No abstract available.
Cartilage, Articular* ; Knee* ; Osmium Tetroxide* ; Synovial Membrane*

The effects of tear gas, o -chlorobenzylidene malononitrile (CS) on the cytoplasmic organelles were studied in the ciliated cell of rat tracheal epithelium. Albino rats (Sprague -Dawley strain), weighing about 150gm, were used as experimental animals. The experimental animals were exposed to 2.0 g/m 3 of CS gas for 20 minutes per day for the succesive 3 days. The experimental animals were sacrified at 1, 3, 6, 12 hours and 1, 3 and 5 days after final exposure to CS gas. Specimens obtained from the trachea were pre -fixed in 2% glutaraldehyde -2.5% paraformaldehyde and post -fixed in the 1% osmium tetroxide for electron microscopic study. The results obtained were as follows: 1. In 1 hour CS gas exposed group, rough endoplasmic reticulum with dilated cisternae, and mitochondria with disrupted double membrane in the ciliated cells are found. 2. In 3 hours and 6 hours CS gas exposed groups, dilated, segmented and sacculated cisterane of rough endoplasmic reticulum, mitochondria with dissolved cristae and disrupted double membrane, and Golgi complex with atrophied cisternae are observed in the ciliated cell. 3. In 12 hours CS gas exposed group, some mitochondria with swollen cristae is found in the ciliated cell. 4. In 1 day CS gas exposed group, mitochondria with dissolved cristae, Golgi complex with hypertrophied cisternae, and autophagic vacuole are found. 5. In 3 day and 5 day CS gas exposed groups, numerous mitochondria, well -developed rough endoplasmic reticulum, and supranuclear Golgi complex are found in ciliated cell. The results of the present study suggest that the o -chlorobenzylidene malononitrile (CS) gas is cytotoxic to the ciliated cells in tracheal epithelium inducing some degenerative changes, which are recovered with the lapse of time.
Animals ; Cytoplasm* ; Endoplasmic Reticulum, Rough ; Epithelium* ; Glutaral ; Golgi Apparatus ; Membranes ; Mitochondria ; Organelles* ; Osmium Tetroxide ; Rats* ; Tear Gases ; Trachea ; Vacuoles

OBJECTIVETo introduce a practical, economical, and time-saving method to stain (with osmic acid) the myelin sheath in normal and regenerated peripheral nerves.

METHODSA total of 12 Sprague Dawley rats, weighing 250-320 g (mean equal to 276 g+/-38 g), were divided into two groups: a normal nerve group (n equal to 6) and a regenerated nerve group (n equal to 6). In the normal nerve group, the ventral and dorsal roots of L(4) to L(6) and their sciatic nerves were harvested for histological analysis. While in the regenerated nerve group, the right sciatic nerves were severed and then repaired with an epineurial microsuture method. The repaired nerves were harvested 12 weeks postoperatively. All the specimens were fixed in 4% paraformaldehyde and transferred to 2% osmic acid for 3-5 days. Then the specimens were kept in 75% alcohol before being embedded in paraffin. The tissues were cut into sections of 3 micromolar in thickness with a conventional microtome.

RESULTSUnder a light microscope, myelin sheaths were clearly visible at all magnifications in both groups. They were stained in clear dark colour with a light yellow or colorless background, which provided high contrast images to allow reliable morphometric measurements. Morphological assessment was made in both normal and regenerated sciatic nerves. The ratios of the myelin area to the fibre area were 60.28%+/-7.66% in the normal nerve group and 51.67%+/-6.85% in the regenerated nerve group, respectively (P less than 0.01).

CONCLUSIONSOsmic acid staining is easy to perform and a very clear image for morphometrical assessment is easy to obtain. Therefore, it is a reliable technique for quantitative evaluation of nerve morphology.

Animals ; Myelin Sheath ; pathology ; Nerve Regeneration ; Osmium Tetroxide ; Peripheral Nerves ; anatomy & histology ; pathology ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve ; pathology ; Staining and Labeling ; methods ; Suture Techniques

BACKGROUND: Stratum corneum lipids are arranged as intercellular membrane bilayers presumed to mediate the epidermal permeability barrier. Acute disruption in barrier function will initiate epidermal lipid synthesis, which can be prevented by occlusive membrane. Whereas, occlusion of the skin is known to cause an increased transepidermal water loss(TEWL) and enhanced percutaneous absorption of a variety of compounds. OBJECTIVE: Previous reports with electron microscopy showed varying sizes of lacunae and disorganized intercorneocyte lipids after tape stripping and occlusion with a water impermeable membrane on the murine skin. Hence we studied the effects on stratum corneum lipids and changes in barrier function after occlusion with a water-impermeable membrane. METHODS: Male hairless mice were occluded with one finger of a Latex glove for 24, 48 and 60 hours. After occlusion, TEWL was measured and biopsy specimens were taken from skin. For electron microscopic examination the samples were treated with osmium tetroxide, ruthenitum tetroxide, and tracer (lanthanum) and infrared spectroscopy were also applied. RESULTS: Occlusion with a water-impermeable membrane on the skin induced higher TEWL Values and greater penetration of the tracer than normal. Alterations of the lipid bilayer membrane and lacunae forwation in the stratum corneum interstices were also induced after 24 hours of occlusion. However, the orderness of the lipid alkyl chain in the stratum corneum was not changed until 60 hours of occlusion. CONCLUSION: These studies indicate that the increased epidermal permeability after occlusion may be due to the abnormal lipid membrane structures and volume expansion of existing lacunar domains in the stratum corneum interstices.
Animals ; Biopsy ; Fingers ; Humans ; Lanthanum ; Latex ; Lipid Bilayers ; Male ; Membranes ; Mice ; Mice, Hairless* ; Microscopy, Electron ; Osmium Tetroxide ; Permeability ; Skin ; Skin Absorption ; Spectrum Analysis ; Water

BACKGROUND: The stratum corneum(SC) has a permeability barrier function which regulates percutaneous absorption by the inhibition of transepidermal water loss(TEWL). Acute mechanical or chemical disruption of the SC induces the impairment of the permeability barrier and so increases the TEWL. The calciumtion has been recognized as an important ion in the recovery of the skin barrier. Recently the main delivery pathway of iontophoretic drugs have been suggested by SC interstices. However the morphologic changes in the SC interstices and calcium after iontophoresis have not been reported. OBJECTIVE: The aim of our study is to confirm that iontophoresis may induce changes in the SC interstices and delay the recovery of the barrier after disruption. MATERIALS AND METHODS: After tape stripping the hairless mouse flank skin, the iontophoresis power supply (6V, 0.6mA) was connected to the patch atiached for 2.5 hours to the stripped site. We checked the THWL 0, 2.5, 6, 12, 18, 24 hours after the tape stripping and obtained specimens and performed osmium tetroxide, ruthenium tetroxide postfixation and calcium ion-capture cytochemical stains for electron microscopic study. RESULTS: The recovery rate of the TEWL in iontophoresis was lower than in the control. This was especially so in the anouse which had received anode iontophoresis for 6 hours. It showed statistically lower TEWL than in the control(p<0.05). Anode iontophoresis induced low calcium in stratum granulosum (SG), but cathode iontophoresis induced high calcium in SC. After iontophoresis there were changes in the SC interstices structures. CONCLUSION: Iontophoresis can induce changes in the SC interstices and calcium distribution and so may help the topical drug delivery system.
Animals ; Calcium ; Coloring Agents ; Drug Delivery Systems ; Electric Power Supplies ; Electrodes ; Iontophoresis* ; Mice ; Mice, Hairless ; Osmium Tetroxide ; Permeability ; Ruthenium ; Skin ; Skin Absorption

BACKGROUND: Prolonged exposure of topical and systemic corticosteroid to skin can result in well-recognized cutaneous abnormalities including cutaneous atrophy, easy bruisibility, increased skin fragility, and increased risk of infection. Skin barrier impairment is also reported as a steroid-induced side effect. A major function of the skin is the formation of a permeability barrier between the external milieu and the organism. Recent studies have shown that chronic corticosteroid negatively impacts epidermal barrier function. As well as this topical corticosteroid not only has antiproliferative actions but also inhibits the differentiation of the epidermis, resulting in structural defects in the epidermis. OBJECT: We wanted to determine whether high dose systemic steroid injection would display adverse effects, specifically on; epidermal functions, permeability barrier homeostasis and stratum corneum integrity and cohesion. The basis for such changes was also to be determined. MATERIAL AND METHODS: Systemic steroid was administered by injecting each hairless mouse, 8-10 week of age, intraperitoneally with 0.3 mg triamcinolone acetonide, two times per week for five weeks. For the controlled hairless mice, 0.9% normal saline was administered by the same method of injection. Every week, transepidermal water loss (TEWL) was checked and skin biopsies were taken. Skin specimens were prepared for electron microscopy using both 0.25% ruthenium tetroxide and 4% osmium tetroxide postfixation. For light microscopy staining hematoxylin-eosin and ion capture cytochemistry was used. RESULTS: The results were as follows; 1. From about 1 week onwards, high dose systemic steroid usage produced visible cutaneous changes and significantly increased the TEWL in the group of 0.3 mg triamcinolone acetate injected hairless mice compared with the control. 2. Light microscopic observations of the steroid-injected hairless mice showed gradual thinning of the epidermis from about 2 weeks onwards, compared with the control. Loss of stratum corneum was also observed in the steroid injected hairless mice. 3. The ruthenium tetroxide staining of high dose systemic steroid treated specimens revealed that the lipid bilayer was impaired and fragmented from about 3 weeks. Intercellular spaces were widened and the lipid bilayer either disappeared or showed damage when compared with the control. 4. From about 3weeks onwards. electron microscopic studies revealed, not only a marked decrease in the number of lamellar bodies, but also an abnormal transformation of lamellar bodies in the steroid injected hairless mice compared with the control. 5. Throughout the five weeks, the calcium gradient gradually disappeared in the 0.3mg triamcinolone injected hairless mice compared with the control. Consequently, high dose systemic steroid use results in barrier dysfunction and morphological abnormalities.
Animals ; Atrophy ; Biopsy ; Calcium ; Epidermis ; Extracellular Space ; Histocytochemistry ; Homeostasis ; Lipid Bilayers ; Mice ; Mice, Hairless* ; Microscopy ; Microscopy, Electron ; Osmium Tetroxide ; Permeability ; Ruthenium ; Skin* ; Triamcinolone ; Triamcinolone Acetonide

BACKGROUND: The stratom corneum lipids, responsible for the epidermal water bar rier, consist mainly of ceramides, cholesterol and free fatty acids. However, little has been studied about the effects of non-polar, polar and mixed organic solvents on the changes of the stratum corneum lipids bilayer. OBJECTIVE: We designed this study in order to investigate the effect of non-polar, polar and mixed organic solvents on the lipids bilayer in hairless mice. METHODS: Twenty four hairless mice were evenly divided into 4 groups; a control group, chloroform treated group, methanol treated group and mixed solvent(chloroform/methanol(2:1) ) treated gr oup. The changes in transepidermal water loss, as measured with an evaporimeter, were recorded after topical application of either chloroform, methanol or mixed solvents at 0, 3, 6, 12, 24 and 48 hours respectiveh. For electron microscopy, the skin samples taken from the mice of ea.:h group were t.rented with osmium tetroxide and ruthenium tetroxide after the treatment with each solvent. RESULTS: The results were as follows ; 1. From 0 to 24 hours after treatment with each solvent transepidermal water loss was significantly increased in the chloroform and the mixed solvent[chloroform/methanol(2:1)] treated groups, compared to the methanol treated group and control group(P<0.001). 2. 48 hours after treatment with each solvent, the differences in the values of transepidermal water loss in all groups were insignificant. 3. On electronmicroscopic examination, separation of intercellular lipid bilayers and a decrease in the numher of lamellar bodies were more severe in the chloroform treated and mixed solvent (chloroform/methanol(2:1)] treated groups than in the methanol treated group. Application of non-polar organic solvents, especially mixed solvents[chloroform/methanol(2:1)] resulted in an increase in transepidermal water loss and greater structural changes than with polar organic solvents. CONCLUSION: Our results suggest that non-polar lipids may play a more important role in the protection of water vaporization of the stratum corneum lipids barrier than polar lipids.
Animals ; Ceramides ; Chloroform ; Cholesterol ; Fatty Acids, Nonesterified ; Lipid Bilayers ; Methanol ; Mice ; Mice, Hairless* ; Microscopy, Electron ; Osmium Tetroxide ; Ruthenium ; Skin* ; Solvents* ; Steam

This experiment was performed to evaluate the morphological responses of 5-fluorouracil or mitomycin C on the spleen of the mice. 5-fluorouracil (60 mg/kg) or mitomycin C (400 microgram/kg) were injected subcutaneously every other day, and the animals were sacrificed at 1 week and 2 weeks following the first injection. Pieces of the tissue were taken from the spleen, fixed in 10% neutral formalin for light microscopy. The paraffin sections stained with hematoxylin-eosin, Masson-trichrome, Bielschowsky's silver impregnation or aldehydefuchsin. For electron microscopy, the tissues were prefixed with 2.5% glutaraldehyde-1.5% paraformaldehyde followed by post-fixed with 1% osmium tetroxide. The ultrathin sections stained with uranyl acetate and lead citrate. The observed results were as follows: 1. On histological study, in the mitomycin C treated group, macrophages which contain pyknotic nuclei were observed more frequently than in those of 5-fluorouracil treated group. 2. In the 5-fluorouracil treated group, positive reactions to Masson-trichrome and Bielschowsky's silver impregnation were observed in the splenic capsule and traculae at the 1 week, and weak postive stains were observed at the 2 weeks. 3. In the mitomycin C and the 5-fluorouracil group, positive staining reaction to aldehyde-fuchsin were observed in splenic capsule, trabeculae and around artery at the 1 week and 2 weeks. 4. On the ultrastructural study, distended cisternae of granular endoplasmic reticula were observed frequently at 1 week. 5. In the mitomycin C treated group, myelin figures in the lymphocytes and reticular cells were observed more frequently than in those of 5-fluorouracil treated group. From the above results, it was concluded that lymphocytes and reticular cells of the spleen were slightly damaged by 5-fluorouracil or mitomycin C, and mitomycin C seems more harmful on the spleen than 5-fluorouracil does.
Animals ; Arteries ; Citric Acid ; Coloring Agents ; Fluorouracil* ; Formaldehyde ; Lymphocytes ; Macrophages ; Mice ; Microscopy ; Microscopy, Electron ; Mitomycin* ; Myelin Sheath ; Osmium Tetroxide ; Paraffin ; Silver ; Spleen*

This experiment was performed to study the morphological responses of the splenic white pulp, the lymphatic tissue of the spleen, of Ehrlich carcinoma cell-implanted mice to three different anticancer drugs (5-fluorouracil, mitomycin C and AG60). Healthy adult ICR mice weighing 20 g each were divided into normal and experimental groups. In the experimental groups, each of mice was inoculated with 1X10(7) Ehrlich carcinoma cells subcutaneously in the inguinal area. From next day, 0.2 ml of saline solution, 5-fluorouracil (30 mg/kg), mitomycin C (400 microgram/kg) or AG60 (30 mg/kg, Taerim Pharm. Co., Seoul, Korea) were injected subcutaneously every other day, and animals were sacrificed at 14th day following the f irst injection. Pieces of the tissues were taken from the spleen, and prefixed with phosphate buffered 2.5% paraformaldehyde-1.5% glutaraldehyde solution (pH 7.3) followed by post-fixation with phosphate buffered 1% osmium tetroxide solution (pH 7.3). Fixed tissue blocks were dehydrated, and embedded in araldite mixture. Ultrathin sections stained with uranyl acetate and lead citrate were observed with a JEM 100CX-II electron microscope. In the experimental control group (carcinoma cell-inoculated mouse), splenic white pulp did not show pronounced morphological alterations, but myelin figures were frequently observed in the cytoplasm of some lymphocytes and reticular cells than those of normal control mice. In the AG60 treated group, splenic white pulp did not show specific morphological defect, but nuclear bodies and severe invaginations of the nuclear envelope of the lymphocytes and reticular cells were observed occasionally. In the mitomycin C treated group, myelin figures, severe invaginations of the nuclear envelope, nuclear protrusions, nuclear bodies and interchromatin granules were frequently observed in the lymphocytes and reticular cells of the white pulp. In the 5-f luorouracil treated group, myelin f igures, severe invaginations of the nuclear envelope, nuclear protrusions, nuclear bodies and interchromatin granules were observed more frequently in the lymphocytes and reticular cells of the white pulp, as compared with those of mitomycin C treated mice. From the above results, 5-f luorouracil or mitomycin C may suppress the splenic immune function of cancerinoculated mice, since they suppress the process of differentiation and maturation of splenic lymphocyte and reticular cells, and 5-fluorouracil was more harmful on the spleen than mitomycin C. Whereas AG60 does not affect remarkably the process of differentiation and maturation of lymphocytes and reticular cells in the splenic white pulp.
Adult ; Animals ; Antineoplastic Agents* ; Citric Acid ; Cytoplasm ; Fluorouracil ; Glutaral ; Humans ; Lymphocytes ; Lymphoid Tissue ; Mice* ; Mice, Inbred ICR ; Mitomycin ; Myelin Sheath ; Nuclear Envelope ; Osmium Tetroxide ; Seoul ; Sodium Chloride ; Spleen

This experiment was performed to study the morphological responses of the spleen of mouse inoculated with Ehrlich carcinoma cells, following administration of Bacillus Calmette-Guerin (BCG). Healthy adult ICR mice weighing 25 g each were divided into normal and experimental groups (experimental control group and BCG treated group). In the experimental groups, each mouse was inoculated with 1X10(7) Ehrlich carcinoma cells subcutaneously in the inguinal area. From next day, 0.2 ml of saline or BCG (0.03X10(8)-0.32X10(8) CFU) were injected subcutaneously to the animals every other day. The day following the 7th injection, each mouse was injected with a single dose of 0.7 microCi/gm of methyl-3H-thymidine (25 Ci/mmol, Amersham Lab., England) through tail vein. Seventy minutes after the 3H-thymidine injection, animals were sacrificed. Pieces of the splenic tissue, fixed in 10% neutral formalin for light microscopy. The sections were coated with autoradiographic emulsion EM-1 (Amersham Lab., England) and the coated sections were exposured for 5 weeks in the dark room. For electron microscopy, tissues were prefixed with phosphate buffered 2,5% glutaraldehyde-1.5% paraformaldehyde (pH 7.3), and post-fixed with phosphate buffered 1% osmium tetroxide solution (pH 7.3). Ultrathin sections of the white pulp area stained with uranyl acetate and lead citrate were observed with a JEM 100CX-II electron microscope. On histological study in the splenic white pulp, BCG treated mice showed more macrophages containing pyknotic nuclei than normal or experimental control mice showed. On autoradiographic study, a large number of the 3Hthymidine labeled cells were seen near the marginal zone, whereas only a small number of labeled cells were seen in the red pulp or the white pulp of the spleen. The number of the labeled cells in experimental control group was similar to that in the normal control mice, whereas that in BCG-treated mice was significantly increased as compared with that of normal control one. On electron microscopic study, in the white pulp of BCG treated mouse, mitotic cells were observed more frequently than in those of the normal or experimental control mice. In the BCG treated mice, macrophages and plasma cells in the white pulp were observed more frequently than in those of the normal or experimental control mice, whereas a few eosinopile leucocytes were observed, and perichromatin granules within the nuclei of the lymphocytes and the reticular cells were observed frequently. From the above results, it was concluded that DNA syntheses were more active in the cells of the marginal zone than in the cells of the white pulp or the red pulp. And repeated treatment with BCG could activate the DNA syntheses of splenic cells and increase the number of the macrophages and the plasma cells in the white pulp.
Adult ; Animals ; Autoradiography ; Bacillus* ; Citric Acid ; DNA ; Formaldehyde ; Humans ; Lymphocytes ; Macrophages ; Mice* ; Mice, Inbred ICR ; Microscopy ; Microscopy, Electron ; Mycobacterium bovis ; Osmium Tetroxide ; Plasma Cells ; Spleen* ; Veins