• Applied Microscopy
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• v.23 no.1
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• pp.35-55
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• 1993

cis-Dichlorodiammineplatinum (II) (cis-Platin), a metallic compound, has widely been used as an effective anticancer chemotherapeutic agent. The precise mechanism of action of this agent is still unknown, but it is postulated that cis-Platin may act on the cancer cell like bifunctional alkylating agents. Although this agent is very beneficial to the patients with cervical cancer, germinoma of testis, neuroblastoma and others, it may also damage to the normal cell so that many side effects; severe hemorrhagic enterocolitis, bone marrow depression, renal damage and liver damage will develope. This experiment has been undertaken to pursue the cytotoxic effects of the cis-Platin on the ultrastructures of the interalveolar septum in the mouse lung. A total of 55 healthy male mice of ICR strain were used as experimental animals and divided into 5 mice of normal control group and 50 mice of cis-Platin treated group. The mice of cis-Platin treated group were sacrificed by carotid exsanguination at 6, 12, 24 hours, 3 days and 7 days after intraperitoneal injection of 6.0 mg of cis-Platin ($Abiplatin^R$ Abic Co. Ltd.) per kg of mouse body weight. The specimen obtained from the lower lobe of left lung were sliced into $1mm^3$ and prefixed with 2% glutaraldehyde -2.5% paraformaldehyde solution prepared with Millonig's phosphatae buffer solution (pH 7.4) at $4^{\circ}C$ for 3-4 hours. After postfixation with 1% osmium tetroxide solution all specimens were embedded in Epon 812. Ultrathin sections about $600-800{\AA}$ in thickness were stained with uranyl acetate and lead citrate and observed with Hitachi-600 electron microscope. The results obtained were as follows: 1. Local swellings with increase of electron density and number of pinocytic vesicles in the cytoplasms of the type I pneumocyte and endothelial cell of the blood air barrier in interalveolar septum of cis-platin treated mice were observed. 2. Cisternae of rough endoplasmic reticulum were dilated and sacculated in association with detachment of membrane bound ribosomes of the type II pneumocyte in interalveolar septum of cis-Platin treated mice. 3. Swollon mitochondria with uneven electron density of their matrix were observed in the type II pneumocyte of interalveolar septum in the cis-Platin treated mice. 4. The lamellae of lammelar bodies in type II pneumocyte of interalveolar septum in cis-Platin treated mice were devoided or transformed into homogeneous electron dense material. It is consequently suggested that cis-Platin would induce the cellular edema of type I pneumocyte and endothelial cell, and degenerative changes of cytoplasmic organelles of the type II pneumocyte in the interalveolar septum of the mouse lung.

• Applied Microscopy
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• v.17 no.1
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• pp.47-64
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• 1987

Morphological difference of the renal glomerulus at different age groups have been studied in young (three month-old), adult (twelve month-old) and old (thirty month-old) Fisher strain 344 rats. Pieces of the tissues were taken from renal corticies prefixed with 2.5% glutaraldehyde-1.5% paraformaldehyde (0.1 M Millonig's phosphate buffer, pH 7.3), following by post-fixation with 1% osmium tetroxide (0.1 M Millonig's phosphate buffer, pH 7.3) and embedded within Araldite. The ultrathin sections contrasted with uranyl acetate and lead citrate were observed under a JEM 100CX electron microscope. The mean thickness of glomerular basal lamina and Bowman's capsule were determined by measuring the thinnest portion of basal lamina, and by taking the average of 50 readings from electron micrographs at different ages. The numerical changes of the slit pores were compared based upon the numbers over the length of 10um of glomerular basal lamina. The results were as follow: 1. The thickness of glomerular basal lamina is increased during aging; 140.4 nm in young rats, 270.0 nm in adult ones, and 437.8 nm in old ones. 2. The thickness of basal lamina of parietal cells of Bowman's capsule is 187.5 nm in young rats, 914.0 nm in adult ones, and 2850.0 nm in old ones. 3. The numbers of the slit pores of basal lamina are reduced during aging, 30.3 slit pores/$10{\mu}m$ in adult ones, and 24.2 slit pores/$10{\mu}m$ in old ones. 4. Accumulation of dense intracytoplasmic filamentous material in the parietal cells of Bowman's capsule is increased in the vicinity of the basal lamina during aging. The proximal tubule-like epithelial cell in Bowman's capsule is observed at one glomerulus in a young rat. 5. The endothelial cells are edematous and form balloon-like structure protruding into capillary lumen in young and old rats. 6. Cytoplasm of the podocyte shows a variety of alteration during aging, such as swelling of mitochondria and of endoplasmic reticulum, and increase of microtubules, microfilaments, lysosomes and lamellated myelin structures, etc. Accumulation of dense intracytoplasmic material in the foot processes is increased in the vicinity of the basal lamina during aging. The podocytic membrane-like structures are seen in young and o]d rats. 7. The mesangial matrices and mesangial cells are increased during aging, and slight swelling of endoplasmic reticulum and Golgi cisternae in young and old rats.

• Applied Microscopy
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• v.17 no.1
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• pp.83-97
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• 1987

In order to discriminate the enteroendocrine cell types in the mucosal epithelium of the normal duodenum of the Korean hedgehog (Erinaceus koreanus). The tissues were fixed in the mixture of 1% paraformaldehyde and 1% glutaraldehyde in phosphate buffer (pH 7.2), and postfixed in 2% osmium tetroxide (phosphate buffer, pH 7.2). They were embedded in Araldite, and the ultrathin sections were made by LKB-V ultratome following the inspection of semithin sections stained with toluidine blue-borax solutions. Ultrathin sections contrasted with uranyl acetate and lead citrate were observed with JEM 100B electron microscope. At least six types of enteroendocrine cells distributed in the mucosal epithelium of the duodenum were identified according to their morphological characteristics mainly based on the size, shape, number and electron density of the secretory granules. Type I cells had moderately developed organelles. The secretory granules were pleomorphic ($370X510nm$), and the granule cores with high electron density were enveloped in limiting membrane and characterized by a narrow halo. Type II cells contained an indented nucleus and well-developed organelles. The secretory granules were round (350 nm) and classified in two kinds by electron density, moderate and high. Both granules were surrounded by limiting membrane and those with high electron density showed often a wide halo. Type III cells had an indented nucleus. The secretory granules with various electron density were round (220 nm) in shape. The granules with high electron density were enveloped in limiting membrane and characterized by a narrow halo, but those with low or moderate electron density had not been observed the limiting membrane. Type IV cells contained an indented nucleus and moderately developed organelles. The secretory granules were round (180 nm) in shape, and the granule cores with high electron density were enveloped in limiting membrane and showed often a wide halo. Type V cells had a large amount of rough endoplasmic reticulum. Secretory granules with low or moderate electron density were round (230 nm) in shape, and surrounded by limiting membrane and showed a narrow halo. Type VI cells contained an oval nucleus and well-developed organelles, especially Golgi complex. The secretory granules with high electron density were round (210 nm) in shape. The granules were enveloped in limiting membrane and showed often a wide halo.

• Applied Microscopy
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• v.25 no.3
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• pp.51-62
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• 1995

It has been well known that ischemia and reperfusion injury to skeletal muscle following an acute arterial occlusion causes significant morbidity and mortality. The skeletal muscle, which contains high energy phosphate compounds, has ischemic tolerance. During the ischemia, the ATP is catalyzed to hypoxanthine anaerobically and hypoxanthine dehydrogenase is converted to xanthine oxidase. During reperfusion, the hypoxanthine is catalyzed to xanthine by xanthine oxidase under $O_2$, presence and that results in production of cytotoxic oxygen free radicals. These cytotoxic free radicals, $O_2^-,\;H_{2}O_2,\;OH^-$, are toxic and make lesions in skeletal muscle during reperfusion. The authors perform the present study to investigate the effects of allopurinol, the inhibitor of xanthine oxidase, on reperfused ischemic skeletal muscles by observing the ultrastructural changes of the muscle fibers. A total of 48 healthy Sprague-Dawley rats weighing from 200 g to 250 g were used as experimental animals. Under urethane(3.0mg/kg., IP) anesthesia, lower abdominal incision was done and the left common iliac artery were ligated by using vascular clamp for 1, 2 and 6 hours. The left rectus femoris muscles were obtained at 6 hours after the removal of vascular clamp. In the allopurinol pretreated group, 50mg/kg of allopurinol was administered once a day for 2 days and before 2 hours of ischemia. The specimens were sliced into $1mm^3$ and prepared by routine methods for electron microscopic observations. All preparations were stained with uranyl acetate and lead citrate, and then observed with Hitachi -600 transmission electron microscope. The results were as follows: 1. In 1 hour ischemia/6 hours reperfused rectus femoris muscles of rats, decreased glycogen particles and electron density of mitochondrial matrix and dilated terminal cisternae are seen. In 2 hours ischemia/6 hours repersed rectus femoris muscles of rats, mitochondria with electron lucent matrix, irregularly dilated triad and spheromembranous bodies are observed. In 6 hours ischemia/6 hours reperfused rectus femoris muscles of rats, irregularly arranged myofibrils, and many spheromembranous bodies, fat droplets and lysosome are seen. 2. In 1 hour ischemia/6 hours reperfused rectus femoris muscles of rats pretreated with allopurinol, decreased glycogen particle and dilated cisternae of sarcoplasmic reticulum and triad are observed. In 2 hours ischemia/6 hours reperfused rectus femoris muscles of rats pretreated with allopurinol decreased electron density of mitochondrial matrix and spheromembranous bodies are seen. In 6 hours ischemia/6 hours reperfused rectus femoris muscles of rats pretreated with allopurinol, mitochondria with electron lucent matrix, spheromembranous bodies and dilated cisternae of sarcoplasmic reticulum and terminal cistern are observed. The results suggest that the allopurinol attenuates the damages of the skeletal muscles of rats during ischemia and reperfusion.

• Journal of Periodontal and Implant Science
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• v.27 no.2
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• pp.317-328
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• 1997

Since laser therapy has been applied to dentistry, many dental practitioners are very interested in laser therapy on various intraoral soft tissue lesions including gingival hyperplasia and aphthous ulcer. The purpose of the present study was to determine the therapeutic effect and the harmful effect of a pulsed-Nd:YAG laser irradiation on human gingival tissue. In twenty periodontal patients with gingival enlargement, the facial gingival surface of maxillary anterior teeth was randomly irradiated at various power of 1.0W(100mJ, 10Hz), 3.0W(100mJ, 30Hz) and 6.0W(l50mJ, 40Hz) for 60 seconds by contact delivery of a pulsed-Nd:YAG laser(EN.EL.EN060, Italy). Immediately after laser irradiation, the gingival tissues were surgically excised and prepared in size of 1mm3. Subsequently the specimens were processed for prefixation and postfixation, embedded with epon mixture, sectioned in $1{\mu}$ thickness, stained with uranyl acetate and lead citrate, and observed under transmission electron microscope(JEM 100 CXII). Following findings were observed; l. In the gingival specimens irradiated with l.OW power, widening of intercelluar space and minute vesicle formation along the widened intercellular space were noted at the epithelial cells adjacent to irradiated area. 2. In the gingival specimens irradiated with 3.0W power, the disruption of cellular membrane, aggregation of cytoplasm, and loss of intercellular space were observed at the epithelial cells adjacent to irradiated area. 3. In the gingival specimens irradiated with 6.0W power, the disruption of nuclear and cellular membrane was observed at the epithelial cells adjacent to irradiated area. The ultrastructural findings of this study suggest that surgical application of a pulsed-Nd:YAG laser on human gingival tissue may lead somewhat delayed wound healing due to damage of epithelial cells adjacent to irradiated area.

• Applied Microscopy
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• v.24 no.2
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• pp.63-77
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• 1994

This experiment was performed to study the morphological responses of the epidermis of the rat scalp, following X-ray irradiation. Male rats were divided into normal and experimental groups. Rats anesthetized with sodium thiopental, were exposed only on their head areas with a single dose of 3,000rads or 6,000rads, respectively. Radiation was produced by Mitsubishi Linea Accelerator ML-4MV at the speed of 200rads/min. The target distance was 80cm. Animals were sacrificed on six hours, two days and six days following irradiation. By the perfusion fixation through the heart, rats were fixed with 1% glutaraldehyde-1% paraformaldehyde solution. Pieces of the tissue taken from the scalp were refixed in 2.5% glutaraldehyde-1.5% paraformaldehyde solution, followed by post-fixation with 1% osmium tetroxide, and embedded within araldite mixture. The sections were cut on a LKB-V ultratome, stained with uranyl acetate and lead citrate, and were observed with JEM 100CX-II electron microscope. The results were as follow; 1. Six hours after exposure to 3,000rads of X-ray. Disrupted intercellular spaces, within which some amorphous materials were filled, disrupted mitochondria, and vacuoles in the keratinocytes were frequently observed, but six days after exposure to 3,000rads of X-ray, Morphology of the keratinocytes was generally restored. 2. Many of the morphological changes were seen on the six days after exposure to 6,000rads of X-ray. 3. Widened intercellular spaces and thickened dense plaques of the desmosomes were frequently observed after exposure to 6,000rads of X-ray. 4. In the experimental groups, the Langerhans and the Merkel cells were damaged, similarly to the keratinocyte. Above results suggest that head irradiation with the dose of 3,000rads temporarily damaged the epidermis of the scalp, though most of the structures recover within six days, whereas with the dose of 6,000rads it severely damaged the epidermis without showing any recovering tendency.

• Applied Microscopy
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• v.24 no.2
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• pp.48-62
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• 1994

The acute irradiation effect on rat Purkinje cell was carried out. Anesthetized rats, weighing 200-250g each, were exposed their heads to the linear accelerator (ML-4MV) with the doses of 3,000 rads or 6,000 rads respectively. Irradiated rats were sacrificed by perfusion fixation under anesthesia, six hours, two days and six days following the irradiations. Rats were perfused with the fixative of 1% glutaraldehyde-1% paraformaldehyde solution (pH 7.4). Small pieces of cerebellar cortices were taken out. Tissue blocks were washed out, and were refixed in the 2% osmium tetroxide solution. After dehydration, tissues were embedded in the araldite mixture. Ultrathin sections stained with uranyl acetate-lead citrate solution, were examined with an electron microscope. The results observed were as follow; 1. Many dark Purkinje cells exhibited most severe cellular alterations on 6 hours. But after the 2 or 6 days, the cells exhibited only some alterations of cytoplasmic organelles. 2. Many granular and agranular endoplasmic reticula exhibited the fusion of cisterns. These reticular alterations were most severe on 6 hours following irradiation. But the alterations were hardly found on 6 days. 3. In the Golgi region, alterations including the adhesion of lamelliform cisterns, enlarged saccules, and increased number of vesicles, etc, were seen on 6 hours. But the Golgi complexes were almost recovered on 6 days. 4. Lysosomes were abundant on 6 hours or 2 days, but some residual bodies were found on 6 days. 5. Mitochondrial changes were also most severe at on hours, and they were recovered thereafter. From the results, it was concluded that the cerebellar Purkinje cells reacted to the high doses of irradiation by hyperactive protein synthesis, autolytic activities and energy metabolism. The reaction was most active in the early stage. It implies that motor-control function of Purkinje cells are severely disturbed in the early stage of irradiation.

• Applied Microscopy
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• v.27 no.3
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• pp.333-346
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• 1997

The ischemia and reperfusion injury of the skeletal muscles is caused by generation of reactive oxygen during ischemia and reperfusion. It is well known that over 4 hours of ischemia injures the skeletal muscles irreversibly. The author has demonstrated the effects of SOD (superoxide dismutase), DMTU (dimethyl thiourea) and ischemic preconditioning on ultrastructural changes of the muscle fibers in the rectus femoris muscles after 4 hours of ischemia and 1 day and 3 days of reperfusion. A total of 72 healthy Sprague-Dawley rats weighing from 200 gm to 250 gm were used as experimental animals. Under urethane(1.15 g/kg, IP, 2 times) anesthesia, lower abdominal incision was done and the left common iliac artery was occluded by using vascular clamp for 4 hours. The left rectus femoris muscles were obtained at 1 and 3 days after the removal of vascular clamp. The SOD (15,000 unit/kg) or DMTU (500 mg/kg) were administered intraperitoneally at 1 hour before induction of ischemia. The ischemic preconditioned group underwent three episodes of 5 minutes occlusion and 5 minutes reperfusion followed by 4 hours of ischemia and 1 day and 3 days of reperfusion. The specimens were sliced into $1mm^3$ and prepared by routine methods for electron microscopic observation. All specimens were stained with uranyl acetate and lead citrate and then observed with Hitachi-600 transmission electron microscope. The results were as follows: 1. SOD or DMTU alone did not affect the ultrastructure of muscle fibers in the rectus femoris muscles. The electron density of mitochondrial matrix was decreased by ischemic preconditioning. 2. Dilated cisternae of sarcoplasmic reticulum, triad, mitochondria and the loss of myofilament in the sarcomere were observed in the 4 hours ischemia and 1 day reperfused rectus femoris muscles. Markedly changed sarcoplasmic reticulum, triad, disordered or loss of myofilament, indistinct A-band and I-band, and irregular electron lucent M -line and Z-line are seen in the 4 hours ischemia and 3 days reperfused rectus femoris muscles. 3. SOD reduced the changes of organelles in the muscle fibers of the 4 hours ischemia and 1 day reperfused rectus femoris muscles of the rats, but SOD did not affect the changes of muscle fibers in the 4 hours ischemia and 3 days reperfused muscles. On the other hand, DMTU markedly attenuated considerably the ultrastructural change of the 4 hours ischemia and 1 day or 3 days reperfused rectus femoris muscles. 4. By the ischemic preconditioning, the change was attenuated remarkably in the 4 hours ischemia and 1 day reperfused rectus femoris muscles. As the ischemic reperfused changes of muscle fibers were regenerated or recovered by ischemic preconditioning, the ultrastructures of them were similar to those of normal control in the 4 hours ischemia and 3 days reperfused rectus formoris muscles. Consequently, it is suggested that DMTU is stronger inhibitor to ischemic reperfused change than SOD. The ischemia and reperfusion-induced muscular damage is remarkably inhibited by ischemic preconditioning.

• Applied Microscopy
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• v.27 no.4
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• pp.357-372
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• 1997

To study the age-related morphological differences of the retinal pigment cells and Bruch's membrane of mouse, retinae of one week-old, five weeks-old, eight weeks-old, six months-old, twelve months-old, eighteen months-old, twenty-four months-old and thirty months-old ICR mice were dissected out under anesthesia. Pieces of the tissue taken from the posterior region of the retina were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde (0.1 M Millonig's phosphate buffer, pH 7.3), and 1% osmium tetroxide (0.1 M Millonig's phosphate buffer, pH 7.3), and embedded in araldite mixture. The ultrathin sections were stained with uranyl acetate and lead citrate, and were observed under a JEM 100 CX-II electron microscope. Observed results were as follows: 1. Retinae of one week old mouse exhibit that some parts of the pigment cell provided with basal foldings, whereas other parts of the one contain without basal foldings. After (ive weeks-old, all retinal pigment cells have the basal infoldings. 2. In the one week-old, stage 1 and stage 2 melanosomes were observed in the retinal pigments cells, but after five weeks-old, most of the retinal pigment cells contain some matured stage melanosomes (stage III and stage IV). 3. The phagosomes in the retinal pigment cells were increased during aging. 4. After eighteen months-old, electron dense materials are observed within the basal infoldings. 5. After eighteen months-old, the thickness of the Bruch's membrane is prominently increased. The thickness of the basal laminae of the pigment cell and the choriocapillary endothelium is more prominently increased as compared with that of the other components of the Bruch's membrane. 6. The thickness of the basal lamina of the pigment cell is more prominently increased as compared with that of the choriocapillary endothelium on aging. From the above results, it was suggested that the pigment cell and Bruch's membrane matures structurally In five weeks, and the function of the pigment cell is prominently suppressed around eighteen months-old, and thereafter the functional suppression is continued on aging.

• Applied Microscopy
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• v.27 no.1
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• pp.13-30
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• 1997

The experiment was performed to study the morphological responses of the thymus of the mice, to antitumour agents (5-Fluorouracil or mitomycin C). Healthy adult mice weighing 25 gm each were divided into normal and experimental groups. 5-Fluorouracil (60 mg/kg) or Mitomycin-C $(400{\mu}g/kg)$ were injected subcutaneously to the animals every other day. Animals were sacrificed at 4 days and 7 days following the first injection. Pieces of the tissue taken from the thymus were prefixed with 2.5% paraformaldehyde-l.5% glutaraldehyde, followed by post-fixation with 1% osmium tetroxide. Ultrathin sections stained with uranyl acetate and lead citrate were observed with a JEM 100 CX-II electron microscope. The observed results were as follow: 1. Apoptoses of T-lymphocytes were observed more frequently in the thymus of the experimental groups than in those of a normal group. 2. In the experimental group, the plasma cells with distended cisternae of the granular endoplasmic reticulum and the eosinophile leukocytes were observed frequently. 3. In the experimental group, newly forming Hassall's corpurscles were observed frequently. 4. In the mitomycin-treated group, the epithelial reticular cells containing distended perinuclear cisternae, distended the granular endoplasmic reticula and pyknotic nuclei were observed in the cortico-medullary junctional area. 5. In the mitomycin-treated group, nuclear bodies with medium electron dense materials were often observed in the T lymphocyte. 6. In the 5-fluorouracil-treated groups, fused and dissolved tonofilament bundles and apoptotic bodies were observed in the some epithelial reticular cells in the medullary area. 7. In the 5-fluorouracil-treated groups, some elongated and bar-shaped lysosomes with electron lucent gap were often observed in the macrophages. 8. In the 5-fluorouracil-treated group, membrane complex of the smooth endoplasmic reticulum were ofen observed in the macrophage. From the above results, it was suggested that 5-fluorouracil or mitomycin could induce rapid involution of the thymus, and disturb maturation and differentiation of T lymphocytes, and, in turn, supress immunity.