Maintenance of the corneal epithelium is carried out by germinative cells of its basal stratum and not by presumed stem cells of the limbus

The purpose of this investigation was to analyze the proliferative behavior of rabbit corneal epithelium and establish if any particular region was preferentially involved in epithelial maintenance. [3H]-thymidine was injected intravitreally into both normal eyes and eyes with partially scraped corneal epithelium. Semithin sections of the anterior segment were evaluated by quantitative autoradiography. Segments with active replication (on) and those with no cell division (off) were intermingled in all regions of the tissue, suggesting that the renewal of the epithelial surface of the cornea followed an on/off alternating pattern. In the limbus, heavy labeling of the outermost layers was observed, coupled with a few or no labeled nuclei in the basal stratum. This suggests that this region is a site of rapid cell differentiation and does not contain many slow-cycling cells. The conspicuous and protracted labeling of the basal layer of the corneal epithelium suggests that its cells undergo repeated cycles of replication before being sent to the suprabasal strata. This replication model is prone to generate label-retaining cells. Thus, if these are adult stem cells, one must conclude that they reside in the corneal basal layer and not the limbal basal layer. One may also infer that the basal cells of the cornea and not of the limbus are the ones with the main burden of renewing the corneal epithelium. No particular role in this process could be assigned to the cells of the basal layer of the limbal epithelium.

Rapid Changes in D1 and D2 Dopamine Receptor Binding in Striatal Subregions after a Single Dose of Phencyclidine

OBJECTIVE: In humans, a single exposure to phencyclidine (PCP) can induce a schizophrenia-like psychosis which can persist for up to two weeks. In rats, an acute dose of PCP increases dopaminergic activity and causes changes in dopamine related behaviours some of which are sexually dimorphic. To better understand the effects of PCP on dopamine receptor adaptations in the short term we examined dopamine D1-like receptors (D1R) and D2-like receptors (D2R) in the mesolimbic and nigrostriatal dopamine pathways, 4 hours after exposure to PCP in female rats. METHODS: Animals received a single dose of 40 mg/kg PCP and were sacrificed 4 hours later. In vitro autoradiography was carried out using [3H] SCH 23390 and [3H] raclopride that target D1R and D2R respectively, in cryostat brain sections. RESULTS: Two way analysis of variance (ANOVA), revealed an overall effect of PCP treatment (F [1,63]=9.065; p=0.004) on D1R binding with an 18% decrease (p<0.01) in binding in the medial caudate putamen. PCP treatment also had an overall effect on D2R binding (F [1,47]=5.450; p=0.024) and a trend for an increase in D2R binding across all the brain regions examined. CONCLUSION: These results suggest opposing D1R and D2R adaptations in striatal subregions of female rats following acute exposure to PCP that may occur through indirect mechanisms.

Effects on prolactin secretion and binding to dopaminergic receptors in sleep-deprived lupus-prone mice

Sleep disturbances have far-reaching effects on the neuroendocrine and immune systems and may be linked to disease manifestation. Sleep deprivation can accelerate the onset of lupus in NZB/NZWF1 mice, an animal model of severe systemic lupus erythematosus. High prolactin (PRL) concentrations are involved in the pathogenesis of systemic lupus erythematosus in human beings, as well as in NZB/NZWF1 mice. We hypothesized that PRL could be involved in the earlier onset of the disease in sleep-deprived NZB/NZWF1 mice. We also investigated its binding to dopaminergic receptors, since PRL secretion is mainly controlled by dopamine. Female NZB/NZWF1 mice aged 9 weeks were deprived of sleep using the multiple platform method. Blood samples were taken for the determination of PRL concentrations and quantitative receptor autoradiography was used to map binding of the tritiated dopaminergic receptor ligands [³H]-SCH23390, [³H]-raclopride and [³H]-WIN35,428 to D1 and D2 dopaminergic receptors and dopamine transporter sites throughout the brain, respectively. Sleep deprivation induced a significant decrease in plasma PRL secretion (2.58 ± 0.95 ng/mL) compared with the control group (25.25 ± 9.18 ng/mL). The binding to D1 and D2 binding sites was not significantly affected by sleep deprivation; however, dopamine transporter binding was significantly increased in subdivisions of the caudate-putamen - posterior (16.52 ± 0.5 vs 14.44 ± 0.6), dorsolateral (18.84 ± 0.7 vs 15.97 ± 0.7) and ventrolateral (24.99 ± 0.5 vs 22.54 ± 0.7 µCi/g), in the sleep-deprived mice when compared to the control group. These results suggest that PRL is not the main mechanism involved in the earlier onset of the disease observed in sleep-deprived NZB/NZWF1 mice and the reduction of PRL concentrations after sleep deprivation may be mediated by modifications in the dopamine transporter sites of the caudate-putamen.

Quantitative Autoradiographic Study on Receptor Regulation in the Basal Ganglia in Rat Model of Levodopa-induced Motor Complications / 华中科技大学学报（医学）（英德文版）

In order to study neurotransmitter receptor regulation in the basal ganglia involved in the functional changes underlying levodopa-induced motor complications,quantitative autoradiography was used to observe receptor bindings of dopamine D1 and D2,N-methyl-D-aspartate (NMDA),amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA) and amino butyric acid (GABA) in the basal ganglia of rats that had unilateral nigrostriatal lesions and had been chronically treated with levodopa until motor complications developed.The rats were randomly assigned to three groups:normal,denervated and treatment-complicated groups.The results showed that response duration to levodopa became progressively shorter and abnormal involuntary movement (AIM) score was progressively increased during the course of levodopa treatment.Chronic treatment augmented DI receptors more than denervation,and reduced D2 receptors that were also increased by dopamine denervation.Striatal NMDA receptors were substantially up-regulated in the treatment-complicated group.Levodopa treatment did not change receptors of nigral AMPA,pailidai GABA,and subthalamic GABA,which remained the same as that in denervation group.However,chronic treatment reversed the increase ofnigral GABA receptors caused by the lesion.It was concluded that a shortening of response duration and AIM mimicked levodopa-induced motor complications of Parkinson's patients.These data suggested that up-regulation of dopamine D1 and NMDA receptors in the striatum leads to an imbalance of stimulation through the striatal output pathways,which is associated with levodopa-induced motor complications.

In vitro distribution and receptor scintigraphy of somatostatin receptor 2 subtypes in small cell lung cancer / 肿瘤

Objective: To investigate the distribution of somatostatin receptor 2 subtype (SSTR2) in small cell lung cancer (SCLC) in vitro and the value of 99mTc-octreotide scintigraphy for SCLC diagnosis in vivo. Methods: The distribution of SSTR2 was detected by electron microscopic autoradiography (EMR) using 125I octreotide. 99mTc-octreotide (0.15 mL, 16.8 MBq) was injected into nude mice via tail veins and 99mTc-octreotide scintigraphy was observed. Results: The tagged rates of cellular sliver grains were 95.0% (19/20) and 85.0% (16/20) at 30 min and 120 min, respectively. The difference was significant (P < 0.05). Sliver grains were distributed in the membranes at 30 min and located in the nucleolus and cytoplasm at 120 min. The numbers of sliver grains in the control group (addition of over Tyr 3-octreotide) were remarkably less than those of group 30 min and 120 min. The scintigraphy of the tumors in 5 nude mice was positively displayed at 4 h postinjection of 99mTc octreotide. Conclusions: SSTR2 is over-expressed in SCLC. Radiolabeled octreotide scintigraphy may become a novel detection method for early diagnosis of SCLC.

Isoproterenol Protects the Demilune Cells of the Sublingual Gland of Mice from Radiation / Oral Science International

In order to examine the influence of X-ray irradiation on the demilune cells of the sublingual gland due to the existence of secretory granules, 10 Gy of X-ray irradiation was applied to the sublingual gland of mice at 3 hours after isoproterenol (IPR) administration. To inspect the influence of irradiation at 3 days after the irradiation, tissue images and results of autoradiography performed at 30 and 120 minutes (min) after ³H-leucine administration of the IPR administration (IPR/10Gy) group were compared with those of the non-IPR administration (nonIPR/10Gy) group. In transmission electron microscope images, swelling and pyknosis were observed in the rough endoplasmic reticulum of the nonIPR/10Gy group. The number of reduced silver grains per unit cell area in nonIPR/10Gy at 30 and 120 min after ³H-leucine administration was less and greater than that in the other 3 groups (nonIPR/0Gy, IPR/0Gy, and IPR/10Gy), respectively, from light microscope autoradiography images. At 120 min after ³H-leucine administration, the ratio of the number of reduced silver grains localized in the secretory granules to the total number of reduced silver grains in the demilune cells of the nonIPR/10Gy group was lower than that of the other 3 groups as indicated by electron microscope autoradiography images. Based on these results, it was apparent that the effect of irradiation was less on the demilune cells that discharged secretory granules than those that did not discharge them.

Electron Microscopic Autoradiographic Study on the Secretion of Atrial Specific Granules from Atrial Cardiocytes in Dehydrated Rats / 대한해부학회지

This study was performed to investigate for the effect of dehydration on the synthesis, secretion and secreted pathway of atrial specific granules contained ANP by electron microscopic autoradiography. Male Sprague-Dawley rats, body weigh of about 50 g (range 47 to 53 g), were divided into control, 1 day dehydration and 3 days dehydration groups. Each group was divided into four groups according to sacrificed time on 20 min, 60 min and 240 min after the injection of L-leucine 3 H. Tissues of the right atrium obtained from animals were processed for typical electron microscopic procedure, and then embedded in Epon 812. Ultrathin sections were followed for electron microscopic autoradiographic method. Atrial specific granules were various in size, and some granules had a lower electron densities and indistinct granular membrane in the dehydration groups compared with the control group. In the electron microscopic autoradiographs of atrial wall, silver grains indicated by means of the positions of labelled L-leucine 3 H over the cell inclusion included atrial specific granules, cell organelles, intercellular spaces and blood vesseles. In the control group, high specific radioactivity was observed in the Golgi apparatus at 20 min, and in the rough endoplasmic reticulums and atrial specific granules at 60 min after the injection of L-leucine3H. And high level of radioactivities were observed in the cell membranes and blood vesseles at 240 min after the injection of L-leucine3H. In the 1 day and 3 days dehydration groups, radioactivities of Golgi apparatus, atrial specific granules, cell membranes and intercellular spaces were high level at 20 min, and radioactivities of rough endoplasmic reticulums and blood vesseles were high level at 60 min after isotope injection. Stored atrial specific granules were increased to 34.1% in the 1 day dehydration group, 27.4% in the 3 days dehydration group compared with the control group. In the 3 days dehydration group, newly formed granules increased 85.02% at 20 min, but those decreased rapidly to 36.87% at 60 min, 20.45% at 240 min after the injection of L-leucine3H in atrial cardiocytes. This results suggest that total ANP increased rapidly in the atrial cardiocytes, and newly formed ANP secreted rapidly into the intercellular space in the condition of dehydration, and ANP from atrial cardiocytes remain in intercellular space for dehydration period.

Feasibility of Reflecting Improvement of Tumor Hypoxia by Mild Hyperthermia in Experimental Mouse Tumors with 18F-Fluoromisonidazole / 대한방사선종양학회지

PURPOSE: The aims of this study were to evaluate the change of [18F]fluoromisonidazole ([18F]FMISO) uptake in C3H mouse squamous cell carcinoma-VII (SCC-VII) treated with mild hyperthermia (42oC) and nicotinamide and to assess the biodistribution of the markers in normal tissues under similar conditions. METHODS AND MATERIALS: [18F]FMISO was producedby our hospital. Female C3H mice with a C3H SCC-VII tumor grown on their extremities were used. Tumors were size matched. Non-anaesthetized, tumor-bearing mice underwent control or mild hyperthermia at 42oC for 60 min with nicotinamide (50 mg/kg i.p. injected) and were examined by gamma counter, autoradiography and animal PET scan 3 hours after tracer i.v. injected with breathing room air. The biodistribution of these agents were obtained at 3 h after [18F]FMISO injection. Blood, tumor, muscle, heart, lung, liver, kidney, brain, bone, spleen, and intestine were removed, counted for radioactivity and weighed. The tumor and liver were frozen and cut with a cryomicrotome into 10-micrometer sections. The spatial distribution of radioactivity from the tissue sections was determined with digital autoradiography. RESULTS: The mild hyperthermia with nicotinamide treatment had only slight effects on the biodistribution of either marker in normal tissues. We observed that the whole tumor radioactivity uptake ratios were higher in the control mice than in the mild hyperthermia with nicotinamide treated mice for [18F]FMISO (1.56+/-1.03 vs. 0.67+/-0.30; p=0.063). In addition, autoradiography and animal PET scan demonstrated that the area and intensity of [18F]FMISO uptake was significantly decreased. CONCLUSION: Mild hyperthermia and nicotinamide significantly improved tumor hypoxia using [18F]FMISO and this uptake reflected tumor hypoxic status.

Study on the dynamic distribution of [~(14)C]benzo(a)pyrene in the corpus striatum / 重庆医科大学学报

Objective:To study the dynamic distribution of BaP in the corpus striatum by r-counting and light microscopic autoradiography,then to explore the neurotoxic mechanism of BaP.Methods:100 SD male rats were divided into control group(n=40)and test group(n=60),experimental animals were given a single intravenous injection of 3.7?105 Bq/kg of 14CBaP while the same doses of Normal Saline were given to the control group.The rats were sacrificed at 1h,1 d,2 d,3 d and 7 d after the administration of radiolabelled BaP.During the experiment,some toxicological symptoms were observed and the ratios of brain-weight/body-weight were detected.Light microscopic autoradiography and r-counting were used to observe the dynamic distribution of BaP in the corpus striatum.Results:The change of toxicological symptoms are observed and the decrease ratios of brain-weight/body-weight are detected.R-counting shows that the percentage dose/g of 14CBaP in striatum is significant higher than in hippocampus and cortex at 1d and 2 d after administration.Light microscopic autoradiography shows that the silver granules in striatum reach the peak in 1d and sharp decrease in 2 d,which can be found even at 7d.Conclusion:BaP can penetrate the blood-brain barrier and distribute in corpus striatum,inducing CNS toxicity in SD rat.

The Temporospatial Distribution of Glutamate Receptors and the Effect of MK-801 on Glutamate Receptors Activation in Kainate-induced Seizure Model: Quantitative Receptor Autoradiography of Ionotropic Glutamate Receptors

BACKGROUND: Excitotoxicity and epileptogenesis have often been associated with glutamate receptor activation. Some evidence indicates that selective down regulation of AMPA receptor may be the mechanism of delayed neuronal cell death in the hippocampus. METHODS: We used in situ hybridization to examine the hybridization density (HD) of NMDA and AMPA receptors on excitotoxicity and epileptogenesis in the hippocampus of the kainic acid (KA)-induced rat seizure model. Some Sprague-Dawley rats were injected with KA, and others with MK-801 prior to KA injection. The rats were killed at 8 hours or 4 weeks after KA or MK-801/KA injection. HD of [3H]MK-801 and [3H]AMPA bind-ing in subfields of the hippocampus was measured by an image analyzer. RESULTS: After 8 hours of KA injection, [3H]MK-801 binding was increased in CA1 and CA3, and decreased in dentate gyrus, and [3H]AMPA binding was decreased in all of CA1, CA3 and fascia dentata, and pretreatment of MK-801 did not affect [3H]AMPA binding in all of CA1, CA3 and dentate gyrus. After 4 weeks, both [3H]MK-801 and [3H]AMPA binding were prominently increased in inner molecular layer of dentate gyrus. CONCLUSIONS: Glutamate receptors, especially NMDA receptor, were associ-ated with excitotoxicity in the hippocampus but the selective down regulation of GluR2 subunit of AMPA receptor without NMDA receptor activation may not be sufficient to cause excitotoxic neuronal cell death in CA1 and CA3. In addition, the synaptic reorganization in inner molecular layer of dentate gyrus was proved to be chronically hyperex-citable in function and may contribute epileptogenesis.

mRNA is Synthesized Mainly at the Phase between the Euchromatin and Heterochromatin: Proposal of a Phase Theory

BACKGROUND: Malignant cell nuclei, in general, have increased amounts of heterochromatin and decreased electron densities of euchromatin, making the chromatin pattern coarser than that of benign cell nuclei. The chromatin pattern in benign and malignant cells, however, is barely explained in terms of molecular structure. In this study, the chromatin pattern of metaplastic and carcinomatous squamous cells of the uterine cervix was correlated with transcriptional activity by ultrastructural autoradiography. METHODS: Punch-biopsied tissues were cultured with 3H-uridine for 5 minutes and processed for electron microscopy. Thin sections of the tissues on nickel grids were covered with photosensitive emulsion and kept cold in a dark room for 10 to 16 weeks. After development and staining, the tissues were observed by electron microscopy. RESULTS: The nuclei of the metaplastic squamous cells consisted mostly of euchromatin. A few silver grains were observed, mainly at the periphery of the nuclei. The nuclei of the carcinomatous cells had increased amounts of heterochromatin along the nuclear membrane, and also in the euchromatin area. Silver grains were observed mainly at the boundary between the heterochromatin and euchromatin. CONCLUSION: These findings suggest that an increased amount of heterochromatin in carcinomatous cells results in an increase of the boundary area between the heterochromatin and euchromatin, an area which may be a transcriptionally active site.

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 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.

Changes of the level of G protein alpha-subunit mRNA by withdrawal from morphine and butorphanol

Morphine or butorphanol was continuously infused into cerebroventricle (i.c.v.) with the rate of 26 nmol/microliter/h for 3 days, and the withdrawal from opioid was rendered 7 hrs after the stopping of infusion. The expression of physical dependence produced by these opioids was evaluated by measuring the naloxone-precipitated withdrawal signs. The withdrawal signs produced in animals dependent on butorphanol (kappa opioid receptor agonist) were similar to those of morphine (mu opioid receptor agonist). Besides the behavioral modifications, opioid withdrawal affected G protein expression in the central nervous system. The G-protein alpha-subunit has been implicated in opioid tolerance and withdrawal. The effects of continuous infusion of morphine or butorphanol on the modulation of G protein alpha-subunit mRNA were investigated by using in situ hybridization study. In situ hybridization showed that the levels of G alphas and G alphai were changed during opioid withdrawal. Specifically, the level of G alphas mRNA was decreased in the cortex and cerebellar granule layer during the morphine and butorphanol withdrawal. The level of G alphai mRNA was decreased in the dentate gyrus and cerebellar granule layer during the morphine withdrawal. However, the level of G alphai mRNA was significantly elevated during the butorphanol withdrawal. These results suggest that region-specific changes of G protein alpha-subunit mRNA were involved in the withdrawal from morphine and butorphanol.

Anticancer Effect of AG60 (Acriflavine-Guanosine Compound) on the Ehrlich Cancer Cells Light Microscopic, Autoradiographic and Electron Microscopic Study / 대한해부학회지

To evaluate the effect and working mechanism of a newly developed anti-cancer drug, AG60 (acriflavine-guanosine compound, Taerim Pharm. Co. Seoul, Korea), histotologic, autoradiographic and electron microscopic studies were carried out. For the histologic study, each Ehrlich carcinoma cells (10(7) cells)-inoculated mouse was subcutaneously injected with saline (0.2 ml), 10 mg/kg of AG60, or 30 mg/kg of AG60, every other day, respectively. Animals were sacrified on the 14th day from the first injection, and tumor masses were fixed in 10% formalin solution. Tissue sections of the tumor were stained with hematoxylin and eosin. For the electron microscopic study, Ehrlich carcinoma (10(7) cells)-inoculated mice were subcutaneously injected every other day with saline (0.2 ml) or 30 mg/kg of AG60, respectively. The day after 7th injection (14th day), animals were sacrified, small piece of tumor masses were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde solution followed by fixation in 2% osmium tetroxide solution. Ultrathin sections were counter stained with uranyl acetate-lead citrate solutions, and observed with JEM 100CX electron microscope. For the autoradiographic study, each Ehrlich carcinoma (10(7) cells)-inoculated mouse was injected every day with 0.2 ml of saline, 5 mg/kg of AG60, or 30 mg/kg of AG60, respectively. The day following the last injection, each animal was given a single dose of 0.7 micricurie/g of methyl-3H-thymidine (Amersham Lab., England) through the tail vein. Seventy minutes after the thymidine injection, animals were sacrified, tumor masses were collected and fixed in 10% neutral formalin. Tissue blocks were washed, dehydrated, embedded and cut in 6 micrometer-thick sections. Deparaffinzied sections were dipped in the autradiographic emulsion E1 (Amersham Lab., England) and dried and stocked in the dark room. Filmed sections were exposured five weeks in the dark room, and were developed in the developer. Labeled indices (mean number of labeled cells per 100 cancer cells) and labeled grain indices (mean number of labeled silver grains per one cancer cell, and total granule numbers per every 100 cancer cell) were observed and calculated. The results were as follows : 1. On histological study, massive apoptosis were occured following the injection of AG60. Only small number of live cancer cells were observed. 2. On electron microscopic study, massive apoptotic figures including fragmentation of nuclei and cytoplasms, multiple nucleoli, condensation of nucleus and cytoplasm, deep invaginations and microcleft formations of nuclei, margination of heterochromatin along the inner nuclear membrane and microcleft , etc. were noticed. Giant cells represent the "tumor cell-tumor cell emperipolesis", and many of them seem to be in process of "cytolytic emperipolesis". 3. On autoradiographic study, labeled grains of 3H-thymidine were suppressed to only 11%~5% of control cancer cells following AG60 administrations. Discussed on the above experiments, it is suggested that severe suppression of DNA, RNA and protein syntheses by AG60 induce massive apoptosis of cancer cells. AG60 is expected as one of most effective anticancer drugs for the cytostatic therapy, the disease stabilization, the improved quality of life, the prolongation of life, and possibly the chemoprevention.

Quantitative Autoradiography on Ontogenic Development of alpha-Adrenoceptor in the Rat Cerebral Cortex / 대한해부학회지

Adrenoceptors mediate response to catecholamines throughout the body. To investigate postnatal ontogenic development of alpha1- and alpha2- adrenoceptors in the rat cerebral cortex, in vitro autoradiography was done on frontal, parietal and temporal cortex in P0, P5, P10, P15, P20, P30 and adult animals. Binding sites for the alpha1-adrenergic receptor ligand, [3H]-prazosin, and the alpha2-adrenergic receptor ligand, [3H]-rauwolscine, were visualized by in vitro autoradiography, and anatomically localized by comparing the autoradiogram to Nissl-stained sections. Nonspecific binding was detected with unlabeled phentolamine (alpha1) and yohimbine (alpha2). There is uniform increase in alpha1- and alpha2- adrenoceptors from birth through first three or four postnatal weeks, followed by a decrease to adult level. Two alpha-adrenoceptors have very different ontogenic patterns of distribution during postnatal development. alpha1- adrenoceptors were expressed differentially in different cortical (frontal, temporal, parietal) regions and in different cortical layers (layers V, II-IV, VI) at same age. alpha2- adrenoceptor was expressed homogenously in throughout regions and layers. These findings may provide evidence that alpha1- adrenoceptors are involved in regulating cortical development or function more specifically than alpha2- adrenoceptors during postnatal development.

Autoradiographic Study on the Effect of AG60 to the DNA Synthesis of Gastric Epithelium of Mouse Inoculated with Ehrlich Carcinoma / 대한해부학회지

To study the tumor-suppression effect of a newly developed anti-tumor agent AG60 [acriflavine (1) : guanosine (1) composition, Taerim Pharm. Co., Seoul, Korea], each Ehrlich carcinoma (10(7) cells)-inoculated mouse received the subcutaneous injection of 0.2 ml of saline, 5 mg/kg of AG60, and 30 mg/kg of AG60 per day for a week. The day following the last injection, each mouse was injected with a single dose of 0.7 microcurie/g of methyl-3H-thymidine (25Ci/mmol, Amersham Lab., England) through tail vein. Seventy minutes after the thymidine injection, animals were sacrificed, and gastric tissues were collected and fixed in 10% neutral formalin. Tissue blocks were washed, dehydrated, embedded and cut in 6 micrometer-thick sections. Deparaffinized sections were coated with autoradiographic emulsion EM 1 (Amersham Lab. England) in a dark room and dried and were placed in a light-tight box. The sections were exposured for 5 weeks in the dark room, and were then developed in D-19 developer. Labeled indices (mean number of labeled cells per 100 epithelial cells in the isthmus) were observed and calculated. The results are as follows; 1. On histological study, gastric mucosa had no morphological changes following the injection of AG60. 2. On autoradiographic study, labeled grains of 3H-thymidine were restricted on the isthmus portion of the gatric gland. 3. On autoradiographic study, labeling indicies of gastric epithelial cells of normal control, experimental control, AG60 (5 mg/kg)-treated and AG60 (30 mg/kg)-treated groups were 21.9+/-0.28%, 18.8+/-0.03%, 21.6+/-1.61% and 6.3+/-0.93%, respectively. These result suggest that AG60 is expected as one of most effective anticancer drugs, and the dosage under 5 mg/kg of AG60 does not result any defect on the DNA synthesis in gastric epithelial cells.

Atrial Natriuretic Peptide System in Eyes

Atrial natriuretic peptide(ANP), a peptide hormone synthesized mainly in the cardiac atrium, has an important physiological role on the regulation of body fluid and electrolyte balance. Extraatrial ANP system has been reported. The presence of ANP in eye has also been reported. ANP in the eye has claimed to control the intraocular pressure. However, the presence of ANP and its receptors in the intraocular tissues are controversial. Therefore, the purpose of the present study was to determine the characteristics of molecular nature of ANP and its receptors in variable intraocular tissues of cow. Immunoreactive ANP was detected in aqueous humor(10+/-1 pg/ml), cornea (3.6+/-0.5 pg/mg), ciliary body(2.62+/-0.6 pg/mg), choroid(2.1+/-0.5 pg/mg), retina (1.7+/-0.2 pg/mg)and iris(1.4+/-0.5 pg/mg). Chromatographic characterization of molecular profile of ANP showed major peak corresponding to small molecular weight forms of ANP and minor peak corresponding to proANP. ANP mRNA was detected in the cornea, retina and ciliary body using reverse transcriptase-polymerase chain reaction. The production of cGMP by the activation of guanylyl cyclase was stimulated by ANP, BNP and CNP in tissue membranes of cornea, ciliary body and iris. Autoradiographic study showed that the corneal endothelium had A, B, and C subtypes of natriuretic peptide receptor. Longitudinal fibers of ciliary muscle and retina had A subtype of natriuretic receptor. These results suggest that the bovine eye has its own ANP system and ANP may have an important paracrine or autocrine function in the eye.

Biodistribution of Iodine-131-Iodomisonidazole and Imaging of Tumor Hypoxia in Mice bearing CT-26 Adenocarcinoma / 대한핵의학회잡지

urpose: Misonidazole is a radiosensitizer that binds in hypoxic cells. The purpose of this study was to find out the feasibility of I-131-Iodomisonidazole (IMISO) for imaging of tumor hypoxia. MATERIALS AND METHODS: Tosyl precursor was dissolved in acetonitrile and I-131-NaI was added to synthesize IMISO. Balb/c mice inoculated with CT-26 adenocarcinoma were injected with IMISO. Mice were sacrificed at 1,2,4,24 hr and % of injected dose per gram of tissue (%ID/g) was determined. For scintigraphy and MRI, mouse bearing CT-26 adenocarcinoma was administered with IMISO and imaging was performed 4 hr after. Then, mouse body was fixed and microtomized slice was placed on radiographic film for autoradiography. RESULTS: %ID/g of tumor was 1.64 (1h), 0.98 (2h), 0.85 (4h) and 0.20 (24h), respectively. At 24h, %ID/g of tumor was higher than that of all other tissues except thyroid. Tumor to muscle ratio increased with time and tumor to blood ratio also increased with time and reached 1.53 at 24 hr. On autoradiogram, tumor was well visualized as an increased activity in central hypoxic area of the tumor which corresponds to the area of high signal intensity on T2-weighted MR image. On scintigraphy, tumor uptake was visualized. CONCLUSION:: This RESULTS suggest that IMISO may have a potential for tumor hypoxia imaging in mouse model. However, further study is needed to improve it's localization in tumor tissue and to achieve acceptable images of tumor hypoxia.

Ontogeny of Adrenergic Receptors in the Rat Amygdala / 대한해부학회지

The mammalian amygdala comprises a heterogeneous complex of cytoarchitectonically, histochemically and connectionally distinct nuclei. To investigate the developmental changes and regional distributions of adrenoceptor binding sites in the adult and postnatal rat amygdala (postnatal days 0, 5, 10, 15, 20, 30), in vitro autoradiography was performed. Binding sites for the alpha1-adrenoceptor ligand [3H]-prazosin, the alpha2-adrenoceptor ligand [3H]-rauwolscine, and the beta-adrenoceptor ligand [125I]-iodocyanopindolol were visualized by the in vitro autoradiography, and anatomically localized by comparing the autoradiograms to Nissl- and acetylcholinesterase-stained sections. On toluidine blue- and acetylcholinesterase-stained sections of the amygdaloid complex of the rat, three major divisions can be distinguished: the cortical-like nuclear group, medial nuclear group, and central nuclear group. The basolateral nuclear group of the cortical-like nuclear group was divided into three subregions, the basal, the basolateral and the basomedial amygdaloid nucleus. Between the medial and the basolateral amygdaloid nucleus, the intercalated nuclei were observed. The highest number of alpha1-adrenoceptor binding sites was detected in the central and the lateral amygdaloid nucleus. The nuclei most strongly labeled by [3H]-rauwolscine were those in the medial part of the amygdaloid complex. The pattern of the beta-adrenoceptor binding was relatively diffuse, the medial amygdaloid nucleus was most strongly labeled among the amygdaloid nuclei. At the postnatal day 0, adrenergic receptor binding sites were only weakly labeled. The expression of a1-adrenoceptor binding was rapidly increased in the central amygdaloid nucleus at the postnatal day 5, and between the postnatal day 10 and 15, the concentration of bindig sites reached the adult levels. The expression of alpha2- and beta-adrenoceptor binding was increased in most amygdaloid nuclei at the postnatal day 10, and higher density was observed at the postnatal day 30. In the adult, the expression of adrenoceptor binding was relatively low in most nuclei when compared to postnatal day 30. These findings may provide evidence that alpha1-adrenoceptor is involved in regulating amygdaloid development and function more specifically than alpha2- and beta-adrenoceptor during postnatal development. These results indicate that the regional distributions of alpha1-, alpha2-, and beta-adrenoceptor show some differences from those of the other mammalian species reported.