This experiment was performed to evaluate the morphological responses of the gastric epithelial cells and the gastric chief cells of the mouse inoculated with Ehrlich carcinoma cells in the inguinal area following administration of acriflavine-guanosine composition (AG60). Healthy adult ICR mice were divided into normal and experimental groups. In the experimental groups, each mouse was inoculated with 1x10(7) Ehrlich carcinoma cells subcutaneously in the inguinal area. The day following the 7th injection of saline or AG60, each mouse was injected with methyl-3H-thymidine through tail vein. Seventy minutes after the thymidine injection, gastric tissues were taken and fixed in 10% buffered neutral formalin. Deparaffinized sections were coated with autoradiographic emulsion EM-1 and dried, and then placed in a light-tight box. The number of labeled epithelial cells in the gastric mucosae were observed and calculated. And for electron microscopic observation, gastric tissues were prefixed with 2.5% glutaraldehyde-1.5% paraformaldehyde solution, followed by post-fixation with 1% osmium tetroxide solution. The ultrathin sections were stained with uranyl acetate and lead citrate. The size of zymogen granules and mitochondria in the gastric chief cells were observed and calculated. On the autoradiographic study, number of labeled cells in the area of 3.5 mm width (6 micrometer thickness) of mouse gastric mucosae of normal control, tumor control and AG60-treated groups were 319.7+/-66.46, 343.7+/-47.72 and 102.3+/-54.99 respectively. On the electron microscopic study, the size of zymogen granule in the gastric chief cells of normal control, tumor control and AG60-treated groups were 0.74+/-0.208 micrometer, 1.18+/-0.291 micrometer and 0.97+/-0.259 micrometer, respectively. And the mitochondrial size of the gastric chief cells of normal control, tumor control and AG60-treated groups were 0.86+/-0.364 micrometer, 1.02+/-0.466 micrometer and 0.92+/-0.390 micrometer, respectively. And in the AG60 treated group, most chief cells did not show any difference in ultrastructure, except that myelin figures were more frequently observed, in comparison with that of nornmal control group. From the above results, AG60 may suppress the DNA synthesis of the gastric epithelial cells, but does not results severe fine structural defect on the gastric chief cells. These results suggest that AG60 is expected as one of the most effective anticancer drugs.
Adult ; Animals ; Chief Cells, Gastric ; Citric Acid ; DNA ; Electrons ; Epithelial Cells ; Formaldehyde ; Gastric Mucosa ; Humans ; Mice ; Mice, Inbred ICR ; Mitochondria ; Mitochondrial Size ; Myelin Sheath ; Organometallic Compounds ; Osmium Tetroxide ; Polymers ; Secretory Vesicles ; Thymidine ; Veins

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

This experiment was performed to evaluate the morphological responses of the mucosa of the mouse appendix, inoculated with Ehrlich carcinoma cells in the inguinal area, following administration of Acriflavine-Guanosine Composition (AG60). Healthy adult ICR mice weighing 25 gm each were divided into normal, experimental control and AG60 treated group. Experimental control and AG60 treated groups, mice were subcutaneously inoculated with 1 x 10(7) Ehrlich carcinoma cells in the inguinal area. From next day after the carcinoma cell inoculations, 0.2 mL of saline or AG60 (5 mg/kg/0.2 mL) were injected subcutaneously to the animals every other day, respectively. The day following the 7 th injection of saline or AG60, 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, and appendix tissues were fixed in 10% formalin solution for light microscopy. The number of the labeled mucosal epithelial cells of the appendix were observed and evaluated. For the electron microscopic study, the tissues were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde solution, followed by post-fixation with 1% osmium tetroxide solution. Ultrathin sections were counter stained with uranyl acetate-lead citrate solutions, and observed. On light microscopic observation of experimental control and AG60 treated mice, did not show any remarkable morphological alterations on the mucosae. On autoradiographic study, number of the labeled cells within 3.5 mm width mucosae of normal control, experimental control, AG60 treated mice were 362.2+/-56.12, 350.7+/-42.65 and 90.7+/-33.48, respectively. On ultrastructural observation of the experimental control and AG60 treated mice, general morphologies of the epithelial cells of appendix were similar. But intranuclear filamentous structures, intramitochondrial dense granules, and myelin figures were occasionally observed in the absorptive cells of AG60 treated mice than control ones. Above results show that AG60 suppress the DNA synthetic activity of the mucosal epithelial cells of mouse appendix, but did show slight ultrastructural alterations in the absortive cells. These results suggest that AG60 is one of effective anticancer drug for the cytostatic therapy.
Adult ; Animals ; Appendix* ; Citric Acid ; DNA* ; Epithelial Cells* ; Formaldehyde ; Humans ; Mice* ; Mice, Inbred ICR ; Microscopy ; Mucous Membrane ; Myelin Sheath ; Osmium Tetroxide ; Robenidine ; Veins

This experiment was performed to evaluate the morphological responses of the gastric chief cells of the mouse, inoculated with Ehrlich carcinoma cells in the inguinal area, following administration of 5-fluorouracil or mitomycin C. Healthy adult ICR mice weighing 25 gm each were divided into normal and experimental groups (experimental control group, 5-fluorouracil-treated group and mitomycin C-treated group). In the experimental group, 1x107 Ehrlich carcinoma cells were inoculated subcutaneously in the inguinal area. From next day after inoculations, 0.2mL of saline (experimental control group), 5-fluorouracil (30 mg/kg, 5-fluorouracil-treated group), or mitomycin C (400 microg/kg, mitomycin C-treated group) were injected subcutaneously to the animals every other day, respectively. The day following the 7th injection, animals were sacrificed. Pieces of the tissue were taken from the gastric mucosa, prefixed with 2.5% glutaraldehyde-1.5% paraformaldehyde solution, followed by post-fixation with 1% osmium tetroxide solution. The ultrathin sections were stained with uranyl acetate and lead citrate. The size of zymogen granule and the size of the mitochondrion in the gastric chief cells were observed and compared. In the 5-fluorouracil treated group, most chief cells did not show any difference in ultrastructure, except myelin figures were more frequently observed, in comparison with those of normal control group. But in the mitomycin Ctreated group, necrotic cells were more frequently observed than in normal control and 5-fluorouracil-treated group. The size of zymogen granule in the gastric chief cells of normal control, experimental control, 5-fluorouracil-treated and mitomycin C-treated groups were 0.98 (+/-0.108)microm, 1.05 (+/-0.092)microm, 0.94 (/-0.123)microm and 0.93 (+/-0.156)microm, respectively. And the size of mitochondrion in the gastric chief cells of normal control, experimental control, 5-fluorouracil-treated and mitomycin C-treated groups were 0.80 (+/-0.130)microm, 0.83 (+/-0.143)microm, 0.87 (+/-0.165)microm and 0.81 (+/-0.083)microm, respectively. From the above results, in the treatment of low therapeutic doses of anticancer drugs into the animals inoculated with Ehrlich carcinoma cells, 5-fluorouracil may not suppress function of the gastric chief cells, but mitomycin C may exert a vicious influence on the function of the gastric chief cells.
Adult ; Animals ; Chief Cells, Gastric* ; Citric Acid ; Fluorouracil* ; Gastric Mucosa ; Humans ; Mice* ; Mice, Inbred ICR ; Mitochondria ; Mitomycin* ; Myelin Sheath ; Osmium Tetroxide ; Secretory Vesicles

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

Prefrontal cortex is called psycological cortex, since it deals with making up of individual personality, regulation of personal depth of feeling, working memory, planning, maintaining attention, etc. Whereas, nucleus accumbens (septi) is called the center of reward and motivation or the center of pleasure, since it deals with feeding, drinking, sex, exploration, appetitive learning, drug addiction, etc. Present study was aimed at the proving the prefronto-accumbens input ultrastructurally. Sprague Dawley rats anesthetized with sodium pentobarbital and were removed their prefrontal cortex with suction instrument. Two days following the operation, heads of rats were fixed by perfusion of with 1% glutaraldehyde-1% paraformaldehyde solution via left ventricle. Peristaltic pump was used during perfusion. Two hours later, brains were removed and refixed for 24 hours in the refrigerator, and small tissues of the nucleus accumbens were punched out with punching needle. Tissue blocks were fixed in 2% osmic acid for 2 hours and were embedded in araldite mixture. Ultrathin sections stained with uranyl acetate-lead citrate solution were observed with JEOL 100 CX II electron microscope. In the nucleus accumbens, some axodendritic terminals and axospinous terminals were found degenerated, and volume of activated glial cytoplasm was increased. The degenerated terminals were seen isolated from intact structures by activated glial processes and removed by glial cytoplasm. The result confirms that axon terminals coming from prefrontal cortex input to the spiny neurons of nucleus accumbens septi, on their dendrites and/or dendritic spines.
Animals ; Brain ; Citric Acid ; Cytoplasm ; Dendrites ; Dendritic Spines ; Drinking ; Head ; Heart Ventricles ; Humans ; Learning ; Memory, Short-Term ; Motivation ; Needles ; Neurons ; Nucleus Accumbens ; Osmium Tetroxide ; Pentobarbital ; Perfusion ; Pleasure ; Prefrontal Cortex ; Presynaptic Terminals ; Rats* ; Rats, Sprague-Dawley ; Reward ; Sodium ; Substance-Related Disorders ; Suction ; Synapses*

This experiment was performed to evaluate the morphological responses of the gastric parietal cells of mouse inoculated with Ehrlich carcinoma cells, following administration of Bacillus Calmette -Guerin (BCG) or acriflavine -guanosine composition (AG60, Taerim Pharm. Co. Seoul, Korea). In the experimental groups, each mouse was inoculated with 1 X 10(7) Ehrlich carcinoma cells subcutaneously in the inguinal area. From next day, 0.2 ml of saline, BCG (0.03 X 10(8) ~0.32 X 10(8) CFU) or AG60 (30 mg/kg) was injected subcutaneously to the animals every other day. Animals were sacrificed on the 14th day from the first injection. Pieces of the tissue were taken from the stomach, prefixed with 2.5% glutaraldehyde -1.5% paraformaldehyde, followed by post -fixation with 1% osmium tetroxide. The ultrathin sections stained with uranyl acetate and lead citrate were observed with a JEM 100CX -II electron microscope. In the experimental control, the BCG and the AG60 treated groups, most parietal cells showed reduced lumenal spaces of the intracellular canaliculi, since microvilli of intracellular canaliculi were very irregularly shaped and crowed with each other. And in the BCG and the AG60 treated mice, myelin figures, lysosomes and multivesicular bodies in the parietal cells were observed more frequently than in those of the experimental control ones. In the BCG treated rats, membranes of the tubulovesicles of the parietal cells were disintegrated, but the similar changes were not observed in the AG60 treated mice,. Above results suggest that the BCG treated animals inoculated with Ehrlich carcinoma cells might suffer from reduced acid secretion of the parietal cell, since the disintegration of the tubulovesicular membranes in the parietal cells are occurred following injections. Whereas AG60 dose not affect remakably defect on the parietal cells.
Acriflavine ; Animals ; Bacillus ; Citric Acid ; Crows ; Gastric Mucosa ; Glutaral ; Lysosomes ; Membranes ; Mice* ; Microvilli ; Multivesicular Bodies ; Mycobacterium bovis* ; Myelin Sheath ; Osmium Tetroxide ; Parietal Cells, Gastric* ; Rabeprazole ; Rats ; Seoul ; Stomach

In cancer therapy, immunological disorder is one of most severe problem. Since thymic cortex is the home of T-cell proliferation and "education", thymic morphology following administration of certain drugs can be used as a parameter of immunological safety of the drug. In this study, morphology of thymic cortex, following administration of 5-fluorouracil or AG60, was studied. AG60 is a newly developed anti-cancer remedy, the compound of acriflavine and guanosine (1 : 1). ICR mice were subcutaneously inoculated with Ehrlich carcinoma cells (10(7) cells/mouse) in their inguinal areas. Each mouse in 5-fluorouracil group was injected subcutaneously with a single dose of 30 mg/kg of 5-fluorouracil every other day, and the mouse in AG60 group, with 30 mg/kg of AG60 (Taerim Pharm. Co., Seoul) every other day. The control mouse was injected with saline. The mice were sacrificed on the day after 7th injection. Tissues of thymic cortices were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde solution (0.1 M Millonig's phosphate buffer, pH 7.3), and refixed in 2% osmium tetroxide solution (0.1 M Millonig's phosphate buffer, pH 7.3). Tissue blocks were dehydrated, and were embedded in araldite mixture. For the overview-comparison, semithin sections stained with toluidine blue solution were photographed. And the typical portions were cut with ultratome, stained and observed with electron microscope. In light microscopy, thymic cortical morphology of AG60-injected mouse was similar with that of control mouse. But the cortical morphology of 5-fluorouracil-injected mouse was impressively different from those of the control or AG60 group mice. Thymocytes in the thymic cortex of 5-fluorouracil-injected mice were severely depleted. In electron microscopy, thymocytes in the thymic cortices of the control or AG60 group mice were crowded, and small groups of thymocytes were surrounded by the cytoplasmic processes of epithelial reticular cells. Mitotic figures were randomly seen. Thymocytes of 5-fluorouracil-injected mouse were naked out from the epithelial reticular cells, and were completely depleted out from the cortex composed mainly of enlarged epithelial reticular cells. Numerous microvilli were protruded from the naked thymocytes. The results were interpreted as that 5-fluorouracil induce leukopenia, and homing of lymphocytes to thymic cortex is severely depressed. 5-fluorouracil also disturb the normal protective and supportive function of epithelial reticular cells for thymocytes. Whereas the complex of acriflavine-guanosine compound (AG60) is immunologically safe, as seen in thymic cortical morphology.
Acriflavine ; Animals ; Cytoplasm ; Fluorouracil* ; Guanosine ; Hydrogen-Ion Concentration ; Leukopenia ; Lymphocytes ; Mice* ; Mice, Inbred ICR ; Microscopy ; Microscopy, Electron ; Microvilli ; Osmium Tetroxide ; T-Lymphocytes ; Thymocytes ; Tolonium Chloride

This experiment was performed to evaluate the morphological responses of 5-fluorouracil or mitomycin C on the gastric parietal cells of mouse. 5 -fluorouracil (30 mg/kg) or mitomycin C (400 micro gram/kg) were injected subcutaneously every other day, and the animals were sacrificed at 4th day and 7th day following the first injection. Pieces of the tissue were taken from the stomach, prefixed with 2.5% glutaraldehyde -1.5% paraformaldehyde, followed by post-fixation with 1% osmium tetroxide. The ultrathin sections were stained with uranyl acetate and lead citrate. In both of the 5-fluorouracil or the mitomycin C treated groups, most parietal cells showed severely reduced luminal spaces of the intracellular canaliculi, since microvilli of intracellular canaliculi were very irregular shaped and nearly contacted with each other, and the cytoplasmic tubulovesicular membranes were disintegrated and indistinct. The changes in the 5-fluorouracil treated group were more indistinct than in those of the mitomycin C treated group. In the 5-fluorouracil treated group, balooning of the cytoplasm, focal cytolysis, myelin figures, lysosomes and multivesicular bodies in the parietal cells were observed more frequently than in those of the mitomycin C treated group. Above results suggest that the 5-fluorouracil or mitomycin C treated animals might suffer from reduced acid secretion of the parietal cell, since the collapsed lumen of the intracellular canaliculi, the disintegration of the tubulovesicular membranes, and the reduction of cell organelles in the parietal cells are occurred within a few days following injections. 5-fluorouracil was proved more harmful on the parietal cell than mitomycin C does.
Animals ; Citric Acid ; Cytoplasm ; Fluorouracil* ; Gastric Mucosa ; Glutaral ; Lysosomes ; Membranes ; Mice* ; Microvilli ; Mitomycin* ; Multivesicular Bodies ; Myelin Sheath ; Organelles ; Osmium Tetroxide ; Parietal Cells, Gastric* ; Phenobarbital ; Rabeprazole ; Stomach

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