Ultrastructural changes associated with the mineralization of deer antler cartilage

The maturation and mineralization of deer antler cartilage were investigated ultrastructurally by using enzymatic digestions and subsequent staining with ruthenium red (RR) or phosphotungstic acid (PTA). RR staining of matrix granules was observed in the immature prechondroblastic matrix and became more intense as the cartilage matured into a mineralized tissue. The granules got larger and more numerically dense in the mature matrix. There were matrix granules that coalesced around matrix vesicles or remnants of such in the mineralized zone. These granules were observed after demineralization, and they were RR and acidic PTA-positive (they were not susceptible to hyaluronidase nor trypsin digestion, however). It appears that the granules were modified such that the matrix vesicle formed a centralized nidus for mineralization. The growth of hydroxyapatite crystals along matrix granules (which in this zone may or may not represent proteoglycan monomers) may have caused the coalescence. Microfibrils associated with matrix granules probably represented the hyaluronic acid core of the large proteoglycan complexes because of their susceptibility to hyaluronidase digestion.     

Ultrastructural cytochemistry of carbohydrates in microfibrils associated with the amorphous elastin in the monkey aorta

Two distinct ultrastructural components of elastic fibers can be identified--namely, the amorphous elastin and the microfibrils. We have examined the tunica adventitia of monkey aortas to demonstrate differential localization of carbohydrates in elastic fibers and collagen fibrils using Thiéry's periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining of thin sections for vicinal-glycol-containing complex carbohydrates, en bloc concanavalin A (Con A) staining specific for alpha-D-mannosyl and alpha-D-glucosyl groups, and en bloc wheat germ agglutinin (WGA) staining specific for N-acetyl-D-glucosamine, N-acetylneuraminic acid, and N-acetyl-D-galactosamine. The PA-TCH-SP method moderately stained microfibrils and weakly stained collagen fibrils, but did not stain the amorphous elastin. Both Con A and WGA staining methods strongly stained microfibrils and moderately stained collagen fibrils, whereas the amorphous elastin lacked staining. Thus PA-TCH-SP, Con A, and WGA staining methods allow differential ultrastructural localization of carbohydrates in elastic fibers and collagen fibrils in monkey aortic adventitia and demonstrate the presence of more carbohydrate components in microfibrils than in collagen fibrils, whereas amorphous elastin lacks carbohydrate staining.     

Ultrastructural cytochemistry of iron absorption.

Conventional ultrastructural autoradiographic and morphologic studies of the duodenal mucosal cell have generally corroborated physiologic observations of iron absorption, but such methods have limited resolution and fail to distinguish ferric and ferrous iron. This study describes the application of the Prussian blue reaction as an electron microscopic cytochemical stain to the investigation of inorganic iron absorption in iron-deficient, normal, and iron-loaded rats. Ferrous iron is converted to ferric iron at the microvillus membrane. Subsequently intraepithelial ferric iron appears bound to a non-heme acceptor substance in microvilli and later appears as small non-membrane-bound stain deposits which are concentrated in the apical cytoplasm. The appearance of larger stain deposits in the lateral intercellular spaces, in the basal extracellular spaces, and along the intraluminal and extraluminal outer plasmalemma of adjacent endothelial cells of the lamina propria suggests passage of iron from epithelial cells through the lamina propria to blood vessels. The extreme sensitivity of the method compared with simultaneous ultrastructural autoradiographic techniques is demonstrated and suggests usefulness of the method in further studies of iron metabolism.     

Ultrastructural features of the shark brain

The ultrastructural characteristics of elasmobranch central nervous system have not been previously reported. This study presents a general assessment of aldehyde perfused brain and spinal cord in three species of shark: tiger, hammerhead and Atlantic nurse. The same distinct cell types are present in the shark that exist in mammals and similar criteria may be used for their differentiation. Neurons have frequently round nuclei with a prominent nucleoli; cytoplasm is abundant and filled with formed elements; somatic synapses and subsurface cisterns are rare. Astrocytes are smaller, and have less cytoplasm and slightly fewer organelles. Glial fibrils occur, but are not invariably present. Separating astrocytes from neurons is the most difficult identification problem. Oligodendrogliocytes are smaller, and have denser cytoplasm and a dark nucleus. The striking feature of capillary morphology is the presence of an appreciable perivascular space containing collagen; many tortuous evanginations of this space occur into surrounding glial processes which completely invest the capillaries. Astrocyte cell bodies frequently lie immediately next to vessels, and capillaries are occasionally totally surrounded by a single astrocyte process, thus being endocellular. Smaller pericapillary processes may be either astrocytic or ependymal. Dendrites, synapses, axons, and myelin have no obvious special characteristics. Sodium, visualized by precipitation techniques, is prominent in the astroglia and neurons.     

Pro-inflammatory properties of shark cartilage supplement

Abstract

The erosion and breakdown of cartilage is generally recognized to be an integral manifestation of arthritic disease, which is often accompanied by the development and progression of inflammation associated with it. Commercial shark cartilage (SC) is a popular dietary supplement taken for the prevention and/or control of chronic disease, including arthritis. The efficacy of SC in maintaining joint health remains questionable; there is a lack of sufficient reliable information on its effect on immunocompetent cells, and the potential health risks involved have not been adequately assessed. Our earlier in vitro studies showed that SC extracts induce a Th1-type inflammatory cytokine response in human leucocytes, and collagen type II alpha 1 protein was shown to be an active cytokine-inducing component in SC. In this study, we further define the cellular response to SC stimulation by classifying leucocytes into primary and secondary responders employing enriched leucocyte subpopulations. Inhibitors of specific signaling pathways were used to verify the functional effect of SC on specific pathway(s) utilized. Results indicate the monocyte/macrophage as the initially responding cell, followed by lymphocytes and the production of interferon-γ. Chemokines, MCP-1 and RANTES, were produced at significant levels in stimulated leucocyte cultures. Initial cellular activation is likely followed by activation of Jun Kinase and p38 mitogen-activated protein kinase signal transduction pathways. This study presents evidence of significant immunological reactivity of components of commercial SC supplement, which could pose a potential health risk for consumers, particularly those with underlying inflammatory disease such as irritable bowel syndrome and arthritis.     

Ultrastructural cytochemical characterization of collagen‐associated proteoglycans in the endometrium of mice

The decidual reaction in mice is characterized by the transformation of a specific population of endometrial fibroblasts into epithelioid cells, known as decidual cells. An important feature of decidualization in mice is a remarkable modification of the endometrial extracellular matrix. The present work is an ultrastructural cytochemical study of matrix with the purpose of analyzing the arrangement of collagen‐associated proteoglycans (PGs) at various regions of nulliparous endometrium and of the antimesometrial decidua of mice using the cationic dye cuprolinic blue associated with enzymatic treatments with chondroitinase ABC, chondroitinase AC, and hyaluronidase. The staining with cuprolinic blue showed PGs as rods and granules of several sizes. Rods measuring 40–60 nm in length (named F2‐rods) were apposed to thin collagen fibrils whereas granules were associated with thick collagen fibrils, particularly in the region occupied by mature decidual cells on the 7th day of pregnancy. The amount of granules was higher than that of F2‐rods. Both F2‐rods and granules were affected by chondroitinase ABC or AC treatment, indicating that they were PGs containing chondroitin sulfate and dermatan sulfate chains. However, the granules associated with thick collagen fibrils were more resistant to chondroitinase AC treatment than F2‐rods, indicating the presence of dermatan sulfate chains that contain both L‐iduronic and D‐glucuronic acid sugar residues. We suggest that the differences of the nature and amount of PGs may be associated with the changes of the thickness of collagen fibrils observed during decidualization of the endometrium in the mouse. Anat Rec 259:413–423, 2000. © 2000 Wiley‐Liss, Inc.     

Ultrastructural radioautography and cytochemistry of lead absorption.

Lead is a universal environmental contaminant absorbed largely through the gastrointestinal tract by unknown mechanisms. Because lead absorption is influenced by iron content in the body and diet, we used ultrastructural radioautography and cytochemistry to study absorption of physiologic lead doses in the rat duodenal epithelial cell and compared these findings to those previously reported for iron absorption. Rat duodenal loops exposed in vivo to 210Pb for 1 minute demonstrated the majority of labels on the microvilli, terminal web, and apical cytoplasm. Specimens exposed to radiolead for 10 minutes demonstrated more abundant labeling with a relative increase in labeling of epithelial cell mitochondria, nuclei and basal cytoplasm, as well as phagocytic cells, endothelial cells, and circulating erythrocytes of the lamina propria. Timm's sulfide-silver method localized trace metals in epithelial cells. After administration of lead, a significant increase in staining was observed in microvilli, mitochondria, non-membrane-bound cytoplasm, and nuclear chromatin. The rapid appearance of absorbed lead in epithelial cell mitochondria and nuclei, as well as phagocytic cells in the lamina propria, was distinctly different from that reported for absorbed iron and suggests different mechanisms for the subcellular transport of these cations. The combination of radioautography and Timm's sulfide-silver staining provides the specificity and resolution needed for ultrastructural evaluation of lead absorption and should be useful in further studies of lead metabolism.     

Ultrastructural morphology and cytochemistry of iron-deficient polymorphonuclear leukocytes

Previous studies have documented decreased activities of certain enzymes and altered function in polymorphonuclear leukocytes (PMN) during iron deficiency. The present study was undertaken to determine if the enzymatic abnormalities could be correlated with morphologic or quantitative change in PMN granules. Ultrastructural examination of primary and secondary granules and assessment of the secondary granule components alkaline phosphatase and vicinal glycol-containing glycoconjugates was performed in rabbit bone marrow, peripheral blood, and peritoneal heterophils. In addition, biochemical quantifications of the secondary granule component alkaline phosphatase and the primary granule marker beta-glucuronidase were performed. The results confirmed that a marked, significant decrease in alkaline phosphatase occurs in iron-deficient animals; however, no biochemical decrease in beta-glucuronidase activity was observed. Ultrastructurally, PMN secondary granules of iron-deficient rabbits tended to be more numerous than in controls when examined with morphometric and glycoconjugate staining methods, but lacked staining in alkaline phosphatase preparations. These results demonstrate that iron-deficient rabbits produce normal to increased quantities of primary and secondary granules, despite a uniform deficiency of alkaline phosphatase, a secondary granule marker.     

Micromechanical spectroscopy of cartilage proteoglycans: hydration

Proteoglycan subunits extracted from calf cartilage have been studied with a high resolving power mechanical spectroscopy: the Thermostimulated Creep (TSC). The influence of hydration on TSC spectra shows the existence of two types of bound water: the weakly bound water increases the inertia of proteoglycan and stiffens their structure; the strongly bound water is responsible to a compensation law indicating the existence of a resonance phenomenon at the physiological temperature. Because of the looseness of bonds in weakly bound water, an increase of the local pressure may induce, in vivo, a release of water in tissues. This hypothesis explains perfectly the role of a water pump of proteoglycans in cartilage.     

An ion-exchange mechanism of cartilage calcification

The provisional calcification of epiphyseal cartilage involves deposition of hydroxyapatite (calcium phosphate) crystals in an extracellular matrix consisting principally of Type II collagen and cartilage proteoglycan. A mechanism is now proposed to explain how epiphyseal cartilage calcification is initiated. Calcium exists at high concentration in cartilage, but is mainly bound to the anionic groups of proteoglycans, and thus is unavailable for precipitation. A local increase in phosphate concentration displaces calcium ions from proteoglycan by an ion-exchange effect, raising the Ca X PO4 product above the threshold for precipitation of hydroxyapatite. Evidence for this hypothesis has been derived from studies of the effect of phosphate on the binding of calcium to cartilage proteoglycan, and on hydroxyapatite formation in the presence of chondroitin sulfate.     

Ultrastructural cytochemistry of trout arterial fibrils as elastic components

The trout arterial wall contains numerous extracellular fibrils that are presumed to be elastic. However, the cytochemical properties of the arterial fibrils have not been studied. Thus, we have ultrastructurally and cytochemically examined these fibrils, utilizing routine uranyl acetate and lead (UA-Pb) double staining, the tannic acid (pH 7.0)-uranyl acetate (TA-UA) method as an electron-dense staining for elastin, and Thiéry's periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method to localize vicinal glycol-containing complex carbohydrates. The arterial fibrils, about 23 nm in thickness, were interwoven at random but frequently showed the circular alignment to the long axis of the aorta. Occasionally they appeared to coalesce side by side, and the coalesced portion tended to lose its affinity for UA-Pb stains. The TA-UA method stained the fibrils moderately to intensely and stained the coalesced parts of the fibrils more intensely. All of those TA-UA positive fibrils were completely removed after elastase en bloc digestion. The PA-TCH-SP method did not stain the arterial fibrils but stained another kind of much thinner interfibrillar filamentous structure. These results suggest that the fibrils in the wall of trout ventral aorta are elastin in nature and do not contain vicinal glycols, although the fibrils usually exist in a fibrillar form, which is unlike mammalian amorphous elastin.     

Ultrastructural cytochemistry of complex carbohydrates in developing feline neutrophils

The feline species provides animal models for at least six congenital lysosomal disorders. Since knowledge of normal feline neutrophils is a prerequisite for studies of their abnormalities, the present report describes the morphology and cytochemistry of normal feline neutrophils and compares the subcellular distribution of sulfate- and vicinal-glycol-containing complex carbohydrates to that of peroxidase and acid phosphatase. Immature feline primary granules, formed in promyelocytes, were stained for peroxidase, acid phosphatase, sulfate, and vicinal glycols. During maturation, primary granules retained strong staining for peroxidase, but staining for vicinal glycols decreased, and acid phosphatase and sulfate reactivity was lost. Secondary granules formed in myelocytes lacked peroxidase, acid phosphatase, and sulfate staining, but stained intensely for vicinal-glycol-containing complex carbohydrates. No analogues of tertiary granules previously described in rabbits and humans were demonstrated in feline neutrophils. However, a new sequential staining technique for peroxidase and vicinal glycols has suggested the formation in myelocytes and late neutrophils of a third granule type that contained peroxidase, acid phosphatase, and vicinal glycols but lacked sulfate staining. Thus, the staining characteristics of primary and secondary granules in cats closely resembled those in humans and rabbits. The third (late-forming) type of granule has not previously been described in other species.     

Ultrastructural cytochemistry of basophils in chronic myelocytic leukemia.

Cytochemical methods were used to determine the ultrastructural distribution of antimonate precipitable cations, anionic complex carbohydrate, acid phosphatase, and peroxidase in blood basophils from patients with chronic myelocytic leukemia and markedly elevated basophil counts. The morphology of these basophils varied but was generally similar to that reported for normal basophils. Antimonate precipitable cations were sparse in specific granules but more abundant in extra-granular cytoplasm and in vesicular structures of many basophils and, although varying with the fixation method, were distributed atypically in nuclei in a pattern similar to that seen in mast cells. The dialyzed iron technique diffusely stained anionic complex carbohydrate in the specific granules of basophils, and high iron diamine staining disclosed a sulfated mucosubstance in these granules. Acid phosphatase and peroxidase were distributed in a pattern similar to that of the acid mucosubstance in the basophil granules, and their presence suggested a lysosomal function for these granules     

Ultrastructural cytochemistry and radioautography of hemoglobin—iron absorption

The subcellular route of hemoglobin-iron absorption by canine intestinal epithelial cells was investigated with cytochemical and radioautographic methods and compared to that observed for inorganic iron absorption. A solution of rabbit [⁵⁹Fe]hemoglobin or an inorganic-iron solution was injected into closed duodenal loops of beagle dogs and mucosal biopsies were obtained 15, 60, and 120 min thereafter. Mucosal retention of radioactivity in dogs given hemoglobin iron was 6.9–10.2% after 120 min, and portal venous blood radioactivity was confined to the inorganic iron fraction. In the same animals diaminobenzidine (DAB)-reactive heme was visualized in microendocytic caveolae at the bases of microvilli and in membrane-bound tubulovesicular structures and granules of the apical cytoplasm. DAB-reactive heme was not identified in the lateral intercellular space or the basal extracellular space. In the same specimens, acid ferrocyanide stained ferric iron in a few apical cytoplasmic vesicles as well as along the outer lateral and basal plasmalemma but failed to identify inorganic iron in microvilli. Both the microvilli and the lateral plasmalemma appeared stained in specimens from animals given inorganic iron. In radioautographic specimens of animals given [⁵⁹Fe]hemoglobin, silver grains were most frequently associated with the apical cytoplasm and the lateral plasmalemma. We conclude that at least one mechanism of hemoglobin-iron absorption involves endocytosis and degradation of hemoglobin in membrane-bound organelles with subsequent conversion of its iron to an inorganic form, which is then transported to the lateral intercellular space where subsequent processing is similar to that observed for absorbed inorganic iron.     

Breakdown of articular cartilage proteoglycans by lymphokine-activated macrophages

Lymphokine supernatants (L_E) prepared from antigen sensitive lymphocytes caused an inhibition of migration of macrophages from capillary tubes. Control supernatants (L_C) had no effect. The lymphokine supernatants, when added to macrophage cultures (the equivalent of 60 × 10⁶ lymphocytes added to 40×l0⁶ macrophages), activated the macrophages so that they secreted the enzyme collagenase after 48 h and 72 h of culture. No collagenase was detected before 48 h or from macrophage supernatants to which L_C was added. The macrophage supernatants (L_E but not L_C) also contained factors (probably enzymes) that, when added to a piece of articular cartilage in medium, caused a partial loss of the hexosamine content of the articular cartilage. These changes were seen as early as after 24 h of culture. Activated macrophages therefore release enzymes that can completely destroy cartilage. Both collagenase and a proteoglycan-hydrolyzing enzyme are released which in vivo might be responsible for the cartilage damage that is found in diseases such as rheumatoid arthritis.