• Journal of Acupuncture Research
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• v.18 no.2
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• pp.51-66
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• 2001

The purpose of this morphological studies was to investigate the central neural pathway projecting to the acupoints $B_{62}$ and $K_6$ using the neuroanatomical method following injection of transsynaptic neurotropic virus, pseudorabies virus(PRV-Ba and PRV-Ga) into the $B_{62}$ and $K_6$. After survival times of 96 hours following injection into the twenty rats with PRV-Ba(Bartha strain) and PRV-Ga(Bartha strain, ${\beta}$-galacidodase insertion). They were perfused, and their spinal cord and brain were frozen sectioned($30{\mu}m$). These sections were stained by X-gal histochemical and PRV immunohistochemical staining method, and observed with light microscope. The results were as follows : 1. In spinal cord, overlaped PRV-Ba and PRV-Ga labeled neurons projecting to the $B_{62}$ and $K_6$ were founded in thoracic, lumbar and sacral spinal segments. In thoracic spinal segments, Densely labeled areas were founded in lamina IV, V, VII(intermediolateral nucleus) and X areas. In lumbar segemnts, labeled areas were founded in lamina II, IV, V and X areas. In sacral spinal segments, labeled areas were founded in lamina IV, V and VI areas. 2. In brain, overlaped PRV-Ba and PRV-Ga labeled neurons projecting to the $B_{62}$ and $K_6$ were founded in the $A_1$ noradrenalin cells/$C_1$ adrenalin cells/caudoventrolateral reticular nucleus, rostroventrolateral reticular nuclens, nucleus tractus solitarius, area postrema, raphe obscurus nucleus, raphe paltidus nucleus, raphe magnus nucleus, lateral paragigantoceltular nucleus, lateral rcticular nucleus, gigantocellular nucleus, locus coeruleus, subcoeruleus nucleus, motor trigeminal nucleus, Kolliker-Fuse nucleus, $A_5$ cell group, central gray matter, oculomotor nerve, paraventricular hypothalamic nucleus, median eminence, amygdaloid nucleus, frontal cortex, forelimb area, hindlimb area, 1, 2 areas of parietal cortex and granular and agranular cortex. This results were suggest that overlaped PRV-Ba and PRV-Ga labeled areas projecting to the $B_{62}$ and $K_6$ may be related to the emotional relay pathway in the central autonomic center.

• Journal of Acupuncture Research
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• v.17 no.2
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• pp.95-117
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• 2000

The purpose of this morphological studies was to investigate the relation between the meridian, acupoints and viscera using neuroanatomical tracers. The common locations of the spinal ganglia, sympathetic chain ganglia, spinal cord and brain projecting to the large intestine meridian were observed following injection of transganglionic tracer, WGA-HRP and transsynaptic neurotropic virus, pseudorabies virus(PRV), Bartha strain(Ba) and PRV-Ba-Gal (Galactosidase)) into the the large intestine(cecum, colon and rectum), ST37 and LI4. After survival times of 96 hours following injection into the thirty rats with WGA-HRP, PRV-Ba and PRV-Ba-Gal. They were perfused, and their spinal ganglia, sympathetic chain ganglia, spinal cord and brain were frozen sectioned($30{\mu}m$). These sections were stained by HRP and X-gal histochemical and PRV immunohistochemical staining method, and observed with a light microscope. The results were as follows : 1. WGA-HRP labeled neurons innervating the large intestine were observed bilaterally within the T13-L4 sympathetic chain ganglia, and T9-11 spinal ganglia. WGA-HRP labeled neurons innervating ST37 were observed within the L3-5 sympathetic chain ganglia, and L2-4 spinal ganglia. WGA-HRP labeled neurons innervating LI4 were observed in the middle cervical ganglion and stellate ganglion, and C5-8 spinal ganglia. 2. In spinal cord, PRV-Ba labeled neurons projecting to the large intestine, ST37 and LI4 were found in thoracic, lumbar and sacral spinal segments. Densely labeled areas of each spinal cord segment were founded in lamina N, V, VII(intermediolateral nucleus), Ⅸ, X and dorsal nucleus. 3. In medulla oblongata, PRV-Ba and PRV-Ba-Gal labeled neurons projecting to the large intestine, ST37 and LI4 were commonly found in the A1 noradrenalin cells/C1 adrenalin cells/caudoventrolateral reticular nucleus, dorsal motor nucleus of vagus nerve, nucleus tractus solitarius, raphe obscurus nucleus, raphe pallidus nucleus, raphe magnus nucleus and gigantocellular nucleus. 4. In pons, PRV-Ba and PRV-Ba-Gal labeled neurons were commonly found in locus coeruleus, Kolliker-Fuse nucieus and A5 cell group. 5. In midbrain, PRV-Ba and PRV-Ba-Gal labeled neurons were commonly found in central gray matter. 6. In diencephalon, PRV-Ba and PRV-Ba-Gal labeled neurons were commonly found in paraventricular hypothalamic nucleus. These results suggest that PRV-Ba and PRV-Ba-Gal labeled common areas projecting to the large intestine may be correlated to that of the large intestine meridian, ST37 and LI4. Especially, These morphological results provide that interrelationship of meridian-acupoints -viscera may be related to the central autonomic pathways.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.16 no.11
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• pp.7575-7581
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• 2015

Corticotropin-releasing factor(CRF), one of the stress driven neuropeptides, was widely proposed to influence hair loss and re-growth. For the development of receptor antagonists, the screening system based on intracellular calcium signal process was developed and optimized. The aequorin parental cells were transfected with CRF1 receptor and alpha 16 promiscuous G protein cDNA to establish HEK293a16/hCRF1, a stable cell line for the human CRF1 receptor. In HEK293a16/hCRF1 cells, the range of sauvagine dose response was 12-fold higher($EC_{50}:15.21{\pm}1.83nM$) than in the transiently expressed cells, hence essential conditions for the antagonist screening experiments such as the robust signals and high solvent tolerance were secured. The standard antagonists for the CRF1 receptor, antalarmin and CP154526, resulted $IC_{50}$ values of $414.1{\pm}5.5$ and $290.7{\pm}1.9nM$, respectively. Similar results were presented with frozen HEK293a16/hCRF1 cells. Finally, our HEK293a16/hCRF1 cells with the aequorin based cellular functional assay can be a model system for the development of functional cosmetics and modulators that can have a clinical efficacy on hair re-growth.

• Journal of Plant Biotechnology
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• v.49 no.3
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• pp.187-192
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• 2022

The repeated monthly or weekly subculture of plant callus is labor intensive and increases the risk of somaclonal variation from the parental callus line. The most effective method for preserving plant callus is cryopreservation, which involves storage in liquid nitrogen. However, this method cannot be applied to the callus of different plant species in the same manner, so it is difficult to develop a standardized cryopreservation method. In addition, the survival rate of the frozen callus after thawing and the regeneration rate after survival are uncertain. Therefore, it is necessary to develop a method to extend the subculture interval of plant callus in an active state. In this study, active plant calli of various species without freezing was incubated at 15℃ for 4 to 12 weeks without subculture. After 12 weeks, 8 lines of plant callus grew less than 2-fold when cultured at 25℃, but at least 2 times as much when cultured at 15℃. Moreover, total antioxidant activity did not differ significantly between plant callus recovered at 25℃ after culturing at 15℃ or at 25℃. These results show that the subculture interval can be extended at a temperature of 15℃ without need for modified medium composition or additional processes. In addition, positive results in all calli of several plant species are expected to reduce labor as well as somaclonal variation by increasing the subculture.

• Korean Journal of Animal Reproduction
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• v.23 no.2
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• pp.133-139
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• 1999

This study was carried out to investigate in vitro/in vivo development of vitrified-thawed immature mouse oocytes. Immature mouse oocytes were vitrified with EFS40 (40% ethylene glycol, 18% ficoll and 0.5 M sucrose). Thawed oocytes were matured for 16 hr in vitro. Matured oocytes with the first polar body were fertilized with the concentration of 1~2$\times$10$^{6}$ $m\ell$ of epididymal sperm. After fertilization, cleavage ($\geq$ 2-cell) and in vitro/in vivo development rates were examined. $\pi$ Ie results were summarized as follows: in vitro maturation rate of immature mouse oocytes in vitrified-thawed group was similar to that in exposed group (67.5%) and control (66.3%), but cleavage rate of vitrified-thawed oocytes (64.9 %) and blastocyst formation rate (59.0%) were significantly different compared to those of exposed group (83.7 and 74.7%) and control (90.7 and 83.7%) (p＜0.05). However, when the blastocysts derived from immature mouse oocytes vitrified-thawed were transferred to pseudopregnant mouse, total implantation (31.3%) was slightly lower than that in control (40.8%), but live fetus formation rate (66.7%)was slightly higher than that in control (58.1%), there was not significantly different. Therefore, when the blastocyts produced in vitro were transferred into recipients, although the development in vitro of oocytes vitrified-thawed was decreased, live fetus formation rate was similar to that of control group. The present results indicate that immature mouse oocytes can be frozen successfully by vitrification with EFS40.

• Applied Microscopy
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• v.30 no.4
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• pp.357-365
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• 2000

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.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.3
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• pp.203-212
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• 2008

Objective: During cryopreservation process, cold shock and cryo-injury affect the fertilizing capacity of the sperm by damaging cell membranes with loss of functional integrity. A longstanding concept for preventing the cryo-damage is to stabilize the plasma membrane by incorporating cholesterol. This study was to determine the effects of cholesterol in freezing media on the motility and functional integrity of human sperm after cryopreservation. Methods: Control group (non-cholesterol treated) and different concentrations of cholesterol-treated sperm (14 healthy males) were frozen and thawed. After freezing and thawing of sperm, the quality of sperm was evaluated by sperm analysis, acrosome reaction test and sperm chromatin structure assay. Results: When human sperm were incubated in sperm freezing medium (SFM) containing $0.5{\mu}g$ cholesterol and then freezing/thawing, the motility of sperm have significantly improved compared to those untreated cholesterol ($33.46{\pm}1.48%$ vs. $30.10{\pm}1.07%$, p<0.05). The rate of calcium ionophore-induced acrosome reactions in post-thawed sperm was significantly higher than that ($53.60{\pm}1.60%$ vs. $47.40{\pm}1.86%$, p<0.05) in SFM containing cholesterol. Sperm chromatin structure assay revealed that DNA damage to the sperm in the cholesterol-treated group was lower than that of non-treated group. Conclusion: These results suggest that increased cholesterol content of sperm plasma membrane by supplementation of cholesterol in SFM improves sperm motility, capacitation status, and DNA integrity. Therefore, addition of cholesterol into SFM could be a useful for protecting human sperm from cold shock and cryo-injury during cryopreservation.

• Journal of the Korean GEO-environmental Society
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• v.15 no.5
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• pp.47-56
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• 2014

In winter season, the pore water inside the ground freezes and thaws repetitively due to the cold air temperature. When the freezing-thawing processes are repeated on the ground, the change in soil particle structure occurs and thus the damage of the infrastructure may be following. This study was performed in order to investigate the stiffness change of soils due to the freeze-thaw by using elastic waves. Sand-silt mixtures are prepared with in the silt fraction of 40 %, 60 % and 80 % in weight and in the degree of saturation of 40 %. The specimens are placed into the square freezing-thawing cell by the temping method. For the measurement of the elastic waves, a pair of the bender elements and a pair of piezo disk elements are installed on the cell, and a thermocouple is inserted into soils for the measurement of the temperature. The temperature of the mixtures is decreased from $20^{\circ}C$ to $-10^{\circ}C$ during freezing, is maintained at $-20^{\circ}C$ for 18 hours, is gradually increased up to the room temperature of $20^{\circ}C$ to thaw the specimens. The shear waves, the compressional waves and the temperature are measured during the freeze-thaw process. The experimental result indicates that the shear and the compressional wave velocities after thawing are smaller than those of before freezing. The velocity ratio of after thawing to before freezing of shear wave is smaller than that of the compressional wave. As silt fraction increases from 40 % to 80 %, the shear and compressional wave velocities are gradually increased. This study suggests that the freezing-thawing process in unsaturated soil loosens the soil particle structure, and the shear wave velocity reflects the effect of freezing-thawing more sensitively than the compressional wave velocity.

• Korean Journal of Veterinary Research
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• v.31 no.2
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• pp.179-187
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• 1991

These studies were carried out to investigate the effects of the size of follicles, semen types, capacitation methods, and additions of hormones, estrous cow serum(ECS), fetal calf serum(FCS), bovine follicular fluid(BFF) and matured cumulus cell(MCC) to the medium on in vitro maturation and fertilization of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluid from the visible follicles of diameter 3~5mm. The follicular oocytes were cultured in TCM-199 medium containing hormones, FCS, ECS, BFF and MCC for 24~48hrs. in an incubator with 5% $CO_2$ in air at $38.5^{\circ}C$ and then matured oocytes were again cultured for 18~20 hrs. with motile capacitated spermatozoas the TCF (Tyrode calcium free) solution containing $100{\mu}g/ml$ of heparin. The results obtained in these experiments were summarized as follows: 1. The oocytes classified as "A,B,C,D and Degenerative" depending morphological integrity and those 61.4%, 12.1%, 19.2%, 4.2% and 3.0% of the total oocytes recovered, respectively. The maturation and fertilization rate of the A, B, C class follicular oocytes, cultured in the TCM-199 medium supplemented with 10% FCS were 89.1%, 78.0%, 52.6% and 78.1%, 66.1, 33.3%, respectively. 2. The average number of the follicular oocytes recovered from follicles size, 1~2mm, 3~5mm and above 5mm in diameter were 67, 98 and 63, respectively. The maturation and fertilization rate of the follicular oocytes, cultured in the TCM-199 medium were 56.7%, 82.5%, 46.0% and 44.8%, 71.4%, 28.6%, respectively. 3. The fertilization and cleavage rate of the follicular oocytes, inseminated with spermatozoas of epididymal cauda, neat and frozen semen were 63.3%, 73.3%, 70.0% and 32.7%, 37.8% 38.3%, respectively. 4. The fertilization and cleavage rate of follicular oocytes, fertilized with capacitated spermatozoas by heparin, BFF and HIS methods were 70.0%, 53.8%, 34.2% and 38.3%, 23.1%. 17.1%, respectively. And the fertilization and cleavage rate were higher method of heparin. 5. The maturation and fertilization rate of follicular oocytes, cultured in the TCM-199 medium supplemented with 5%, 10%, 15%, 20% and FSH, HCG, 17, $\beta$-estradiol were 76.0~82.3% and 26.2~70.0%, and those values were higher the supplementation than non-supplementation. 6. The maturation and fertilization rate of the follicular oocytes cultured in TCM-199 medium supplemented with 5~20% ECS and FCS were 74.0~80.6%, 26.2~30.0% and 71.7~76.9%, 51.9~58.0%, and the values were higher the supplement of ECS than FCS. 7. The maturation rate (68.0~64.6%) and fertilization rate(59.6~60.4%) of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 20~30% BFF were higher than those of follicular oocytes cultured TCM-199 medium supplemented with 10% FCS and 10% and 50% BFF. 8. The maturation rate(76.5%) and fertilization rate (61.7%) of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and $1{\times}10^6/ml$ MCC were higher than those of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and $1{\times}10^{{4}{\sim}{5}}/ml$ and $1{\times}10^8/ml$ cumulus cells.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.25 no.4
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• pp.281-294
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• 1999

Vasospasm causes microvascular surgery to fail as a main factor in the loss of transferred flap dye to the diminution of blood flow in reconstruction surgery. Although there has been extensive research to resolve the vasospasm problem, no one has reached an ideal solution to date. However, cryotherapy, which is often used for destruction of tumor lesions, is being presented as a new way of releasing vasospasm. After making a histomorphometric measurement on vasodialation during the course of 1, 3 and 7 days, 2 and 4 weeks, and 5 months periods and observing the change of blood vessel in a histologic, immunohistochemical, and scanning electronic microscopic approach, the results were as follows : 1. Vascular inner diameters of the experimental 1 and 3 days groups were measured $476.3{\pm}28.20{\mu}m$, $497.15{\pm}48.79{\mu}m$ respectively showing statistically meaningful vasodilation(P<0.05), which continued by the experiment 4 weeks group. However, in the experimental 5 months group, the vascular inner diameter appeared similar to the control groups. Even though the thickness of smooth muscular layers come out to be thinner in all the experimental groups compared to the control group, it was difficult to find any statistical meaningfulness. In addition, the vascular external diameters of every experimental groups were shown to be longer than the control group. 2. In light microscopic view, severe injury was evident on the smooth muscular layer cell from the experimental 1 day group, started recovering partially from the experimental 7 days group, and was mostly restored in the experimental 4 weeks group and layer of adventitial stripping were nearly recoverd 2 weeks group. 3. The PCNA positive cells of smooth muscular layer were observed from the experimental 7 days group and had a tendency to increase by the experimental 2 weeks group. In the experimental 4 weeks and 5 months group, the number of PCNA possitive cells observed was comparable to the control group. 4. ${\alpha}$-SMA level of smooth muscular layer cells, having been significantly lower than the control group in the severly damaged experimental 1 day group. It was seen to be increased in the experimental 7 days group and turned out to show similar ${\alpha}$-SMA level in 4 weeks to the control group. 5. In the view of SEM, the endothelial cells were destructed and falling off, and also present the appearance of flattening in the experiment 1 day group. The endothelial layer cells started partially recovering from the 7 days group after the freezing injury. On 4 weeks and 5 months, the endothelial cells were fully coverd the damaged area, also it's appearance is similar to control group. In conclusion, the vascular freezing after the removal of adventitia caused damages to smooth muscular layer cells, and brought about vasodilation, which continued by the 4th week. The smooth muscular layer cells started partially reviving from the 7rd day after the damage by vascular freezing, and recovered their similar figure to the control group's 4 weeks later. This was considered the result of cells which surround the damaged blood vessel being influxed into the smooth muscular layers. Therefore, this local freezing injury on the blood vessel was thought to be applied clinically to relieve severe vasospasm which cannot be treated by vasodilation drug, a microvascular surgery.