• Applied Microscopy
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• v.29 no.1
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• pp.105-124
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• 1999

Histological and ultrastructural differentiations of the neuroepithelial cells in the mouse embryo during neurulation were observed. The neural plates and grooves consisted of pseudostratified columnar epithelium in the embryonic day (ED) 8 embryo were developed. In the ED 9 embryo, the neural tube was developed in all body length of embryo except both the cephalic and caudal ends. Secondary neurulation was shown at the tail bud of the ED 10 embryo. In the ED 8 embryo, the primitive streak was shown in the posterior end of the embryonic disc. The neuroepithelium, notochord and mesenchyme were well differentiated in the cephalic and cervical portions. In the ED 9 and 10 embryos, the roof plates of neural tubes were constituted of the closing of the surface ectodermal cells in the hindbrain and the neuroepithelial cells in the spinal cord. The floor plate of neural tube were consisted of the low pseudostratified columnar epithelium. The spinal motor nerve fibers were initially differentiated in the ED 10 embryo. According to the electron density of the cell and the differentiation of tell organelles, the neuroepithelial cells in the ED 9 and 10 embryos were classified into three types: dark, intermediate and light types. All types in the ED 9 embryo were observed but the dark cell in the ED 10 embryo was not done. The free ribosomes and polysomes in all neuroepithelial cells were developed. The RER and lipid droplets in the dark cell and the Golgi complex in the intermediate and light cells were observed. Many microfilaments in the cytoplasmic processes of intermediate cell and the microfilaments and microtubules in the light cell processes were observed to be well differentiated.

• Applied Microscopy
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• v.36 no.2
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• pp.83-91
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• 2006

The number and structure of synapses are dynamically changed in response to diverse physiological and pathological conditions. Since strength of synaptic transmission is closely related to the synaptic density on a neuron, both synaptogenesis and synapse loss may play important roles in controlling neuronal activity. Thus it is essential to estimate the number of synapses using an accurate quantitative method for better understanding of the numerical alteration of synapses under terrain experimental conditions. We applied physical disector principle to estimating the number of synapses per neuron in the dentate gyrus of adult mice. First, we measured the numerical density of granule cells using the physical disector principle. Second, the density of medial perforant path to granule cell synapses was estimated using the bidirectional physical disector. Then, the volume ratio of molecular layer to granule cell layer was measured. With these numerial values, we successfully calculated the number of synapses per neuron. Individual granule cells have approximately 6500 synapses in the dentate gyrus of adult mice $(6,545{\pm}330)$, which are comparable to those of other researchers. Our results showed that the estimation of synapse numbers per neuron using the physical disector principle would provide accurate and precise information on the numerical alteration of synapses in diverse physiological and pathological conditions. Following analyses of synapse numbers using this method will contribute to the better understanding of structural synaptic plasticity in a variety of experimental animal models.

• Journal of Korean Ophthalmic Optics Society
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• v.20 no.3
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• pp.391-396
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• 2015

Purpose: The objective of this study was to investigate the visual system of the greater horseshoe bat (Rhinolophus ferrumequinum) by location analysis of some major neurotransmitters glutamate, ${\gamma}$-aminobutyric acid (GABA), acetylcholine, and their receptors, and $m{\ddot{u}}ller$ glial cells in retina. Methods: Standard immunocytochemical techniques were used after vibratome section of retinal tissues of adult greater horseshoe bat for this study. Immnoreactions in immunofluorescence images were analyzed using confocal microscope. Results: Anti-glutamate-immunoreactive neurons were mainly localized in the ganglion cell layer (GCL). The majority of anti-GABA-immunoreactive cells distributed in the inner nuclear layer (INL), and GABAA receptors were localized in the inner plexiform layer (IPL). Anti-choline acetyltransferase-immuoreactive cholinergic neurons were mainly located in the INL and GCL, and most of nicotinic acetylcholine receptors were localized in the IPL. The $m{\ddot{u}}ller$ cells in the retina of the greater horseshoe bat stretched theirs range from the GCL to outer nuclear layer (ONL). Conclusions: This study revealed that the retinas of the greater horseshoe bats contain the same major neurotransmitters and receptors, and glial cell in visually functional mammalian retinas. The present results may suggest that the greater horseshoe bats have the functional retinas for visual analysis through the organized retinal neural circuits.

• Journal of Acupuncture Research
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• v.25 no.3
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• pp.53-69
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• 2008

목적 : 이 연구는 Vipera lebetina turanica의 사독약침액(蛇毒藥鍼液)(Snake venom toxin, SVT)이 인간 신경아세포종의 암세포주인 SK-N-SH 세포에서 암세포성장의 억제 및 그 기전에 대하여 살펴보고자 하였다. 방법 : SVT를 처리한 후 SK-N-SH의 성장억제를 관찰하기 위해 CCK-8 assay와 LDH assay를 시행하였고, apoptosis 평가에는 세포형태의 관찰과 DAPI, TUNEL, Annexin V-PI double staining assay 및 cell detachment assay를 시행하였다. 세포자멸사 관련 세포기전을 보기 위하여 세포주기, 세포내 칼슘량, 세포내 활성산소량 및 미토콘드리아의 세포막전위 변화를 측정하였고, DNA fragmentation assay를 시행하였으며, 세포자멸사 조절 단백인 Bax, Bcl-2, caspase-3, -9의 발현 변화 관찰에는 western blot analysis를 시행하였다. 결과 : SK-N-SH 세포에 SVT를 처리한 후, 신경아세포종 세포의 성장, Apoptosis의 유발 및 기전의 영향을 관찰하여 다음과 같은 결과를 얻었다. 1. SVT를 처리한 SK-N-SH 세포 관찰에서 세포독성은 농도의존적으로 증가하는 경향이 있는 반면 암세포성장의 유의한 억제는 $20{\mu}g/m{\ell}$ SVT에 의해서만 나타났다. 2. 세포자멸사 평가에서 SVT를 처리한 SK-N-SH 세포는 세포자멸사의 특징적 형태를 나타내었다. TUNEL assay에서는 세포자멸사 활성세포가 미약하게 나타난 반면 cell detachment assay와 Annexin V-PI double staining에서는 각각 세포박리와 세포자멸사 활성세포의 유의한 증가를 나타내었다. 3. 세포자멸사 관련 세포기전연구에서 SVT를 처리한 SK-N-SH 세포의 세포주기, 세포 내 칼슘양 및 DNA fragmentation에는 유의한 변화가 관찰되지 않은 반면 세포내 활성산소 양은 유의한 증가를 나타내었고, 그에 따른 미토콘드리아 세포막 전위의 유의한 변동이 관찰되었다. 4. SVT를 처리한 SK-N-SH는 세포자멸사 관련 단백 발현에서 Bax에 대해 유의한 증가를 나타내지 않았으나 caspase-3 및 caspase-9의 유의한 증가와 Bcl-2의 유의한 감소를 나타내었다. 결론 : 이상의 결과는 SVT가 세포 내 활성산소를 증가시킴으로써 미토콘드리아의 세포막전위에 변화를 일으켜 인간 신경아세포종 세포주인 SK-N-SH의 세포박리와 유관한 세포자멸사를 유발함으로써 증식억제 효과가 있음을 입증한 것이다.

• Journal of Acupuncture Research
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• v.25 no.2
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• pp.41-57
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• 2008

목적 : 이 연구는 Vipera lebetina turanica의 사독약침액(蛇毒藥鍼液)(Snake venom toxin, SVT)이 인간 신경아세포종의 암세포주인 SK-N-MC 세포에서 암세포성장의 억제 및 그 기전에 대하여 살펴보고자 하였다. 방법 : SVT를 처리한 후 SK-N-MC의 성장억제를 관찰하기 위해 CCK-8 assay와 LDH assay를 시행하였고, apoptosis 평가에는 세포형태의 관찰과 DAPI, TUNEL, Annexin V-PI double staining assay 및 cell detachment assay를 시행하였다. 세포자멸사 관련 세포기전을 보기 위하여 세포주기, 세포내 칼슘량, 세포내 활성산소량 및 미토콘드리아의 세포막전위 변화를 측정하였고, DNA fragmentation assay를 시행하였으며, 세포자멸사 조절 단백인 Bax, Bcl-2, caspase-3, -9의 발현 변화 관찰에는 western blot analysis를 시행하였다. 결과 : SK-N-MC 세포에 SVT를 처리한 후, 신경아세포종 세포의 성장, Apoptosis의 유발 및 기전에 영향을 관찰하여 다음과 같은 결과를 얻었다. 1. SVT를 처리한 SK-N-MC 세포 관찰에서 $20{\mu}g/m{\ell}$ SVT 처리가 암세포성장의 유의한 억제를 나타내었다. 세포독성 관찰에서 SVT처리는 처리하지 않은 것에 비하여 증가를 나타내었다. 2. 세포자멸사 평가에서 SVT를 처리한 SK-N-MC 세포는 세포자멸사의 특징적 형태를 나타내었다. TUNEL assay에서는 세포자멸사 활성세포가 미약하게 나타난 반면 cell detachment assay와 Annexin V-PI double staining에서는 각각 세포박리와 세포자멸사 활성세포의 유의한 증가를 나타내었다. 3. 세포자멸사 관련 세포기전연구에서 SVT를 처리한 SK-N-MC 세포의 세포주기, 세포내 칼슘량 및 DNA fragmentation에는 유의한 변화가 관찰되지 않은 반면 세포내 활성산소 양은 유의한 증가를 나타내었고, 그에 따른 미토콘드리아 세포막 전위의 유의한 변동이 관찰되었다. 4. SVT를 처리한 SK-N-MC는 세포자멸사 관련 단백 발현에서 caspase-9에 대해 유의한 증가를 나타내지 않았으나 Bax 및 caspase-3의 유의한 증가와 Bcl-2의 유의한 감소를 나타내었다. 결론 : 이상의 결과는 SVT가 세포내 활성산소를 증가시키므로 미토콘드리아의 세포막전위에 변화를 일으켜 인간 신경아세포종 세포주인 SK-N-MC의 세포박리와 유관한 세포자멸사를 유발하므로 증식억제 효과가 있음을 입증한 것이다.

• Journal of Life Science
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• v.25 no.6
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• pp.656-662
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• 2015

Natural products and natural product structures play a general and highly significant role in drug discovery and development process because it has various merits and potentials for new drug source that have extensive clinical experience, development time contraction, excellent stability and safety. In several neurological disorders, neuronal death and excessive activation of microglia (neuro-inflammation) are observed. A number of drug discovery-related neuronal cell death and neuro-inflammation was studied from natural products, respectively. However, until now, it has not been possible to study dual-protection molecules recorded in the Natural Product library. In the present study, using the natural product-derived library of the Institute for Korea Traditional Medical Industry, we investigated dual-protective molecules against glutamate (a classical excitatory neurotransmitter)-induced oxidative stress mediated neuronal cell death and LPS-induced excessive activated microglial cells (immune cells of the brain). Chrysophanol, extracted from Rheum palmatum, had dual-protective effects against both glutamate-induced neuronal cell death and LPS-induced NO production, triggering proinflammatory cytokines and microglia activation and resulting in neuroinflammation. Flow-cytometry analysis revealed that chrysophanol had a scavenger effect, scavenging glutamate- and LPS-induced reactive oxygen species (ROS) produced by neuronal and microglial cells, respectively. Based on the present study, chrysophanol may have an important protective role against neuronal cell death and neuroinflammation in the brain. The results may be helpful for studying drug development candidates for treating central nervous system disorders.

• Korean Journal of Acupuncture
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• v.25 no.1
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• pp.155-164
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• 2008

목 적 : 본 연구의 목적은 양릉천 침 처치 시 C57BL/6 생쥐의 중뇌 흑질에 위치한 도파민성 신경세포 사멸 억제 효과를 조직화학 염색법을 이용하여 Tyrosine hydroxylase(TH)와 laminin의 발현으로 관찰하고자 한다. 실험방법 : 실험에 이용한 동물은 C57BL/6 생쥐로, 매일 25mg/kg의 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)를 5일간 주사하였고, 매일 MPTP 주사한 뒤 2시간 후에 양릉천에 침치료를 시행하였으며 MPTP 주사를 종료한 뒤 침치료는 7일동안 계속 시행하였다. 마지막 MPTP 주사 7일 후에 동물을 희생하여 뇌를 적출하고 고정하였다. 침효과를 확인하기 위해 Thyrosine hydroxylase(TH), laminin의 발현 변화 정도를 조직염색화학법으로 이용하여 확인하였다. 각 그룹간의 유의성 검증은 one-way ANOVA를 이용하였다. 결 과 : 도파민성 신경세포 선택적인 신경독소인 MPTP에 대한 양릉천 침처치에 의한 신경보호 효과를 도파민신경세포의 표지자인 TH 발현을 면역화학조직염색법으로 관찰하였다. 대조군에 비해 MPTP 처치 군의 신경세포 사멸이 유의적으로 감소하였고(P <0.05), MPTP + 침처치 군에서 증가되는 양상을 확인하였다 (P <0.05). 또한 도파민성 신경세포내에 존재하는 laminin의 발현정도 역시 대조군보다 MPTP 처치 군에서 유의적으로 감소하였고, MPTP + 침처치 군에서 증가되는 양상을 확인하였다 (P <0.05). 결 론 : MPTP에 의한 도파민성 신경세포 손상에 대한 양릉천 침처치의 신경보호 효과는 세포외 기질중의 하나인 laminin의 발현 정도를 조절하여 비롯되는 것으로 사료된다.

• Journal of Korean Ophthalmic Optics Society
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• v.18 no.4
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• pp.541-548
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• 2013

Purpose: Marrow stromal cells (MSCs) have been known for their potential to trans-differentiate into neural and glial cells in vitro and in vivo. To investigate the influence of the developing host environment on the survival and morphological and molecular differentiation, murine MSCs transplanted into the eye of Brazilian opossum (Monodelphis domestica). Methods: Enhanced green fluorescent protein (GFP) - expressing MSCs were transplanted into developing Brazilian opossums. Animals were allowed to survive for up to 4 weeks after transplantation, at which time the eyes were prepared for immunohistochemical analysis. Results: Some transplanted MSCs survived and showed morphological differentiation into neural cells with some processes within the host vitreous chamber. Some transplanted cells expressed class III ${\beta}$-tubulin (TuJ1, a marker for neuronal cells) or glial fibrillary acid protein (GFAP, a marker for glial cells) or Nestin (a marker for neural stem cells). In addition, some transplanted cells were located in ganglion cell layer but did not show morphological and molecular differentiation. Conclusions: Our result show that the most effective stage of development for transplantation into the retina was postnatal day 16, which retinas developmentally corresponded to postnatal day 4-5 days mouse retina based on cell differentiation and lamination patterns. The present findings suggest that the age of the host appears to play a key role in determining cell fate in vivo.

• Applied Microscopy
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• v.42 no.1
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• pp.27-33
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• 2012

Transient receptor potential ankyrin 1 (TRPA1), responding to noxious cold (${\leq}17^{\circ}C$) and pungent compounds, is implicated in nociception, but little is known about the coexpression of TRPA1 and other channels or receptors involved in the nociception in craniofacial regions. To address this issue, we characterized the TRPA1-immunopositive (+) neurons in the rat trigeminal ganglion (TG) and investigated their colocalization with other proteins known to be expressed in nociceptive neurons, such as transient receptor potential vanilloid (TRPV1) and $P2X_3$ receptor, using light microscopic immunofluorescence labeling method with TRPA1 and TRPV1 or $P2X_3$ antisera. The majority of TRPA1+ neurons costained for TRPV1 (TRPV1+/TRPA1+; 58.8%, 328/558) and 41.2% only expressed TRPA1 but not TRPV1. The TRPV1+/TRPA1+ neurons were small and medium sized. In addition, we investigated the colocalization of TRPA1 with $P2X_3$, a nonselective cation channel activated by ATP that may be released in the extracellular space as a result of tissue damage and inflammation. Among all TRPA1+ TG neurons, 26.1% (310/1186) costained for $P2X_3$, whereas 73.9% (876/1186) of TRPA1+ neurons did not coexpress $P2X_3$. $P2X_3$+/TRPA1+ neurons were predominantly small and medium sized. These results suggest that TRPA1+ neurons coexpressing TRPV1 or $P2X_3$ are involved in specific roles in the transmission and processing of orofacial nociceptive information by noxious cold, heat, and inflammation.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.20 no.2
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• pp.286-293
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• 2019

Alzheimer's disease (AD) is a neurodegenerative disease caused by accumulation of amyloid beta ($A{\beta}$) in the brain. In the present study, we investigated the neuroprotective effects of four flavonoids such as kaempferol, kaempferol-3-O-glucoside, quercetin, and quercetin-3-${\beta}$-D-glucoside against neuronal apoptosis induced by $A{\beta}$ in SH-SY5Y neuronal cells. Treatment with $A{\beta}$ decreased cell viability compared to the non-treated normal group. However, treatment with the four flavonoids increased cell viability in SH-SY5Y cells treated with $A{\beta}$. In addition, we measured the expression of apoptosis-related proteins such as Bcl-2-associated X protein (Bax) and cleaved caspase-9. Treatment with the four flavonoids down-regulated Bax and cleaved caspase-9 in $A{\beta}$-treated SH-SY5Y neuronal cells. Overall, the results of the present study demonstrated the neuroprotective effect of flavonoids by anti-apoptotic activity in $A{\beta}$-induced SH-SY5Y neuronal cells. These results suggest that these four flavonoids would be useful therapeutic and prevention agents for AD.