Ultrastructure Of Connective Tissue Research Articles

Vascular histopathology and connective tissue ultrastructure in spontaneous coronary artery dissection: pathophysiological and clinical implications.

Aims Spontaneous coronary artery dissection (SCAD) is a cause of acute coronary syndromes and in rare cases sudden cardiac death (SCD). Connective tissue abnormalities, coronary inflammation, increased coronary vasa vasorum (VV) density, and coronary fibromuscular dysplasia have all been implicated in the pathophysiology of SCAD but have not previously been systematically assessed. We designed a study to investigate the coronary histological and dermal collagen ultrastructural findings in SCAD.Methods and resultsThirty-six autopsy SCAD cases were compared with 359 SCAD survivors. Coronary and myocardial histology and immunohistochemistry were undertaken. Transmission electron microscopy (TEM) of dermal extracellular matrix (ECM) components of n = 31 SCAD survivors and n = 16 healthy volunteers were compared. Autopsy cases were more likely male (19% vs. 5%; P = 0.0004) with greater proximal left coronary involvement (56% vs. 18%; P < 0.0001) compared to SCAD survivors. N = 24 (66%) of cases showed no myocardial infarction on macro- or microscopic examination consistent with arrhythmogenic death. There was significantly (P < 0.001) higher inflammation in cases with delayed-onset death vs. sudden death and significantly more inflammation surrounding the dissected vs. non-dissected vessel segments. N = 17 (47%) cases showed limited intimal fibro-elastic thickening but no features of fibromuscular dysplasia and no endothelial or internal elastic lamina abnormalities. There were no differences in VV density between SCAD and control cases. TEM revealed no general ultrastructural differences in ECM components or markers of fibroblast metabolic activity.Conclusions Assessment of SCD requires careful exclusion of SCAD, particularly in cases without myocardial necrosis. Peri-coronary inflammation in SCAD is distinct from vasculitides and likely a reaction to, rather than a cause for SCAD. Coronary fibromuscular dysplasia or increased VV density does not appear pathophysiologically important. Dermal connective tissue changes are not common in SCAD survivors.

Spontaneous coronary artery dissection: novel pathophysiological insights from histological and ultrastructural tissue analysis

Abstract Introduction Spontaneous coronary artery dissection (SCAD) is a cause of acute coronary syndromes and rarely sudden cardiac death (SCD). SCAD is characterised by medial false lumen haematoma formation and periadventitial inflammatory cell infiltrate. Although SCAD has been linked to connective tissue disorders, its pathophysiology remains poorly understood and the role of inflammation unknown. Purpose We sought to establish the definitive histopathological features of SCAD and explore pathophysiological mechanisms through assessment of dermal connective tissue ultrastructure. Methods N=36 SCD cases diagnosed as SCAD on autopsy were identified in pathology archives at four international centres. Their demographic and clinical characteristics were compared with n=359 survivors recruited in a SCAD survivors cohort. Haematoxylin &amp; eosin sections were examined under light microscope. Immunohistochemistry (IHC) was employed for quantification of inflammatory cell infiltrate (CD68, CD3) and vasa vasorum density (CD31) of SCAD cases (n=20) compared to age- and sex-matched controls (n=10). Dermal extracellular matrix components (EMC) of n=32 SCAD survivors and n=16 healthy volunteers (HV) were compared using electron microscopy (EM). Results The autopsy series cases were more likely to be male (p=0.0256) and had higher incidence of left main stem (p=0.0475) and proximal left anterior descending (p&amp;lt;0.001) disease compared to SCAD survivors. N=24 (66%) of SCAD autopsy case showed no evidence of myocardial necrosis. N=17 (47%) showed mild-moderate atherosclerotic changes but no features of fibromuscular dysplasia. There were no differences in vasa vasorum density between SCAD and control cases (A). The degree of inflammatory cell infiltrate varied greatly but significantly higher than controls (B), comprising CD68+ macrophages, eosinophils and CD3+ positive T-cells. There was a statistically significant association (p=0.006) between the degree of inflammatory cell infiltrate and the length of time from onset of symptoms to death (Panel C), as well as significantly (p&amp;lt;0.001) denser inflammatory cell infiltrate adjacent to the dissection plane (D, exemplary sections E&amp;F). EM revealed no differences between SCAD and HV in dermal fibroblast size &amp; activity or elastin size &amp; damage indicators, but possible changes in subgroups with more extreme clinical phenotype or pregnancy-related SCAD (G). Conclusions To our knowledge this is the largest SCAD pathology case series so far. We show for the first time that periadvential inflammation in SCAD appears to be time-dependent and localising to the dissected coronary segment, suggesting healing response to injury rather than causal contribution. We found no evidence to suggest increased vasa vasorum density is pathophysiologically important. Connective tissue changes were only linked to a small proportion of cases. These novel findings may have important implications for the management of SCAD patients. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): British Heart Foundation, Leicester NIHR Biomedical Research Centre

Tenderization and fragmentation of myofibrillar proteins in camel semitendinosus muscle of high connective tissue content using proteolytic extract from ginger rhizome and papain

This study was conducted to develop a method for improving tenderness and overall qualities of tough camel's meat using plant proteolytic enzymes. 3x3 cm uniform-sized from Semitendinosus muscle of old female camel's meat Camelus dromedaries (over 7 years) were marinated with 7% freshly prepared ginger extract, 0.7% papain extract and mixture of papain extract (0.6%) and freshly prepared ginger extract (5%) then kept at 4°C for 3 days. The tenderized as well as the control samples were subjected to analysis in the due time in terms of sensory, bacterial, physicochemical, ultrastractural and electrophoretic examinations. Improvement of sensory scores and significant (P< 0.05) decreased APC, Coliforms, Fecal Coliforms were observed in all enzyme treated samples. Physico-chemical parameters showed significant(P< 0.05) reduction in collagen content, MFD, TBA value, shear force and significant (P< 0.05) increase total soluble protein, collagen solubility %, MFI and SL in all enzyme treated samples compared to control. Electrophoretic pattern of muscle proteins also revealed extensive proteolysis and reduction in number of protein bands in all treated samples. While, histological evaluation showed significant changes in the ultrastructure of connective tissue and muscle fiber. Mixture of both enzymes induced the most obvious effect on camel's meat tenderness followed by papain extract and finally ginger extract.

Ultrastructure of connective tissue of eye drainage system in ophthalmic hypertension associated with primary juvenile glaucoma

In primary juvenile glaucoma, the connective tissue of the juxtacanalicular zone and sclera undergo constant reorganization because of incessant synthesis and degradation of extracellular matrix components with pronounced fluctuations of electron density of collagen fibrils. Destructive changes in the perivascular nerve trunks were detected at all stages of the glaucoma process, including the initial stages: numerous large foci of cytoplasmic organelle destruction with the formation of autophagosomes and residual bodies predominated in the cytoplasm of myelinated axons of nerve cells.

Etiology of inguinal hernia: ultrastructure of rectus sheath revisited

In the last decade, in the search for abdominal-wall hernia etiology, attention has been brought to alterations in the connective tissue ultrastructure as the probable etiological factor. These may cause weakening of connective tissue, which in turn may form ground for hernia formation. To investigate this hypothesis in depth, we compared the ultrastructure of the connective tissue in hernia patients and the control group. The study group consisted of five patients with primary inguinal hernia (Nyhus II = 4, Nyhus IIIa = 1). Another five patients posted for emergency appendectomy created the control group. Tissue specimens, harvested intraoperatively from the rectus muscle sheath (RAMS) and fixed in 4% glutaraldehyde, underwent staining by the Masson, H-E and methylene blue techniques and were assessed by microscopy (light and scanning electron). The examinations showed significant differences in the rectus sheath ultrastructure. They included altered architecture, placement and quantity of collagen and elastic fibers, differences in the caliber of individual fibers and disrupted ground matter-to-fiber ratio. In patients with hernias, chaotic arrangement of collagen fibers was seen, as well as their thinning and a decrease in the general amount of elastic fibers, replaced by ground matter. Our research has shown significant differences in the structure of the RAMS between patients with hernias and healthy individuals. This supports the theory linking connective tissue alterations with the etiology of hernia, and stating that these alterations include connective tissue at locations distant from the hernia site as well, as the rectus sheath itself does not form a hernial defect.

The ultrastructure of periductal connective tissue and distinctive populations of collagen fibrils associated with ductal epithelia of exocrine glands.

The ultrastructure of the connective tissue around the intraglandular ducts was observed in rat exocrine glands. Connective tissue with a dense population of collagen fibrils was found either around the ducts and blood vessels (perivasculoductal connective tissue; PVDCT) as in the lacrimal and salivary glands and liver, or solely surrounding the ducts (periductal connective tissue; PDCT) as in the exocrine pancreas, whereas the interlobular and intralobular interstitium of the glands-except for the liver-contained substantially fluid-filled spaces without collagen fibrils. The PVDCT and PDCT of these glands contained two populations of collagen fibrils-fibroblast-associated and epithelium-associated-although the development and density of these fibrils varied considerably in individual glands. Both populations of collagen fibrils were most developed in the lacrimal glands, in which the basal aspects of the ductal epithelium and the basement membrane showed considerable undulation associated with a distinctive peribasement membrane zone with amorphous matter and a small population of the fibrils. In the parotid and submandibular glands, both populations were distinct, though poorly developed. In the exocrine pancreas and hepatic Glisson's sheath, the two populations of collagen fibrils were moderately developed, and the basal aspects of the ductal epithelium were characterized by prominent invaginations in which the multilaminar basement membranes and the epithelium-associated collagen fibrils were frequently engulfed. These observations provide evidence that the two populations of collagen fibrils around the ducts are found universally in exocrine glands, and support the hypothesis of the collagen fibril-synthesizing and -secreting ability of ductal epithelial cells.

The Ultrastructure of the Connective Tissue Matrix is Significantly Improved Using Cryo-Fixation At High Pressure

The investigation of connective tissue ultrastructure has historically relied on chemical fixation to stabilize micro architecture. These fixatives are not effective in retaining many matrix molecules, including proteoglycans, and allow precipitation of many other components in subsequent processing steps. The result is a preponderance of open space between matrix components. Cells shrink considerably and a precipitation of molecules in highly concentrated regions leads to artifacts including the lamina lucida and lamina densa of basement membranes. Cryo-fixation at high pressure followed by freeze substitution seeks to immobilize tissue components within vitreous (non-crystalline) ice, which is later substituted with acetone at temperatures below the recrystalization temperature of water. Formation of large ice crystals during any part of the preparation protocol significantly distorts structure and can be recognized in the compound microscope. Recognition of smaller freezing artifacts, including microcrystalline ice, is imperative for interpretation of resulting images.

Paraurethral connective tissue in stress-incontinent women after menopause.

To study whether stress urinary incontinence after menopause is correlated to changes in the paraurethral connective tissue ultrastructure and metabolism. Transvaginal biopsies were obtained from the paraurethral connective tissue in stress urinary incontinent women after menopause with and without estrogen replacement therapy, and from comparable controls. All the stress-incontinent women underwent urodynamic investigation. In the specimens, collagen concentration, measured as hydroxyproline, and the degree of extractability by pepsin digestion, were quantified. Proteoglycan composition and concentration were analyzed using Alcian Blue precipitation, followed by electro-phoretic separation and quantification. Using Northern blots, mRNA levels for the collagens I and III, the small proteoglycans decorin and biglycan, and the large proteoglycan versican, were quantified. Collagen structure was examined with transmission electron microscopy, and the diameters of collagen fibrils were analyzed with an interactive image analysis system (IBAS, Zeiss/Kontron). No significant difference in paraurethral connective tissue biochemistry or ultrastructure was registered between women with stress incontinence and controls. Estrogen replacement therapy resulted in a lower collagen concentration both between the controls (p = 0.02) and between the incontinent women (0.02). In the women with stress incontinence also the extractability by pepsin digestion was higher in the group with estrogen treatment (p = 0.004), indicating a decrease in cross-linking. The proteoglycan/collagen ratio was higher in the control group with estrogen treatment compared to untreated (p = 0.02), but no difference was found between estrogen treated and untreated incontinent women. The median collagen fibril diameter was 15% larger in the incontinent group of women without estrogen therapy compared to the control group and 5% larger when comparing the incontinent group on estrogen replacement therapy to the corresponding control group. The extracellular matrix of paraurethral connective tissue in stress urinary incontinent women after menopause reacted differently to estrogen replacement therapy compared to continent controls. In contrast to incontinent women of fertile age no major changes in collagen metabolism were found in stress urinary incontinent women after menopause.

Different organization of collagen fibrils in stress-incontinent women of fertile age.

The objective was to test the hypothesis that stress urinary incontinence in women is correlated to changes in the paraurethral connective tissue ultrastructure and metabolism. Transvaginal biopsies were obtained from the paraurethral connective tissue in women of fertile age with stress urinary incontinence and in matched continent controls. All the stress-incontinent women were characterized with urodynamic investigation. In the biopsies, collagen concentration, measured as hydroxyproline, and the degree of extraction by pepsin digestion were quantified. Proteoglycan composition and concentration were analyzed using Alcian blue precipitation, followed by electrophoretic separation and quantification. Using Northern blots mRNA levels for the collagens I and III, the small proteoglycans decorin and biglycan, and the large proteoglycan versican, were quantified. Collagen organization was examined with transmission electron microscopy and the diameters of collagen fibrils were analyzed with an interactive image analysis system (IBAS, Zeiss/Kontron). The biochemical and morphological analyses exposed a significant difference in the paraurethral connective tissue between stress urinary incontinent women before menopause and comparable controls. The collagen concentration was almost 30% higher and the diameters of the collagen fibrils were 30% larger in the incontinent group of women. Also the organization of the collagen fibrils differed, with considerably higher cross-linking. A higher level of mRNA for collagen I and III in the incontinent group indicates that the differences can be related to an altered collagen metabolism. No change of proteoglycan amount or composition was observed, resulting in a significantly lower proteoglycan/collagen ratio in the incontinent group of women. Stress urinary incontinence in fertile women is associated with a change in collagen metabolism resulting in an increased concentration of collagen and larger collagen fibrils. These alterations should result in a more rigid form of extracellular matrix, suggesting a connective tissue with impaired mechanical function.

Lysyl oxidase activity, collagen cross-links and connective tissue ultrastructure in the heart of copper-deficient male rats

Copper deficiency is characterized by multiple connective tissue manifestations, such as skeletal and joint abnormalities, and vascular lesions that lead to aneurysms and aortic rupture. However, the rupture of the heart observed in cooper-deficient male rats is not fully understood. We demonstrated the effect of copper deficiency on cardiac collagen content and solubility, regional differences in cardiac lysyl oxidase activity, collagen cross-links, and on the ultrastructural morphology of collagen in the myocardium. Weaned male rats fed copper-deficient or copper-adequate diets were examined. Copper-deficient rats died prematurely of heart rupture at the apex, and those who survived exhibited heart enlargement, decreased cardiac lysyl oxidase activity with increasing heart soluble collagen content. Mature collagen cross-links, as assessed by the concentrations of pyridinoline and deoxypyridinoline, were lower both in the apex and in the right and left ventricles of copperdeficient rats as compared with copper-adequate controls. However, in copper-deficient rats, cross-links levels were significantly lower at the apex than at the left and right ventricles. In addition, severe ultrastructural abnormalities in the size and shape of endo-and epimysium collagen fibers were observed in the apex of copper-deficient rats. Transmission electron microscopy showed giant, spiralled or frayed, and spiny collagen fibers. These findings allow us to postulate that the reduced cardiac lysyl oxidase activity accompanied by altered collagen cross-links and an abnormal connective tissue ultrastructure play a significant role in the manifestation of copper deficiency in the male rat.

On the theory of the spatial organization of macromolecules in connective tissue.

The radial distribution function characterizing the spatial organization of the long fibrils of connective tissue is obtained by mathematical analysis of molecular models. The models are based on the assumption that polymeric chains form bridges between the fibrils, thereby providing the long range interactions responsible for the quasi-ordered spatial disposition of the fibrils. The theory is applied to rabbit cornea for which an empirical radial distribution has been obtained previously by analysis of electron micrographs. General agreement is found between theory and experiment for parameter values that are thought to be representative of stroma. The analysis constitutes a step toward the development of the physical basis of the ultrastructure of connective tissue and the way in which that structure affects physiological behavior.