Use of scanning electron microscopy in the cochlea of guinea pigs / Microscopia eletrônica de varredura em cócleas de cobaias

Abstract Introduction: The use of electron microscopy in the study of the inner ear has allowed us to observe minute details of the hair cells, especially in ototoxicity studies; however, the preparation of this material is a difficult and delicate task. In an attempt to simplify the handling of these materials, two agents, toluidine blue and ethylenediamine tetra-acetic acid were tested, in addition to the elimination of osmium tetroxide during the preparation of albino guinea pig cochleae. We also tested the applicability of these methodologies in an ototoxicity protocol. Objective: To verify the quality of the images obtained with and without the use of ethylenediamine tetra-acetic acid, toluidine blue and osmium tetroxide in the preparation of cochleae of albino guinea pigs for the scanning electron microscopy. Methods: Three groups of cochleae were used. In Group 1, 10 cochleae were prepared with the usual methodology, dissecting the optical capsule without decalcification and using osmium tetroxide as a post-fixative agent. In Group 2, we prepared 10 cochleae decalcified with ethylenediamine tetra-acetic acid, injecting toluidine blue in the endolymphatic space to facilitate the identification of the organ of Corti. In Group 3, we used 4 cochleae of guinea pigs that received 3 doses of cisplatin (7.5 mg/kg, D1-D5-D6), two prepared according to the methodology used in Group 1 and two with that used in Group 2. Scanning electron microscopy images were obtained from the organ of Corti region of the basal turn of each cochlea. Results: The organ of Corti was more easily identified with the use of toluidine blue. The dissection of the cochlea was more accurate in the decalcified cochleae. The quality of the images and the preservation of the organ of Corti obtained with the two methodologies were similar. Conclusion: The proposed modifications resulted in images of similar quality as those observed using the traditional methodology.

Resumo Introdução: O emprego da microscopia eletrônica no estudo da orelha interna permitiu observar detalhes minuciosos das células ciliadas especialmente em estudos de ototoxicidade. Entretanto, o preparo desse material é trabalhoso e delicado. Para simplificar a manipulação desses materiais, testou-se o uso de dois agentes, azul de toluidina e ácido etilenodiamino tetra-acético, além da retirada do tetróxido de ósmio na preparação de cócleas de cobaias albinas. Testamos também a aplicabilidade dessas metodologias em um protocolo de ototoxicidade. Objetivo: Verificar a qualidade das imagens obtidas com e sem o uso de ácido etilenodiamino tetra-acético, azul de toluidina e tetróxido de ósmio na preparação de cócleas de cobaias albinas para a microscopia eletrônica de varredura. Método: Foram utilizados três grupos de cócleas. No Grupo 1 preparou-se 10 cócleas com a metodologia usual, dissecando a cápsula ótica sem descalcificac¸ão e utilizando tetróxido de ósmio como pós-fixador. No Grupo 2 preparamos 10 cócleas descalcificadas com ácido etilenodiamino tetra-acético, injetando azul de toluidina no espac¸o endolinfático para facilitar a identificação do órgão de Corti. No Grupo 3 utilizamos 4 cócleas de cobaias que receberam 3 doses de cisplatina (7,5 mg/kg, D1-D5-D6), duas preparadas com a metodologia do Grupo 1 e duas com a do Grupo 2. Foram obtidas imagens da microscopia eletrônica de varredura da região do órgão de Corti do giro basal de cada cóclea. Resultados: O órgão de Corti foi mais facilmente identificado com o azul de touidina. A dissecção da cóclea foi mais precisa nas cócleas descalcificadas A qualidade das imagens e a preservac¸ão do órgão de Corti obtidas com as duas metodologias foi similar. Conclusão: As modificações propostas resultaram em imagens de qualidade similar as observadas com o uso da metodologia tradicional.

Effects of AG60 on Gastric Chief Cells of the Mouse Implanted with Ehrlich Carcinoma Cells / 대한해부학회지

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.

synovirthesis: a reappraisal

The evolution of the rheumatologic practice involved a handing-over in question of the place and methods of application of the synoviorthesis. The last innovations, in particular the appearance of the biotherapies, allowed a better control of inflammatory rheumatisms thus making it possible to better select arthritis likely to profit precociously from a synoviorthesis before the installation of major articular destruction. Through a general review of the literature, we recall in this work the various means of synoviorthesis, their current indications and their results. An extensive electronic search of the relevant literature was carried out using medline. Key words used for the final search were: synoviorthesis, osmical acid, radiosynoviorthesis, arthritis, traitment. this systematique review allowed us to conclude that fields of application of the synoviorthesis is in addition widens because of the interesting results to see spectacular this technique in some other affections such as the haemophilia. In addition we have compared the efficiency and the tolerance of the different methods of synoviorthesis. The synoviorthesis constitutes a tempting therapeutic alternative of share its effectiveness and its good tolerance so much so that it constitutes an undeniable factor of articular protection. Its fields of application widened. Thus on the good knowledge of the indications and the precautions necessary to its realization its success willdepend

Glândula submandibular de ratos com envelhecimento: observações ao microscópio eletrônico de varredura de alta resolução / Submandibular gland of rats with ageing: observations with high resolution scanning electron microscopy

As características tridimensionais dos componentes intracelulares de células acinares e de ductos foram reveladas usando o método ósmio-DMSO-ósmio. As amostras foram maceradas em solução de tetróxido de ósmio diluído após a fratura na solução de dimetil sulfoxido. As lamelas do retículo endoplasmático granular são reveladas entremeadas por várias mitocôndrias. As lamelas do retículo endoplasmático granular são localizados ao redor dos núcleos na porção basal e estas estruturas são observadas em imagens tridimensionais de microscopia eletrônica de alta resolução.

The three-dimensional characteristics of the intracellular components of acinar and ductal cells were revealed using the osmium-DMSO-osmium method. The samples were macerated in diluted osmium after fractured in DMSO solution. The stacks of the rough endoplasmic reticulum are revealed intermingling by several mitochondria. The lamellae of the rough endoplasmic reticulum are located around the nuclei at basal portion and these structures are shown in three-dimensional HRSEM images.

Osmic acid staining of myelin sheath in normal and regenerated peripheral nerves / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To introduce a practical, economical, and time-saving method to stain (with osmic acid) the myelin sheath in normal and regenerated peripheral nerves.</p><p><b>METHODS</b>A 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.</p><p><b>RESULTS</b>Under 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).</p><p><b>CONCLUSIONS</b>Osmic 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.</p>

Effects of Acriflavine-Guanosine Composition (AG60) on the DNA Synthesis and Ultrastructure of Epithelial Cells of the Appendix of Mice Inoculated with Ehrlich Carcinoma Cells / 대한해부학회지

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.

Ultrastructural Alterations in the Gastric Chief Cells of Mouse, induced by 5-Fluorouracil or Mitomycin C / 대한해부학회지

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.

Zinc Iodide-Osmium Tetroxide (ZIO) Reactive Golgi Apparatus in the Principal Cells of Dog Epididymal Epithelium / El Tetróxido de Zinc Iodide-Osmio (ZIO) Reactiva el Aparato de Golgi en las Células Principales del Epitelio Epididimal del Perro

The cisternae of the Golgi apparatus of dog epididymal principal cells were labeled by the zinc iodide-osmium tetroxide method (ZIO). These cisternae were observed in the supranuclear region of the cytoplasm of the epididymal principal cells. Abundant endoplasmic reticulum cisternae, multivesicular bodies, mitochondria, lysosomes and vesicular elements of variable size were also found in this region, all associated with the sacks of the well-developed Golgi apparatus. The use of the ZIO method facilitates the observation and identification of the cisternae of the Golgi apparatus, thus permitting a correlation between structure and function in the so-called Golgi area. These ultrastructural characteristics support the secretory role of epididymal principal cells in the dog.

La cisterna del aparato de Golgi de las células principales del epidídimo del perro, fueron tratados con el método de tetróxido de zinc iodide-osmium (ZIO). Estas cisternas fueron observadas en la región supranuclear del citoplasma de las células principales del epidídimo. Abundante cisternas del retículo endoplasmático, cuerpos multivesiculares, mitocondrias, lisosomas y elementos vesiculares de tamaño variable, fueron encontrados en esta región, todos asociados con los sacos del aparato de Golgi maduro. El uso del método de ZIO facilita la observación e identificación del aparato de Golgi, permitiendo efectuar una correlación entre estructura y función en el área de Golgi. Estas características estructurales suponen el rol secretorio de las células epididimarias principales en el perro.

The Effect of High Dose Systemic Steroid and its Long-term Usage on the Skin Barrier of Hairless Mice / 대한피부과학회지

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.

An alternative technique to reveal polysaccharides in Toxoplasma gondii tissue cysts

Ultrathin sections of tissue cysts isolated from the brain of Toxoplasma gondii infected mice were submitted to two different methodologies derived from the periodic acid - Schiff's reagent (PAS) technique. The use of osmium tetroxide vapor as a developing agent of the aldehyde oxidation to reveal polysaccharides with periodic acid resulted in positive reaction in amylopectin granules in bradyzoites, as well as in the wall and matrix of the cysts, with excellent increment of the ultrastructural morphology. This technique can be used for study of T. gondii-host cell intracellular cycle, the differentiation tachyzoite-bradyzoite, and also for the formation of cysts into the host cells.

Comparative effect of osmium tetroxide and ruthenium tetroxide on Penicillium sp. hyphae and Saccharomyces cerevisiae fungual cell wall ultrastructure / Efecto comparativo del tetróxido de osmio y del tetróxido de rutenio como fijadores sobre la ultraestructura de la pared celular de hongos

The fungal cell wall viewed through the electron microscope appears transparent when fixed by the conventional osmium tetroxide method. However, ruthenium tetroxide post-fixing has revealed new details in the ultrastructure of Penicillium sp. hyphae and Saccharomyces cerevisiae yeast. Most significant was the demonstration of two or three opaque diverse electron dense layers on the cell wall of each species. Two additional features were detected. Penicillium septa presented a three-layered appearance and budding S. cerevisiae yeast cell walls showed inner filiform cell wall protrusions into the cytoplasm. The combined use of osmium tetroxide and ruthenium tetroxide is recommended for post-fixing in electron microscopy studies of fungi.

Ultrastructure of Fronto-Accumbens Synapses after Prefrontal Decortication of the Rat / 대한해부학회지

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.

Morphological Study on the Thymic Cortex of the Ehrlich Carcinoma-Inoculated Mice, Treated with 5-Fluorouracil and Acriflavine-Guanosine Complex (AG60) / 대한해부학회지

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.

Effects of BCG or AG60 on Gastric Parietal Cells of the Mouse Implanted with Ehrlich Carcinoma Cells / 대한해부학회지

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.

Ultrastructural Alterations Induced by 5-Fluorouracil or Mitomycin C on the Gastric Parietal Cells of Mouse / 대한해부학회지

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.

Morphological Study on the Mouse Spleen following the Administration of Mercuric Chloride or Potassium Dichromate / 대한해부학회지

This experiment was performed to study the morphological changes of the spleen of mice following injection of sodium dichromate (K2Cr2O7) or mercuric chloride (HgCl2). Male mice were divided into normal and experimental groups. The mice were subcutaneously injected with mercuric chloride (5 mg or 10 mg/kg) or sodium dichromate (10 mg or 20 mg/kg). Animals were sacrificed on 6 hours, 1 day, 3 days, 1 week and 2 weeks after injections. Pieces of splenic tissue were taken from each mouse, and fixed in 10% neutral formalin for light microscopy. The paraffin sections were stained with hematoxylin-eosin, Masson-trichrome, Bielschowsky's silver impregnation or aldehyde-fuchsin stain. For electron microscopy, the tissues were fixed in 2.5% glutaraldehyde-1.5% paraformalde-hyde, and post-fixed in 1% osmium tetroxide. Dehydrated blocks were embedded in araldite mixture. The ultrathin sections stained with uranyl acetate and lead citrate were observed with JEM 100CX-II electron microscope. On histological study, in the early stage (6 hours) of experimental groups, splenic white pulp exhibited numerous vacuoles containing pyknotic nuclei were observed as compared with those of normal control group. But after 3 days(sodium dichromate, 10 mg/kg or 20 mg/kg; mercuric chloride, 5 mg/kg) and 1 week (mercuric chloride, 10mg/kg), the morphology was recovered to normal one. In the experimental groups, positive reactions to Bielschowsky's silver impregnation, Masson-trichrome or aldehyde-fuchsin stain were similar to those of normal control group. On the ultrastructural study, in white pulps of experimental groups, nuclear bodies were observed frequently in the nuclei of the lymphocytes and the reticular cells, and myelin figures were observed in the nucleus or in the cytoplasm of the lymphocytes and the reticular cells. The plasma cells showed many irregularly distended cisternae of granular endoplasmic reticula and the macrophages containing phagosomes, were observed frequently. From the above results, it was concluded that potassium dichromate or mercuric chloride could disturb the normal differentiation or maturation of the lymphocytes and the reticular cells of the spleen, especially in the early stage of treatment. But histological changes occurred in the spleen following injection of the potassium dichromate or mercuric chloride were recovered to normal appearance in 3 days (potassium dichromate) or 1 week (mercuric chloride). Mercuric chloride was more harmful than potassium dichromate on the spleen.

Effects of Bacillus Calmette-Guerin (BCG) on the Spleen of Mouse Inoculated with Ehrlich Carcinoma Cells: A Morphological Study / 대한해부학회지

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.

Effects of Antitumor Agents on Ultrastructure of the Splenic White Pulp of the Mouse Implanted with Ehrlich Carcinoma Cells / 대한해부학회지

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.

Effects of Lower Dose-Injections of Acriflavine-Guanosine Compound (AG60) on the DNA Synthesis and Ultrastructure of the Ehrlich Carcinoma Cells Inoculated to the Mouse / 대한해부학회지

AG60, a recently introduced anti-cancer compound, was reported to show highly effective anti-cancer activities, when injected with doses from 30 mg/kg to 5 mg/kg.The purpose of this study was to know the lower effective doses of AG60, and to give the informations for preparing more advanced therapeutic tools for anti-cancer war. Ehrlich cancer cells were inoculated in the subcutaneous tissue of inguinal region of ICR mice, and saline (treated control groups) or AG60 (experimental groups) were injected daily. Animals of experimental groups were injected subcutaneously with doses of 0.2 mg/kg, 0.5 mg/kg, 1.0 mg/kg, or 2.0 mg/kg body weight, according to their subgroups. Five mice from each subgroup were sacrificed on 1 week, 2 weeks, 3 weeks following the first injection. Seventy minutes before sacrifice, each mouse was injected with 0.7 microCi/g body weight of 3H-thymidine (Amersham Lab.) through tail vein. After sacrifice, cancer masses were fixed in 10% formalin solution for autoradiography and light microscopy, and in 2.5% glutaraldehyde-1.5% paraformaldehyde solution, followed by post-fixation in 2% osmium tetroxide solution, for electron microscopy. The observed results were as follows: Autoradiographic observations show, 1. Labelled cancer cell indices of the experimental groups received AG60 were decreased around to 80% (0.2 mg/kg), to 74% (0.5 mg/kg), to 75~60% (1.0 mg/kg), and to 70~50% (2.0 mg/kg), as compared with those of the controls. 2. The contents of silver grains were dramatically decreased nearly to 35% (0.2 mg/kg), to 20% (0.5 mg/kg), to 21~16% (1.0 mg/kg), and to 20~15% (2.0 mg/kg), as compared with those of the controls. 3. Total granular content in 100 cancer cells on the third week of the experiment decreased nearly to 30% (0.2 mg/kg), to 15% (0.5 mg/kg), to 10% (1.0 mg/kg), and to 8% (2.0 mg/kg), as compared with those of the controls. Histological observations show, 1. AG60 induces large amount of apoptosis on Ehrlich cancer cells. 2. Following the treatment with AG60, multinuclear cells or giant cells were increased in number. Comparing by autoradiography and histology, multinuclear or giant cells were interpreted as those cells supplied by poor amounts of thymidine, or almost no new DNA content. Electronmicroscopic readings show, 1. AG60 induces numerous macroclefts and microclefts within the nuclei of Ehrlich cancer cells. 2. AG60 induces numerous apoptosis among Ehrlich cancer cells. 3. Apoptotic bodies are phagocytosed by adjacent cancer cells or by macrophages. From the above results, AG60 is expected to be a very successful anti-cancer candidate. And it is suggested that combined or cocktail therapy including AG60 may greatly improve the anti-cancer therapy on certain kind of cancer.