Number Of Secondary Lysosomes Research Articles

Ultrastructural analysis of human umbilical cord derived MSCs at undifferentiated stage and during osteogenic and adipogenic differentiation

ABSTRACTMesenchymal stem cells (MSCs) are considered as an important tool for regenerative medicine and experimental treatments. Unveiling the ultrastructural changes during the differentiation of MSCs might help us to understand the nature of the process and to develop novel therapeutic approaches. For this purpose, human umbilical cord (hUC) was chosen as MSC source. In the first place, MSCs were isolated from sub-amniotic, intervascular and perivascular areas of hUC by enzymatic and tissue explant method to determine the most favorable region of hUC and technique for further processing. Therefore, microscopic and growth kinetics analyses showed that there was no clear difference in the morphologies and proliferation rates among the hUC-MSC groups. Flow cytometric analysis showed that CD44 and CD90 MSC markers were highly expressed, while CD34 and CD45 hematopoietic stem cells markers were expressed at low degree. Because our preliminary results showed that there was no conspicuous superiority among the hUC-MSCs groups, whole UC was utilized as a source, and tissue explant method was applied to isolate MSCs for further differentiation analysis. At the 1st and 3rd week of osteogenic and adipogenic differentiation, ultrastructural analysis showed an increase in the number of secondary lysosomes in comparison with the undifferentiated status. Increase in the mitochondrial content was also detected at the 1st week of adipogenic differentiation. Consequently, ultrastructural changes including increase in the number of mitochondria and secondary lysosomes during the adipogenic and osteogenic differentiation could be attributed to the switch in energy metabolism of the MSCs and increment in the lysosomal activity respectively.

Ultrastructural Changes and Corneal Wound Healing After SMILE and PRK Procedures

ABSTRACTPurpose: To compare keratocyte activation, cellular morphologic changes and wound healing after SMILE and PRK procedures using transmission electron microscope (TEM).Methods: In this study, 22 New Zealand white rabbits (10- to 15-week old) were used. The right eyes of all animals underwent SMILE procedure and the left eyes underwent PRK procedure. Cornea samples taken 1 day and 1 week postoperatively were examined using TEM.Results: Using TEM 1 day after SMILE procedure, the organization of collagen fibers seemed to have been preserved without thermal alterations. Keratocyte activation was observed in the anterior stroma. Disrupted collagen arrangement and debris of cells are visible in the area of damage, and some phagocytic cells and a large number of secondary lysosomes are visible in those cells. At the perimeter zone of the interface, many coenocytes and collagen fragments could be found within the phagocytic cell. One week after SMILE procedure, potential lacuna could be discerned. A large part of the interface of the lenticule extracted had an appearance of clearly being adhered to some mucus secretions. One day after PRK procedure, an irregular epithelial surface was visible using TEM. Keratocytes had been activated and the rough endoplasmic reticulum in those cells had expanded. One week after PRK procedure, the epithelial surface still was irregular and keratinization of the epithelium was still visible in some areas. Corneal endothelium cells were mildly damaged and some vacuoles within the cytoplasm could be discerned. In the anterior stroma, some unhealthy activated keratocytes could still be observed. New collagen fibrils were found present near the activated keratocytes.Conclusion: Using TEM, keratocyte activation could still be observed after SMILE compared to after PRK procedure. Fewer cellular ultrastructural changes were seen after SMILE procedure. Unlike in PRK procedure, no damaged epithelium and endothelium were found after SMILE.

Developmental competence and ultrastructural changes of heat-stressed mouse early blastocysts produced in vitro

Abstract Mouse early blastocysts were exposed to temperatures of 39°C and 41°C for 2 h, respectively, to determine their developmental competence and ultrastructural changes. The results showed that heat stress at 41°C for 2 h, significantly reduced the percentages of expanded and hatched blastocysts, but not at 39°C for 2 h. The average cell numbers in expanded blastocysts, which developed from early blastocysts heat-stressed at temperatures of 39°C and 41°C, were significantly reduced. The average cell numbers in hatched blastocysts subjected to heat stress were no different from those in the control group cultured at 37°C. The mitochondria of the early blastocysts heat-stressed at 39°C for 2 h, were slightly swollen, but they had recovered after culturing at 37°C for 2 h. However, the mitochondria in the blastocysts heat stressed at 41°C for 2 h were severely swollen, and their number increased. The ribosomes shed from the rough endoplasmic reticulum, and the number of secondary lysosomes in the plasma increased. The integrity of desmosomes was disrupted. The space between the nuclear envelope and the perivitelline membrane enlarged. The fibre fraction and the particulate fraction of nucleoli were separated. The heterochromatin in nucleoli was also increased in its quantity. There were some lamellar-shape structures and heterogeneous dense materials exhibiting in the cytoplasm. The ultrastructural changes induced by heat shock at 41°C for 2 h were not reversible. In conclusion, the damage of heat stress to mitochondria, lysosomes, ribosomes and cell nucleus, may be one of the most important factors that inhibit the normal development of mouse early blastocysts.

Immunohistochemical and ultrastructural investigation of the human vestibular dark cell area: Roles of subepithelial capillaries and T lymphocyte‐melanophage interaction in an immune surveillance system

Background The aim of the present study was to morphologically characterize the structure of the subepithelial blood vessels in the dark cell area of the human vestibular organs, and to determine whether immunocompetent cells such as macrophages and lymphocytes could be found around these small blood vessels. Materials and Methods All 31 surgical specimens (semicircular canals and utricles) were obtained from patients with vestibular schwannoma. Formalin fixed specimens were stained with hematoxylin and eosin (H&E), and with antibodies to von Willebrand Factor (vWF), leukocyte common antigen (LCA), and UCHL-1, and were examined with light microscope. Specimens fixed with glutaraldehyde were examined with a transmission electron microscope (TEM). Observations: Subepithelial blood vessels stained positive for vWF. By TEM observation, these blood vessels were observed to be capillaries that consisted of non-fenestrated endothelium, occasional pericytes, and a basement membrane. They were usually accompanied by melanophages with a number of secondary lysosomes containing phagocytosed degraded melanosomes and lipid droplets. Moreover, melanocytes and their cell processes directly surrounded these subepithelial capillaries. The fact that cells which were positively stained with LCA and UCHL-1 were present both in the intra- and subepithelial layer of the specimens, and that by TEM the intra- and subepithelial mononuclear cells with a lymphoid appearance had clustered dense bodies in their cytoplasm, suggested that they were a population of T lymphocytes. Conclusions Results suggested the possibility of a T lymphocyte-melanophage (macrophage) interaction, both originating from and harbored around subepithelial capillaries, which suggests the presence of an immune surveillance system in the human vestibular organs. Anat. Rec. 249:153–162, 1997. © 1997 Wiley-Liss, Inc.

Immunohistochemical and ultrastructural investigation of the human vestibular dark cell area: roles of subepithelial capillaries and T lymphocyte-melanophage interaction in an immune surveillance system.

The aim of the present study was to morphologically characterize the structure of the subepithelial blood vessels in the dark cell area of the human vestibular organs, and to determine whether immunocompetent cells such as macrophages and lymphocytes could be found around these small blood vessels. All 31 surgical specimens (semicircular canals and utricles) were obtained from patients with vestibular schwannoma. Formalin fixed specimens were stained with hematoxylin and eosin (H&E), and with antibodies to von Willebrand Factor (vWF), leukocyte common antigen (LCA), and UCHL-1, and were examined with light microscope. Specimens fixed with glutaraldehyde were examined with a transmission electron microscope (TEM). Subepithelial blood vessels stained positive for vWF. By TEM observation, these blood vessels were observed to be capillaries that consisted of non-fenestrated endothelium, occasional pericytes, and a basement membrane. They were usually accompanied by melanophages with a number of secondary lysosomes containing phagocytosed degraded melanosomes and lipid droplets. Moreover, melanocytes and their cell processes directly surrounded these subepithelial capillaries. The fact that cells which were positively stained with LCA and UCHL-1 were present both in the intra- and subepithelial layer of the specimens, and that by TEM the intra- and subepithelial mononuclear cells with a lymphoid appearance had clustered dense bodies in their cytoplasm, suggested that they were a population of T lymphocytes. Results suggested the possibility of a T lymphocyte-melanophage (macrophage) interaction, both originating from and harbored around subepithelial capillaries, which suggests the presence of an immune surveillance system in the human vestibular organs.

Fine structure and detoxification kinetics in kupffer cells after injection of endotoxin in rats

The aim of this study was to clarify the role of Kupffer cells in the mechanism of endotoxin-induced liver injury. The study on fine structure of Kupffer cells was performed after the injection of endotoxin. The endotoxin(Escherichia coli lipopolysaccharide 026 : B6. 1.5mg/100 g of body weight) was intraperitoneally injected in Sprague-Dewley rats. Animals were sacrificed at 1/4, 1/2, 1, 2, 4, 8, 16, 24, 72 and 120 hours after the injection of endotoxin. Livers were extirpated and processed to be examined by light and electron microscopy. The results obtained were summerized as follows: Early changes observed in liver after endotoxin injection included the increased number and hypertrophy of Kupffer cells, infiltration of neutrophils and presence of fibrin thrombi within the sinusoids. The continuous increase of the Kupffer cells in number with hypertrophy, congestion and infiltration of inflammatory cells within the sinusoids were observed. Hepatocytes showed fatty change and occasional necrosis. At 72 hours the congestion decreased. At 120 hours the number of Kupffer cells was increased, but the morphology of Kupffer cells became similar to that of the control group. The numbers and sizes of primary and secondary lysosomes and amount of euchromatin of Kupffer cells increased. Swellings and increase in number of mitochondria, Golgi complex, smooth endoplasmic reticulum, rough endoplasmic reticulum were evident. Microthrombi were present within the sinusoids. The swelling of rough endoplasmic reticulum and mitochondria, decrease of glycogen particles, fatty change, hypoxic vacuoles, pyknotic nuclei and occasional necrosis were observed in hepatocytes. At 72 hours the number of secondary lysosomes in Kupffer cells decreased. At 120 hours the morphology of Kupffer cells became similar to that of the control group. According to these results, it was postulated that the endotoxin was initially taken up by pinocytosis into Kupffer cells and degraded in secondary lysosomes of activated Kupffer cells. Kupffer cells may play an important role in the defense mechanism of liver during endotoxemia. The dysfunction of Kupffer cells and ischemia by sinusoidal microthrombi may cause liver injury.

Induction and exacerbation of hyaline droplet formation in the proximal tubular cells of the kidneys from male rats receiving a variety of pharmacological agents

Kidneys from male and female Wistar rats dosed with 1 of 3 chemically unrelated pharmacological agents, a pyrazoline BW540C, a napthoquinone BW58C and the levoisomer of tetramisole, levamisole or the light hydrocarbon Decalin ®, were examined by light and electron microscopy. Paraffin histology showed that all 4 agents induced and exacerbated hyaline droplet accumulation in the renal proximal tubular cells of the male rats. Resin histology at both the light and electron microscope level, along with cytochemical procedures for acid phosphatase and the protein ‘alpha 2U globulin’, helped further in the characterisation of these cytoplasmic inclusions. These techniques confirmed that the accumulation of hyaline droplets seen by paraffin histology represented an increase in the size and number of secondary lysosomes which have been shown to be involved in protein uptake and metabolism. Time course studies showed that increased numbers of small dense lysosomes appear first, which then increase in size, presumably by fusion. Crystalloid bodies form in these large lysosomes eventually giving rise to rectilinear bodies. The accumulation of these protein laden secondary lysosomes took place primarily in the cells of the S 1 and S 2 segments of the proximal tubules. In extreme cases of lysosomal accumulation however, loading of the S 3 segments was noted. In tubules where cellular inclusion loading was heavy, there was evidence of increased cell turnover. The kidneys of female rats dosed with any one of the 4 agents appeared normal.

Acute hepatocellular effects of erythromycin, gentamicin, and trospectomycin in the perfused rat liver: lack of correlation between lamellar body induction potency and cytotoxicity.

The formation of multilamellar inclusion bodies in cytoplasm is a generalized cellular response to treatment with a variety of chemical agents. The present study was conducted to determine if a correlation exists between acute lamellar body induction potency and cytotoxicity in the perfused rat liver. Livers were perfused for 3 hrs with various concentrations of erythromycin, gentamicin, sulfate, or trospectomycin sulfate, all of which are known to produce lamellar bodies in the rat in vivo. At the end of the experiments, the livers were perfusion fixed for transmission electron microscopy. Based on the bile flow rate, perfusion rate at constant pressure, and cytoplasmic enzyme release, neither gentamicin nor trospectomycin was hepatotoxic at concentrations up to 1.8 mM, whereas erythromycin was toxic at 0.1 mM. Gentamicin caused no ultrastructural changes compared to controls, but trospectomycin caused the dose-dependent formation of lamellar bodies in hepatocytes without other cytoplasmic alterations. Erythromycin caused cellular degeneration accompanied by an increase in the number of secondary lysosomes, but these lacked lamellated inclusions. It is concluded that hepatic lamellar bodies can be induced in acute ex vivo experiments, but that their formation does not appear to be linked with acute cytotoxicity.

Ultrastructure of mucocartilage in the larval anadromous sea lamprey, Petromyzon marinus L.

The fine structure of mucocartilage, a tissue unique to larval lampreys, was examined in Petromyzon marinus L. This tissue is surrounded by a perichondrium of vascularized, dense connective tissue composed of fibroblasts, collagen fibrils, and elastic-like microfibrils, but it is avascular itself and consists of elastic-like microfibrils, ground substance, and a few diffusely scattered fibroblasts. Fibroblasts possess rough endoplasmic reticulum, may free ribosomes, a well-developed Golgi apparatus, a tubulo-vesicular network, and a number of secondary lysosomes containing crystalline material. The appearance of the organelles suggests the involvement of the cell in the synthesis and secretion of the ground substance and microfibrils. Tubular microfibrils, 11-13 nm in diameter, comprise the major portion of the matrix, and they are similar to those described in developing mammalian elastic tissue (Ross and Bornstein, 1969). The retention of the microfibrils may represent either a primitive form of elastic fiber in this "primitive" vertebrate or reflect the larval condition of the lampreys under examination. Scattered spherical to polyhedral-shaped mitrix granules and intergranular filaments make up the remainder of the matrix. It was concluded that mucocartilage in larval lampreys is not a conventional type of vertebrate connective tissue.

Lysosomal enzyme activities during ageing of adult human liver cell lines

Ten lysosomal enzyme activities have been compared during the growth and ageing of adult human liver cell lines. Arylsulfatase A, β-D-galactosidase and β-D-glucuronidase activities were significantly lower and arylsulfatase B activity was significantly higher in senescent cells than in actively growing cells. Furthermore, hexosaminidase activity was lower and acid phosphatase activity higher in old cells in every cell line tested, but the differences were not significant. On the other hand, no change occurred in α-L-fucosidase, α-D-mannosidase, α-D-galactosidase and α-D-glucosidase activities. These results demonstrate that the increase in size and number of secondary lysosomes during ageing is accompanied for a few lysosomal enzymes by an increase or a decrease in activity depending on the enzyme.

Cytochemistry of Gastrodermal Autophagy Following Starvation in Schistosoma mansoni

The gastrodermis of adult Schistosoma mansoni was examined by electron microscopy to determine the effects of starvation and the effects of hycanthone, administered in vitro. Special attention was focused on the relationship of the Golgi complexes with the process of autophagy. In general, autophagy was increased in the gastrodermis when it was exposed to stress conditions such as starvation and hycanthone. Acid phosphatase and thiamine pyrophosphatase activities were used as enzyme markers for the Golgi complexes and lysosomes. During the early stages of starvation, there was a 4-fold increase in the number of Golgi complexes per unit area in the gastrodermis. A progressive increase in the number of secondary lysosomes was evident as starvation time was increased. Hycanthone accelerated the effects of starvation. It was hypothesized that acid hydrolases are passed to the Golgi complexes via ER-derived vesicles. The enzymes are subsequently released as primary lysosomes from the Golgi complex to fuse with cytosegresomes and form secondary lysosomes (cytosomes).

The cytological effect of ecdysterone on the midgut cells of the flesh-fly Sarcophaga bullata.

Larvae of the flesh-fly, Sarcophaga bullata, were injected with the synthetic moulting hormone ecdysterone or saline at the beginning of the third and final larval instar. One group was left untreated. The ecdysterone-injected larvae showed an increase in number of secondary lysosomes in the midgut epithelial cells similar to that observed at the onset of metamorphosis, an event which would normally occur about 48 hr later in these larvae.