• Title/Summary/Keyword: Nerve tissue

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Changes of CGRP immunoreactivity in rat trigeminal ganglion neurons during tooth movement (백서 삼차신경절내 신경세포체의 치아이동에 따른 CGRP 면역염색성의 변화)

  • Park, Chyo-Sang;Park, Guk-Phil;Sung, Jae-Hyun
    • The korean journal of orthodontics
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    • v.27 no.4 s.63
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    • pp.607-621
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    • 1997
  • GRP was known as the modulator of Pain transmission in central nervous system and local effector to peripheral tissue causing vasodilation, increased blood flow, modulation of immune sysem, stimulation of endothelial cell proliferation, and stimulation of bone formation. Numerous study, therefore, were done to elucidate involvement of CGRP to tooth movement. To investgate the response of CGRP immunoreactive nerve cells according to cell size in trigeminal ganglion during tooth movement, immunohistochemical study was performed using rat. Experimental rats(9 weeks old, 210 gm) were divided as six groups(normal(n=6), 3 hour group(n=5), 12 hour group(n=4), 1 day group(n=5), 3 day group(n=5), 7 day group(n=5)), and were applied orthodontic force (approximately 30 gm) to upper right maxillary molar. After frozen sections of trigeminal ganglions were immunostained using rabbit antisera, the changes of CGRP immunoreactive cells in regard to cell size distribution(small cell(upto $20{\mu}m$), medium cell($20-35{\mu}m$), large cell(above $35{\mu}m$)) were observed. The results were as follows 1. The percentage of CGRP immunoreactive cells to all nerve cells in trigeminal ganglion was 33.0% in normal control group, was decreased to 24.5% in 1 day group, and was increased to 41.8% in 7 day group. 2. The percentage of small, medium, and large cells expressing CGRP immunoreactivity in normal trigeminal ganglion to all CGRP immunoreactive cells were 51.3%, 44.0%, 4.7%, respectively. 3. The percentage of small cells with CGRP immunoreactivity to all CGRP immunopositive cells was increased in 3 hour and 12 hour groups. 4. The percentage of medium cells with CGRP immunoreactivity was increaed in 3 day and 7 day groups. 5. The percentage of large cells with CGRP immunoreactivity was increaed in 7 day group. Conclusively, the small cells with CGRP immunoreactivity in trigeminal ganglion respond to orthodontic force during initial phase of tooth movement, and later the medium and large cells with CGRP immunoreactivity respond

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Mammalian Cloning by Nuclear transfer, Stem Cell, and Enzyme Telomerase (핵치환에 의한 cloning, stem cell, 그리고 효소 telomerase)

  • 한창열
    • Korean Journal of Plant Tissue Culture
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    • v.27 no.6
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    • pp.423-428
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    • 2000
  • In 1997 when cloned sheep Dolly and soon after Polly were born, it had become head-line news because in the former the nucleus that gave rise to the lamb came from cells of six-year-old adult sheep and in the latter case a foreign gene was inserted into the donor nucleus to make the cloned sheep produce human protein, factor IX, in e milk. In the last few years, once the realm of science fiction, cloned mammals especially in livestock have become almost commonplace. What the press accounts often fail to convey, however, is that behind every success lie hundreds of failures. Many of the nuclear-transferred egg cells fail to undergo normal cell divisions. Even when an embryo does successfully implant in the womb, pregnancy often ends in miscarriage. A significant fraction of the animals that are born die shortly after birth and some of those that survived have serious developmental abnormalities. Efficiency remains at less than one % out of some hundred attempts to clone an animal. These facts show that something is fundamentally wrong and enormous hurdles must be overcome before cloning becomes practical. Cloning researchers now tent to put aside their effort to create live animals in order to probe the fundamental questions on cell biology including stem cells, the questions of whether the hereditary material in the nucleus of each cell remains intact throughout development, and how transferred nucleus is reprogrammed exactly like the zygotic nucleus. Stem cells are defined as those cells which can divide to produce a daughter cell like themselves (self-renewal) as well as a daughter cell that will give rise to specific differentiated cells (cell-differentiation). Multicellular organisms are formed from a single totipotent stem cell commonly called fertilized egg or zygote. As this cell and its progeny undergo cell divisions the potency of the stem cells in each tissue and organ become gradually restricted in the order of totipotent, pluripotent, and multipotent. The differentiation potential of multipotent stem cells in each tissue has been thought to be limited to cell lineages present in the organ from which they were derived. Recent studies, however, revealed that multipotent stem cells derived from adult tissues have much wider differentiation potential than was previously thought. These cells can differentiate into developmentally unrelated cell types, such as nerve stem cell into blood cells or muscle stem cell into brain cells. Neural stem cells isolated from the adult forebrain were recently shown to be capable of repopulating the hematopoietic system and produce blood cells in irradiated condition. In plants although the term$\boxDr$ stem cell$\boxUl$is not used, some cells in the second layer of tunica at the apical meristem of shoot, some nucellar cells surrounding the embryo sac, and initial cells of adventive buds are considered to be equivalent to the totipotent stem cells of mammals. The telomere ends of linear eukaryotic chromosomes cannot be replicated because the RNA primer at the end of a completed lagging strand cannot be replaced with DNA, causing 5' end gap. A chromosome would be shortened by the length of RNA primer with every cycle of DNA replication and cell division. Essential genes located near the ends of chromosomes would inevitably be deleted by end-shortening, thereby killing the descendants of the original cells. Telomeric DNA has an unusual sequence consisting of up to 1,000 or more tandem repeat of a simple sequence. For example, chromosome of mammal including human has the repeating telomeric sequence of TTAGGG and that of higher plant is TTTAGGG. This non-genic tandem repeat prevents the death of cell despite the continued shortening of chromosome length. In contrast with the somatic cells germ line cells have the mechanism to fill-up the 5' end gap of telomere, thus maintaining the original length of chromosome. Cem line cells exhibit active enzyme telomerase which functions to maintain the stable length of telomere. Some of the cloned animals are reported prematurely getting old. It has to be ascertained whether the multipotent stem cells in the tissues of adult mammals have the original telomeres or shortened telomeres.

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STUDY OF RAT EPIGASTRIC VESSELS ACCORDING TO THE FREEZING TIME : HISTOLOGIC, HISTOMORPHOMETRIC, IMMUNOHISTOCHEMICAL & SCANNING ELECTRON MICROSCOPIC STUDY (백서 상복부 혈관의 동결시간에 따른 변화에 대한 연구)

  • Kim, Woo-Chan;Lee, Chong-Heon;Kim, Kyung-Wook;Kim, Chang-Jin
    • Maxillofacial Plastic and Reconstructive Surgery
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    • v.21 no.2
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    • pp.89-109
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    • 1999
  • Vascular spasm which has been reported to occur in 25% of clinical cases continues to be a problem in microvascular surgery; When prolonged and not corrected, it can lead to low flow, thrombosis, and replant or free flap failure. Ischemia, intimal damage, acidosis and hypovolemia have been implicated as contributors to the vascular spasm. Although much work has been done on the etiology and prevention of vasospasm, a spasmolytic agent capable of firmly protecting against or reversing vasospasm has not been found. Therefore vascular freezing was introduced as a new safe method that immediately and permanently relieves the vasospasm and can be applied to microsurgical transfers. Cryosurgery can be defined as the deliberate destruction of diseased tissue or relief the vascular spasm in microvascular surgery by freezing in a controlled manner. 96 Sprague Dawley rats each weighing within 250g were used and divided into 2 group, experimental 1 and 2 group. In the experimental 1 group, right epigastric vessels (artery and vein) were freezed with a cryoprobe using $N_2O$ gas for 1 min. In the experimental 2 group, after freezing for 1 min, thawing for 30 secs and repeat freezing for 30 secs. Left side was chosen as control group in both group. We sacrified the experimental animals by 1 day, 3 days, 1 week, 2 weeks, 4 weeks & 5 months and observed the sequential change that occur during regeneration of epigastric vessels using a histologic, histomorphometric, immunohistochemical and SEM study after the vascular freezing. The results were as follows1. In epigastric arteries, internal diameters had statistically significant enlargement in 1 day, 3 days of Exp-1 group and 1 day, 3 days, 1 week & 2 weeks of Exp-2 group. Wall thickness had statistically significant thinning in 2 weeks of Exp-2 group. 2. In epigastric veins, internal diameters had enlargement of statistical significance in 1 day of Exp-1 and Exp-2 group. 3. The positive PCNA reactions in smooth muscle appeared in 1 week and increased until 2 weeks, decreased in 4 weeks. There was no statistical significance between Exp-1 and Exp-2 group. 4. The positive ${\alpha}$-SMA reaction in smooth muscles showed weak responses until 1 week and slowly increased in 2 weeks and showed almost control level in 4 weeks. 5. The positive S-100 reactions in the perivascular nerve bundles showed markedly decrease in 1 day, 3 days and increased after 1 week and showed almost control level in 4 weeks. Exp-1 group had stronger response than Exp-2 group. 6. In SEM, we observed defoliation of endothelial cell and flattening of vessel wall. Exp-2 group is more destroyed and healing was slower than Exp-1 group. To sum up, relief of vasospasm (vasodilatation) by freezing with cryoprobe was originated from the damage of smooth muscle layer and perivascular nerve bundle and the enlargement of internal diameter in vessels was similar to expeimental groups, but Exp-2 group had slower healing course and therefore vessel freezing in microsurgery can be clinically used, but repeat freezing time needs to be studied further.

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Determination of plasma ketone body following oximation-trimethylsily| derivatization using gas chromatography-mass spectrometry selected ion monitoring (혈장 중 케톤체의 옥심-TMS 유도체화 후 GC-MS/SIM을 이용한 분석)

  • Yoon, Hye-Ran
    • Analytical Science and Technology
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    • v.29 no.1
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    • pp.49-55
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    • 2016
  • A ketone body (acetoacetic acid, β-hydroxybutyric acid, and acetone) increases from blood or urine when bio-energy dependence pays more fatty acid than glucose. However, in case oxidation of fat is greater than the capacity of the citric acid cycle the fatty acid oxidation is made from acetoacetyl CoA to acetoacetate then, again form β-hydroxyburytic acid to acetone, the diffusion take place into the blood. Enzymes that oxidize ketone body in the brain and nerve tissue blood ketone dody is increased during prolonged fasting, brain used it as energy. In this study, we developed the rapid two step derivatization method for sensitive detection of the ketone body by GC-MS/SIM. The plasma was deproteinized and then the hydroxy and carboxyl groups of ketone body are subjected to extraction and drying then, keto-group were derivatized with hydoxylamine at 60℃ for 30 min for oximation. Then it was trimetyl-silylated with BSTFA at 80℃ for 30 min and analyzed using a GC-MS. The linear ranges were in between 0.001 μg/mL and 250 μg/mL for β-hydroxy butyrate, and acetoacetate. The method detection limits were below 0.1 pg over each target compound determined. The mean recoveries (%) of target compounds were ranged from 88.2 % to 92.3 % at 1 µg/mL, from 89.5 % to 94.8 % at 10 μg/mL, with RSD of 6.3-9.4 %. This method could be applied to quantification of ketone bodies which are seen in the keto-acidosis in children and adults from a variety of diseases that cause ketones in the blood and urine.

Simultaneous Maxillo-Mandibular Distraction Osteogenesis in Hemifacial Microsomia: a Case Report (상하악에 동시 다발성 골신장술을 이용한 반안면왜소증의 치험례)

  • Kim, Il-Kyu;Park, Jong-Won;Lee, Eon-Hwa;Yang, Jung-Eun;Chang, Jae-Won;Pyun, Yeong-Hun;Ju, Sang-Hyun;Wang, Boon
    • Maxillofacial Plastic and Reconstructive Surgery
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    • v.32 no.5
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    • pp.447-453
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    • 2010
  • The hemifacial microsomia is characterized by variable underdevelopment of the craniofacial skeleton, external ear, and facial soft tissues. So, patients with hemifacial microsomia have an occlusal plane canting and malocclusion with facial asymmetry. Distraction osteogenesis (DO) with an intraoral or extraoral device is a technique using tension to generate new bone with gradual bone movement and remodeling. DO has especially been used to correct craniofacial deformities such as a hemifacial microsomia, facial asymmetry, and mandible defect that could not adequately be treated by conventional reconstruction with osteotomies. It has a significant advantage to lengthen soft and hard tissue of underdeveloped site without bone graft and a few complication such as nerve injury or muscle contracture. A 13-years old girl visited our clinic for the chief complaint of facial asymmetry. She had a left hypoplastic maxilla and mandible, occlusal plane canting and malocclusion. We diagnosed hemifacial microsomia and lanned DO to lengthen the affected side. Le Fort I osteotomy, left mandibular ramus and symphysis osteotomy were performed. The internal distraction devices fixed with screw on maxillary and mandibular ramus osteotomy sites. External devices were adapted to lower jaw for DO on symphysis osteotomy site and to upper jaw for rapid maxillary expansion (RME). At 7days after surgery, distraction was started at the rate of 1mm per day for 13days, and after 4months consolidation periods, distraction devices were removed. Simultaneous multiple maxillo-mandibular distraction osteogenesis with RME resulted in a satisfactory success in correcting facial asymmetry as well as occlusal plane canting for our hemifacial microsomia.

Intraoperative Neurophysiological Monitoring : A Review of Techniques Used for Brain Tumor Surgery in Children

  • Kim, Keewon;Cho, Charles;Bang, Moon-suk;Shin, Hyung-ik;Phi, Ji-Hoon;Kim, Seung-Ki
    • Journal of Korean Neurosurgical Society
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    • v.61 no.3
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    • pp.363-375
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    • 2018
  • Intraoperative monitoring (IOM) utilizes electrophysiological techniques as a surrogate test and evaluation of nervous function while a patient is under general anesthesia. They are increasingly used for procedures, both surgical and endovascular, to avoid injury during an operation, examine neurological tissue to guide the surgery, or to test electrophysiological function to allow for more complete resection or corrections. The application of IOM during pediatric brain tumor resections encompasses a unique set of technical issues. First, obtaining stable and reliable responses in children of different ages requires detailed understanding of normal age-adjusted brain-spine development. Neurophysiology, anatomy, and anthropometry of children are different from those of adults. Second, monitoring of the brain may include risk to eloquent functions and cranial nerve functions that are difficult with the usual neurophysiological techniques. Third, interpretation of signal change requires unique sets of normative values specific for children of that age. Fourth, tumor resection involves multiple considerations including defining tumor type, size, location, pathophysiology that might require maximal removal of lesion or minimal intervention. IOM techniques can be divided into monitoring and mapping. Mapping involves identification of specific neural structures to avoid or minimize injury. Monitoring is continuous acquisition of neural signals to determine the integrity of the full longitudinal path of the neural system of interest. Motor evoked potentials and somatosensory evoked potentials are representative methodologies for monitoring. Free-running electromyography is also used to monitor irritation or damage to the motor nerves in the lower motor neuron level : cranial nerves, roots, and peripheral nerves. For the surgery of infratentorial tumors, in addition to free-running electromyography of the bulbar muscles, brainstem auditory evoked potentials or corticobulbar motor evoked potentials could be combined to prevent injury of the cranial nerves or nucleus. IOM for cerebral tumors can adopt direct cortical stimulation or direct subcortical stimulation to map the corticospinal pathways in the vicinity of lesion. IOM is a diagnostic as well as interventional tool for neurosurgery. To prove clinical evidence of it is not simple. Randomized controlled prospective studies may not be possible due to ethical reasons. However, prospective longitudinal studies confirming prognostic value of IOM are available. Furthermore, oncological outcome has also been shown to be superior in some brain tumors, with IOM. New methodologies of IOM are being developed and clinically applied. This review establishes a composite view of techniques used today, noting differences between adult and pediatric monitoring.

Evaluation of Embryotoxic Potential of Olaquindox and Vitamin A in Micromass Culture and in Rats

  • Kang, Hwan-Goo;Ku, Hyun-Ok;Jeong, Sang-Hee;Cho, Joon-Hyoung;Son, Seong-Wan
    • Toxicological Research
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    • v.26 no.3
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    • pp.209-216
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    • 2010
  • Limb bud (LB) and central nerve system (CNS) cells were prepared from 12.5 day old pregnant female Crj:CD (SD) rats and treated with olaquindox and vitamin A. Cytotoxicity and inhibition on differentiation were measured in each cell. Three doses of olaquindox (4, 21 and 100 mgkg), and 0.2 and 75 mg/kg of vitamin A were administered to pregnant rat for 11 days from $6^{th}$ to $16^{th}$ of pregnancy. $IC_{50}$ values of olaquindox for proliferation and differentiation in CNS cell were 22.74 and $28.32\;{\mu}g/ml$ and 79.34 and $23.29\;{\mu}g/ml$ in LB cell and those values of vitamin A were 8.13 and $5.94\;{\mu}g/ml$ in CNS cell and 0.81 and $0.05\;{\mu}g/ml$ in LB cell, respectively. Mean body weights of pregnant rats were decreased at high dose of olaquindox (110 mg/kg) but relative ovary weight, number of corpus lutea, and number of implantation were not changed. Resorption and dead fetus were increased at high dose of olaquindox, and relative ovary weight, the number of corpus lutea and implantation, and sex ratio of male to female were not significantly changed in all dose of olaquindox. Mean fetal and placenta weights were significantly (p < 0.01) decreased in rats of high group. Seven fetuses out of 103 showed external anomaly like bent tail, and 10 out of 114 fetuses showed visceral anomalies at high group. The ossification of sternebrae and metacarpals were significantly (p < 0.01) increased by low and middle dose of olaquindox but it was significantly (p < 0.01) prohibited by high dose of olaquindox. In rats treated with vitamin A, the resorption and dead fetus were increased by high dose. Mean fetal weights were significantly (p < 0.01) increased by low dose but significantly (p < 0.01) decreased by high dose. Thirty four fetuses out of 52 showed external anomaly; bent tail (1), cranioarchschisis (14), exencephaly (14), dome shaped head (22), anophthalmia (15), brcahynathia (10) and others (19). Forty five fetuses out of 52 showed soft tissue anomaly; cleft palate (42/52) and anophthalmia (22/52) by high dose of vitamin A. Sixty one fetuses out of 61 (85.2%) showed skull anomaly; defect of frontal, partial and occipital bone (21/61), defect of palatine bone (52/61) and others (50/61). In summary, we support that vitamin A is strong teratogen based on our micromass and in vivo data, and olaquindox has a weak teratogenic potential in LB cell but not in CNS cell. We provide the in vivo evidence that a high dose of olaquindox could have weak embryotoxic potential in rats.

A study on Hand Injury in Industrial Complex Workers (공단 근로자의 수부 손상에 관한 연구)

  • Kim, Soo-Min;Gu, Kyung-Sook;Lee, Geun-Hee;Kang, Ho-Hyun;Hyung, In-Han;Sung, Kwang-Jun
    • Journal of Korean Physical Therapy Science
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    • v.6 no.1
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    • pp.905-913
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    • 1999
  • The hand is a part of the body which has various functions and a sophisticated structures bone, Joint, tendon, ligament and logical nerve organ are aggregated densely in the hand of anatomy its functions are delicate. The hand's functions are very important for doing ADL and a worker's As the hand is structure which is used often and commonly it is a site that can be easily damaged by a accident on work. This study was investigated and analyzed by the results from 238 workers who had hand injury and physical therapy from January, 1997 to June, 1998 at Shin-Pung, Jang-Lim industrial complex which is located in Pusan. 1. A hand injury most frequertly happened in worker in their 30's and 20's(66 case, 28%), second was 57 case(24%) their 40's. 2. Among workers suffering from injury, who had one or less than 2 years on the job, a injury most frequently occurred. 3. Scale of workers, A injury most frequently happend in a place where had workers from 10 to 50. 99 workers from that place were 42% of total. 4. Month of occurrence rate in March were the highest compared to other months 40 injury workers in march were 17% of the total. Second was July, and the least was April that injury appeared with 10 workers. (4.2% of the total) 1. Frocture was the most common lesion 82 workers had that lesion(34%) soft tissue rupture was 71 workers who were 30% of total in type of lesion. 2. Of the site of lesion multiple case was the most common in which 57 workers had that lesion it were 24% of the total. The next was the index finger site in which 44 workers were 19% of the total. The index finger was the most common mono-site that can be lesion 36 workers were the middle finger lesion workers who were 15% of the total. Of the joint lesion, PIP was the most in which 76 workers were 44% and in MC 27 workers were 15% of the total. 3. Interval of physical therapy 79 workers from one month to 2 months, was 33% of the total and 174 workers who had therapy below 3 months were 71% 4. Post injury of disablity of the site, phalangeous disability was the most in which 148 workers had that injury and they were 62% of the total of the grade of disability 110 workers who were 46% of the total state from less than 25% disability in hand-function and 59 patients who were 25% had no disability. There were 6 workers (3% of the total) who had over -75%-dibability in the hand-function.

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Effect of Adenosine on the Mechanical and Electrical Activities of Guinea-pig Stomach (기니피그 위 평활근의 기계적 및 전기적 활동에 대한 아데노신 효과)

  • Kim, Heui-Jeen;Ko, Kwang-Wook;So, In-Suk;Kim, Ki-Whan
    • The Korean Journal of Physiology
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    • v.21 no.2
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    • pp.225-239
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    • 1987
  • The effects of adenosine on the mechanical contractions and electrical activities were investigated in guinea-pig stomach. Spontaneous contractions of the antral region were recorded with force transducer, and the phasic contractions of fundic region were induced by electrical field stimulation. Electrical responses of smocth muscle cells were recored using glass capillary microelectrodes filled with 3M-KCl. Field stimulation was applied transmurally by using a pair of platinum wire (0.5 mm in diameter) placed on both sides of tissue. All experiments were performed in tris-buffered Tyrode solution which was aerated with 100% $O_2$ and kept at $35^{\circ}C$. The results obtained were as follows. 1) Adenosine suppressed the spontaneous contractions of antrum in a dose-dependent manner. 2) The inhibitory effect on antral spontaneous contractions was not influenced by the administration of guanethidine $(5{\times}10^{-6}\;M)$ and atropine $10^{-6}\;M$, or in the presence of dipyridamole $10^{-7}\;M$. 3) The phasic contractions of fundus induced by electrical field stimulation, which disappeared rapidly by the addition of tetrodotoxin $(3{\times}10^{-7}\;M)$, were potentiated by adenosine in the presence of guanethidine. 4) Adenosine decreased the amplitude and the maximum rate of rise of slow waves, and the increased amplitude and rate of rise evoked in the high calcium solution or in the presence of TEA were decreased by adenosine. 5) The non-adrenergic, non-cholinergic inhibitory junction potential (IJP) was inhibited by adenosine in the antral region, while the excitatory junction potential (EJP) in the fundic region was potentiated. From the above results, the following conclusions could be made. 1) Adenosine suppresses the spontaneous contractions of antrum strip by the decrease in amplitude and rate of rise of slow waves. 2) The release of neurotransmitter(s) from non-adrenergic, non-cholinergic nerve terminals is inhibited by adenosine.

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Tactics and Pitfalls of MED(Micro Endoscopic Discectomy) System for Lumbar Disc - For Surgeons Who Wish to Attempt - (요추간반 탈출증에 있어 MED(Microendoscopic Discectomy) System을 이용한 수술의 효율성과 수기)

  • Hong, Hyun Jong;Oh, Seong Hoon;Bak, Kwang Hum;Kim, Jae Min;Kim, Choong Hyun;Kim, Young Soo;Ko, Yong;Oh, Suck Jun;Kim, Kwang Myung;Lee, Sang Gu;Kim, Nam Kyu
    • Journal of Korean Neurosurgical Society
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    • v.29 no.1
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    • pp.35-43
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    • 2000
  • Objective : Percutaneous lumbar approaches such as arthroscopic discectomy, laser discectomy, and nucleotome remain controversial and have technical limitations to free fragment disc, bony pathology and access to L5-S1, The purpose of this study was to determine efficacy of this new endoscopic system and to report techniques and tactics. Methods : From July 1997 to May 1998, we treated 40 consecutive patients(43 levels) with the MED system. Mean age was 32 years(range ; 18 to 62). There were 30 males and 10 females. All patients had sciatica with SLRT limitation. There were 23 patients with disc herniation at L4-5 and 14 patients at L5-S1. Three patients had 2 level disc herniations. There was one far lateral disc herniation at L4-5. Results : Using modified MacNab criteria, there were 37 excellent results and 3 good result. Most patients were discharged within 3-4 days except 2 patients with dural tearing. There were no other complications. Mean operation time was 1.5 hours(range : 40 minutes to 2.5 hours). Conclusion : The MED system is a reliable approach to lumbar disc herniations. This system combines the advantages of conventional open surgery and a minimally invasive technique. As tactics for the doctors who wish to attempt, "palpate" the lamina by first dilator, identification of interlaminar space by removal of overlying soft tissue and confirmation of the shoulder portion of nerve root before discectomy are important to this procedure. We conclude that lumbar disc herniations can be successfully treated with MED approach.

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