation of their role. Using Tcf4, a newly identified fibroblast marker, and Pax7, a satellite cell marker, we found that after injury satellite cells and fibroblasts rapidly proliferate in close proximity to one another. To test the role of satellite cells and fibroblasts in muscle regeneration in vivo, we created Pax7(CreERT2) and Tcf4(CreERT2) mice and crossed these to R26R(DTA) mice to genetically ablate satellite cells and fibroblasts. Ablation of satellite cells resulted in a complete loss of regenerated muscle, as well as misregulation of fibroblasts and a dramatic increase in connective tissue. Ablation of fibroblasts altered the dynamics of satellite cells, leading to premature satellite cell differentiation, depletion of the early pool of satellite cells, and smaller regenerated myofibers. Thus, we provide direct, genetic evidence that satellite cells are required for muscle regeneration and also identify resident fibroblasts as a novel and vital component of the niche regulating satellite cell expansion during regeneration. Furthermore, we demonstrate that reciprocal interactions between fibroblasts and satellite cells contribute significantly to efficient, effective muscle regeneration.     

Constitutive connective tissue growth factor expression in scleroderma fibroblasts is dependent on Sp1

Fibrotic diseases such as scleroderma (systemic sclerosis, SSc) are characterized by an excessive production of extracellular matrix and profibrotic proteins such as connective tissue growth factor (CTGF). In normal dermal fibroblasts, CTGF is not expressed unless induced by proteins such as tumor growth factor-beta (TGFbeta). Conversely, in fibroblasts cultured from fibrotic lesions CTGF mRNA and protein are constitutively expressed, even in the absence of exogenously added TGFbeta. Thus, studying the mechanism underlying CTGF overexpression in SSc fibroblasts is likely to yield valuable insights into the basis of the fibrotic phenotype of SSc and possibly other scarring disease. CTGF overexpression is mediated primarily by sequences in the CTGF promoter. In this report, we identify the minimal promoter element involved with the overexpression of CTGF in SSc fibroblasts. This element is distinct from the element necessary and sufficient for the induction of CTGF expression by TGFbeta in normal fibroblasts. Within this region is a functional Sp1 binding site. Blocking Sp1 activity reduces the elevated, constitutive levels of CTGF promoter activity and protein expression observed in SSc fibroblasts. Relative to those prepared from normal dermal fibroblasts, nuclear extracts prepared from SSc fibroblasts possess increased Sp1 binding activity. Removal of phosphate groups from nuclear extracts enhanced Sp1 binding activity, suggesting that phosphorylation of Sp1 normally reduces Sp1 binding to DNA. Thus, the constitutive overexpression of CTGF in SSc fibroblasts seems to be independent of TGFbeta signaling but dependent at least in part on Sp1.