Autometallography for Zinc Detection in the Central Nervous System

조승묵,김성준,박승국,강태천,원무호,Jo, Seung-Mook,Gorm, Danscher,Kim, Sung-Jun,Park, Seung-Kook,Kang, Tae-Cheon,Won, Moo-Ho Korean Society of Electron Microscopy 2000 Applied microscopy Vol.30 No.4

Zinc is one of the most abundant oligoelements in the living cell. It appears tightly bound to some metalloproteins and nucleic acids, loosely bound to some metallothioneins or even as free ion. Small amounts of zinc ions (in the nanomolar range) regulate a plentitude of enzymatic proteins, receptors and transcription factors, thus rolls need accurate homeostasis of zinc ions. Zinc is an essential catalytic or structural element of many proteins, and a signaling messenger that is released by neural activity at many central excitatory synapses. Growing evidences suggest that zinc may also be a key mediator and modulator of the neuronal death associated with transient global ischemia and sustained seizures, as well as perhaps other neurological disease stoles. Some neurons have developed mechanisms to accumulate zinc in specific membrane compartment ('vesicular zinc') which can be evidenced using histochemical techniques. Substances giving a bright colour or emitting fluorescence when in contact with divalent metal ions are currently used to detect them inside cells; their use leads to the so called 'direct' methods. The fixation and precipitation of metal ions as insoluble salt precipitates, their maintenance along the histological process and, finally, their demonstration after autometallographic development are essential steps for other methods, the so called 'indirect methods'. This study is a short report on the autometallograhical approaches for zinc detection in the central nervous system (CNS) by means of a modified selenium method.

Zinc 대사와 관련된 Paneth 세포활성의 변화에 관한 조직화학적 연구

조승묵,김성준,박승국,강태천,원무호,Jo, Seung-Mook,Kim, Sung-Jun,Park, Seung-Kook,Kang, Tae-Cheon,Won, Moo-Ho 한국현미경학회 2000 Applied microscopy Vol.30 No.4

Paneth cells have been suggested to contribute to the elimination of excess metals into the intestinal lumen. The purpose of this study wat to investigate the changes of the zinc pools in rats subjected to functional loading with zinc salt by mean of both light and electron microscopical autometallography (AMG). Wistar rats 4 were administrated with zinc chloride (20 mg/kg body weight) intraperitoneally dissolved in 1 ml distilled water. The control group received 1 ml saline IP. After further one hour the animals were transcardially perfused with 0.4% sodium sulphide dissolved in 0.1 M PB fellowed by 3% glutaraldehyde solution for 10 minutes. Pieces of ileum were frozen with solid $CO_2$ and sectioned on a cryostat. The sections $(20{\mu}m)$ were autometallographically developed. Sections selected for EM were reembedded on top of a blank Epon block, from which ultrathin sections (100 nm) were cut. The ultrathin sections were double stained with uranyl acetate (30 min) and lead citrate (5 min), then examined under electron microscope. Studies of comparable sections from control and zinc loaded animals with the AMG selenium method gave quite different results. The control animals demonstrated a weakly positive staining in the cytoplasm of the Paneth cells. In the electron microscope the AMG silver grains were found to be located in the cytoplasm, while the electron dense secretary granules and other cell organelles were void of staining. Few AMG grains were located at the apical surface of the Paneth cells. In sections from zinc loaded rats, the AMG grains were seen in abundance in the lumen of the Lieberkuhn crypts at light microscopic levels. At EM levels the zinc revealing silver grains were located in the cytoplasm as in the controls, but much more AMG grains were shifted into the secretary granules. Furthermore, profound AMG grains were found in the lumen of the crypts and surrounding vessels. And a few grains were seen in the endothelium. The AMG technique demonstrated a pattern of AMG grains in the Paneth cells that strongly suggests a transport of zinc ions through these cells.

랫드 척수신경 결찰에 따른 척수신경절세포의 퇴행성변화

김이석,조승묵,Kim, Yi-Suk,Jo, Seung-Mook 한국현미경학회 2009 Applied microscopy Vol.39 No.3

This study aim to disclose a possible mechanism for the neuronal cell death induced by peripheral nerve injury following a spinal nerve ligation (SNL) as a neuropathic pain model. Male Sprague-Dawley rats (270~290 g) were used for this study. Pain threshold was evaluated for their response to mechanical (von Frey hairs) stimuli 1, 3, and 7 days after a tight ligation of L5 ventral ramus. In control group, the small ganglion cells were strongly stained with routine toluidine blue (TB), whereas the large ganglion cells showed a little bit weak stainity. Each large ganglion cell is surrounded by perineuronal satellite cells. In experimental groups, small ganglion cells showing apparent degenerative changes increased on 1 day, and showed a peak in degenerative cell number at 3 days group, and decreased gradually at 7 days group. We also found a small number of large-sized ganglion cells showing mild degenerative changes. However their satellite cells ware relatively intact with no typical findings throughout this experiment. Under the electron microscope, small ganglion cells showed various stage and typical features of the dark degeneration including mitochondrial swelling.

Histochemically-reactive Zinc in the Rat Dorsal Root Ganglion (DRG) Neurons: Zinc Selenium Autometallography

김이석,조승묵,Kim, Yi-Suk,Jo, Seung-Mook Korean Society of Microscopy 2010 Applied microscopy Vol.40 No.1

The present study was designed to demonstrate ionic zinc in the rat DRG by means of zinc selenium autometallography($ZnSe^{AMG}$). Ganglion cells varied in size from 15 to 100 ${\mu}m$. The smaller neurons were strongly stained with AMG, whereas the larger cells were weakly stained. Each large ganglion cell was surrounded by perineuronal satellite cells, showing apparent AMG staining. We demonstrated for the first time the existence of zinc-containing satellite cells in the rodent DRG. Using electron microscopy, fine AMG grains were observed scattered in the somata of the DRG neurons, especially small cells. However, much lower concentrations of the AMG grains occupied in the large cells, and these were mostly localized in lysosome-like organelles. These results indicate that zinc may be involved in sensory transmission in the DRG level.

5,7-Dihydroxytryptamine의 세포독성에 의한 고양이 망막내 미세아교세포의 반응양상

주우현,남성안,조승묵,조현후,신민철,원무호,최창도,Joo, Woo-Hyun,Nam, Seong-Ahn,Jo, Seung-Mook,Cho, Hyon-Hoo,Shin, Min-Cheol,Won, Moo-Ho,Choi, Chang-Do 한국현미경학회 1998 Applied microscopy Vol.28 No.4

This study was designed to investigate the microglial reactions to the neurodegenerative changes in the cat retina. All experiments were performed using adult cats of both sex, weighing $2,500g\sim3,500g$. 5,7-DHT $(100{\mu}g)$ dissolved in 0.1% ascorbic acid was injected into the vitreous body. All injections were performed in one-side eye; the other side served as the control, which was injected only with 0.1% ascorbic acid. Cats were sacrificed at 1, 3, 7, 14 and 21 days after intravitreal injection of 5,7-DHT For light microscopy, retinae were fixed with 4% paraformaldehyde and processed using NDPase histochemistry. Same retinae were fixed with 1% para(formaldehyde-2.5% glutaraldehyde and processed for electron microscopy. NDPase-positive microglial cells were mainly distributed in the inner plexiform layer of the retina, and characterized by a small somata with a few slender processes, which were also extended in the ganglion cell layer (GCL) and inner nuclear layer (INL). The intensity of the microglia stained for NDPase was abruptly increased at 7 day as compared with that of the control, and thereafter continuously sustained until 21 day, the last experimental group in this study. Under the electron microscopical observation, microglial cells in the control group exhibited elongate nucleus with perinuclear chromatin condensation, and the perikaryon was scanty. However, a few hypertrophic glial cells were frequently found at 3 days after the drug injection. By 7 day, most microglial cells directed toward the degenerated neurons in the GCL, and the number of microglial cells was slightly increased as compared with the former group. At the 14 day, most microglial cells wrapped the degenerated cells in the GCL, and a few cells showed phagocytotic features. By 21 day, most microglial cells were engaged in phagocytotic activity, and their cytoplasm was filled with the phagorytosed material. Based on the results, 5,7-DHT may act as a specific neurotoxin to the cat retina, and microglial reactions to the neuronal death are already induced in early experimental stage. These results indicate that the microglial cells in the cat retina show characteristic features as a protective effect of neural tissue.

The Oxytocinergic Neurons in Hypothamo-hypophysial Tract Contributes to CNS Pathway Innervating Ovary in Rat

변경희,오지현,조승묵,이봉희,Byun, Kyung-Hee,Oh, Jee-Hyun,Jo, Seung-Mook,Lee, Bong-Hee Korean Society of Microscopy 2010 Applied microscopy Vol.40 No.4

The mammalian ovary is innervated by sympathetic and sensory neurons which contribute to regulating several aspects of ovarian function, including blood flow, steroidogenesis and follicular development. The existence of a neural connection between central neurons and the ovary has been rarely reported, but the mechanism underlying integration of ovarian activity to broader neuroendocrine responses has not been reported. We have now used a viral transneuronal tracing technique combined with a conventional retrograde labeling procedure of CT-HRP to demonstrate that oxytocin-producing neurons of the hypothalamus are synaptically connected to the ovary. Since ovarian activity is suppressed but the activity of oxytocin neurons is increased during breast feeding. Our finding that the oxytocinergic neural connection is likely to provide a direct transsynaptic mechanism by which the central nervous system maintains the state of infertility that accompanies lactation in mammals.

5, 7-DHT가 흰쥐 등쪽솔기핵내 신경아교세포에 미치는 영향

채제명(Je Myung Chae),조승묵(Seung Mook Jo),남성안(Seong Ahn Nam),윤상선(Sang Seon Yoon),고병문(Byung Moon Ko),최창도(Chang Do Choi),최월봉(Wol Bong Choi) 대한해부학회 1998 Anatomy & Cell Biology Vol.31 No.1

신경독성물질인 5, 7-dihydroxytryptamine (5, 7-DHT)이 중추신경계통내 serotonin 신경세포에 미치는 영향과 이에 따른 신경아교세포들의 반응을 형태학적으로 규명하고자 본 연구를 시행하였다. 흰쥐의 가쪽뇌실에 5, 7-DHT(200μg)를 투여한 후면역세포화학적 염색을 시행한 후 광학 및 전자현미경하에서 등쪽솔기핵내 serotonin 신경세포에 초래되는 퇴행성변화와 주위 신경아교세포들의 반응양상 등을 경시적으로 (1, 3, 5, 10 및 20일) 관찰하였던 바 아래와 같은 결과를 얻었다. 흰쥐 등쪽솔기핵내 5, 7-DHT의 세포독성에 반응을 보인 신경아교세포로는 미세아교세포와 별아교세포였으며, 이들 세포 들의 반응양상은 매우 특징적이었으며, 상당한 차이를 보였으며, 전반적으로 미세아교세포가 별아교세포에 비해 빠른 변화소견을 보였다. 즉, 미세아교세포의 경우 실험 3일군 및 5일군에서 이미 그 수의 증가와 함께 활발한 포식작용을 보였던 반면, 별아교세포는 후기 실험군인 10일군 및 20일군에 이르러서야 세포질의 비대 및 수적 증식을 통하여 손상된 신경조직을 보상할 뿐 아니라 밀집된 미세아교세포들을 분산시키는 소견이 관찰되었다. 그러나 희소돌기아교세포는 본 연구의 전 실험군을 통하여 특별한 변화소견이 관찰되지 않았다. 이상의 결과로 부터 5, 7-DHT가 흰쥐 등쪽솔기핵내 신경세포에 강한 세포독성을 유발하며, 이에 따른 주위 신경아교세 포들 특히 미세아교세포와 별아교세포는 시간경과에 따라 매우 다양한 반응양상을 보인다는 사실을 알게 되었다 This study was designed to clarify the cytotoxic effects of 5, 7-dihydroxytryptamine (5, 7-DHT) on the serotonergic neurons in the dorsal raphe nucleus, and to investigate the glial reaction during the neurodegenerative changes by light and electron microscopy. Adult male rats (Sprague-Dawley strain) weighing from 250 g to 350 g each were used as experimental animal. 5, 7-DHT(200 μg dissolved in 0.9% NaCl) was injected into the lateral ventricle of the rat brain with the Hamiton syringe fixed on stereotaxic apparatus. The control rats were given with the similar volume of 0.9% NaCl. The rats were sacrified on the 1st, 3rd, 5th, 10th and 20th day after the injection of 5, 7-DHT. The results were as follows : Glial reactions induced by 5, 7-DHT were also observed in DRN. In early experimental stage, microglial reactions prevailed, whereas astroglial reactions were prevailing in later stage. In addition, microglial cells phagocytosed and removed the degenerated cells. However, astrocytes in DRN did not show phagocytotic activities such as microglial cells. Based on the results, author thought that 5, 7-DHT act as a specific neurotoxin to serotonergic neurons in DRN, and induces severe neurodegenerative changes. The glial reactions in DRN are activated during the neurodegerative changes, and show characteristic patterns of glial reactions.

모래쥐 흑색질의 도파민성 신경세포의 분포와 미세구조

최월봉,윤상선,고병문,조승묵,남성안,최창도,Choi, Wol-Bong,Yoon, Sang-Seon,Ko, Byoung-Moon,Jo, Seung-Mook,Nam, Seong-Ahn,Choi, Chang-Do 한국현미경학회 1997 Applied microscopy Vol.27 No.4

The substantia nigra of the Mongolian gerbil was studies by tyrosine hydroxylase immunohistochemistry and immunoelectron microscopy with preembedding method. The purpose was to obtain information on the distribution and ultrastructure of the Tyrosine hydroxylase immunoreactive and dopaminergic neurons in the substantia nigra, in order to provide the necessary background for the gerbil. Large number of tyrosine hydroxylase immunoreactive neurons were located in the compact part of substantia nigra. Findings in the gerbil, compared to observations in the other species, included the presence of prominent bundles of tyrosine hydroxylase immunoreactive cytoplasmic processes passing in the dorsoventral direction from pars compacta into pars reticulata at middle and caudal levels of the substantia nigra, and the presence of a distinct tyrosine hydroxylase immunoreactive substantia nigra pars lateralis. Tyrosine hydroxylase immunoreactive neurons had well-developed cell organelles, especially rough endoplasmic reticulum, free ribosome and poly-ribosome, and showed the infoldings of the nuclear envelope. We anticipate that the present description of the cellular organization of the tyrosine hydroxylase immunoreactive dopaminergic area in the substantia nigra of gerbil will be useful for the animal experimental model of Parkinson's disease.

Detection Methods of Histochemically-reactive Zinc in the CNS at the Light Microscopical Level

김이석,김상현,이법이,이현숙,김성주,조승묵,Kim, Yi-Suk,Kim, Sang-Hyun,Lee, Beob-Yi,Lee, Hyun-Sook,Kim, Sung-Joo,Jo, Seung-Mook Korean Society of Microscopy 2008 Applied microscopy Vol.38 No.1

Small amounts of zinc ions regulate a plentitude of enzymatic proteins, receptors and transcription factors, thus cells need accurate homeostasis of zinc ions. Some neurons have developed mechanisms to accumulate zinc in specific membrane compartment ("vesicular zinc"), which can be evidenced using histochemical techniques. These neurons are the socalled zinc enriched (ZEN) neurons, which accumulate glutamate and zinc inside their synaptic vesicles and release it during synaptic transmission. In the present paper we have studied the distribution of the ZEN terminals in the rat hippo-campus using ZnSe autometallography, Neo-Timm staining, ZnT3 immunohistochemistry and TSQ fluorescence staining.

랫드 발바닥 염증부위에서 관찰된 zinc함유 비만세포의 미세구조: 조직화학적 염색을 중심으로

이보예,김이석,이법이,이현숙,탁계래,이영일,이정열,조승묵,Lee, Bo-Ye,Kim, Yi-Suk,Lee, Boeb-Y.,Lee, Hyun-Sook,Tak, Gye-Rae,Lee, Young-Il,Lee, Jeoug-Yeol,Jo, Seung-Mook 한국현미경학회 2006 Applied microscopy Vol.36 No.4

Mast cells (MCs) are granulated cells that play a pivotal role in allergic reaction and inflammation. The granules of mast cells are known to be rich in zinc (Zn). Male Sprague-Dawley rats were used. We injected $200{\mu}L$ of complete Freund's adjuvant (CFA) subcutaneously in the dorsal aspect of one hindpaw Finally, zinc selenium autometallography(AMG) was done by Danscher's method. The present study showed the ultrastructures of zinc-containing mast cells found in inflammatory area following an complete freund's adjuvant (CFA) inoculation into the rat hindpaw. At light microscopic level, mast cells were round or oval, at average $12{\mu}m$ in diameter, with many filopodia extending from the cell surface. Because the rather small and spherical nucleus was centrally placed; it was frequently obscured by the cytoplasmic granules, it sometimes could not be seen. Mast cells were distributed chiefly in the vicinity of small blood vessels. In most preparation many mast cells were ruptured and their granules escaped into the surrounding tissue. In electron micrographs, The secretory granules were at average $0.5{\mu}m$ in diameter and were limited by a membrane. The cell surface contained numerous microvilli and folds. Their interior was heterogenous in appearance. The nucleus was surrounded by large numbers of prominent vesicels and a well developed Golgi apparatus, but scant endoplasmic reticulum.