• Journal of Biomedical Engineering Research
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• v.36 no.6
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• pp.296-301
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• 2015

Explant culture condition of dorsal root ganglion have been used to investigate the pathophysiology of peripheral nerve injury, while applying for the various clinical symptom such as trauma, pressure, and stretch. However, explant culture is usually contaminated by mitotic cells, which may observed as a newly divided cells including fibroblast or glia. The mitotic cells could be able to interrupt and change the cell signaling that make it difficult to avoid detrimental effects during the experiments. To eliminate mitotic cells, anti-mitotic reagents like mixture of uridine and 5-fluorodeoxyuridine or cytosine arabinoside were added to the cultures on the following day, but there is no research that investigate viability of anti-mitotic reagent in dorsal root ganglion explant culture. In this study, we investigate inhibition effect of cytosine arabinoside to mitotic cells in dorsal root ganglion explant culture. Also we visualized and analyzed anti-mitotic effect and toxicity of cytosine arabinoside in various concentration condition. This dorsal root ganglion explant culture condition can be applied to research that effect and mechanism of various stimulation and chemical application which affect peripheral nerve regeneration.

• Korean Journal of Veterinary Service
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• v.27 no.4
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• pp.345-354
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• 2004

Two cases of classical swine fever (CSF) disease have broken out in Cheolwon (7 April, 2002). The suspected pig herds were huddled together because of high fever (over $40^{\circ}C$) and showed remarkable decrease of the leukocytes. The staggering gait related to posterior weakness, constipation and lethargy, hyperemia, hemorrhagic lesions (on the skin, muzzle, ears, limbs, tail and inner part of legs) and conjunctivitis with dirty streaks below the eyes were observed. The inflammation in the lung, infarction in the spleen, swelling and hemorrhage in lymph nodes, kidney, intestine, heart and cheese like purulent inflammation of the tonsil were observed. The ulcers of the colon were also detected. Several clinical and laboratory techniques including blood test, histo-pathological examinations, indirect fluorescent antibody (IFA) test and RT-PCR test were applied to diagnose the disease. Inoculation test on PK-15 cell was also performed. The necrosis of the lymphatic cells and infiltration of the vessel circumferential cells in the brain and lymph organs were commonly viewed. The proliferation of the glia cell (gliosis) in the lymph was particular. Cytopathogenic effect (CPE) and specific fluorescent-bright-green areas (with IFA) appeared in PK-15 cells inoculated with suspected blood plasma. The IFA test on the epithelial and mucous membrane cells of tonsil was positive. RT-PCR technique required more working hours and labor than other techniques in this examination but it was useful because of the sensitivity to the CSF viral gene.

• The Korean Journal of Physiology and Pharmacology
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• v.25 no.4
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• pp.365-374
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• 2021

The mammalian target of rapamycin (mTOR) plays a role in various cellular phenomena, including autophagy, cell proliferation, and differentiation. Although recent studies have reported its involvement in nociceptive responses in several pain models, whether mTOR is involved in orofacial pain processing is currently unexplored. This study determined whether rapamycin, an mTOR inhibitor, reduces nociceptive responses and the number of Fos-immunoreactive (Fos-ir) cells in the trigeminal nucleus caudalis (TNC) in a mouse orofacial formalin model. We also examined whether the glial cell expression and phosphorylated p38 (p-p38) mitogen-activated protein kinases (MAPKs) in the TNC are affected by rapamycin. Mice were intraperitoneally given rapamycin (0.1, 0.3, or 1.0 mg/kg); then, 30 min after, 5% formalin (10 μl) was subcutaneously injected into the right upper lip. The rubbing responses with the ipsilateral forepaw or hindpaw were counted for 45 min. High-dose rapamycin (1.0 mg/kg) produced significant antinociceptive effects in both the first and second phases of formalin test. The number of Fos-ir cells in the ipsilateral TNC was also reduced by high-dose rapamycin compared with vehicle-treated animals. Furthermore, the number of p-p38-ir cells the in ipsilateral TNC was significantly decreased in animals treated with high-dose rapamycin; p-p38 expression was co-localized in microglia, but not neurons and astrocytes. Therefore, the mTOR inhibitor, rapamycin, reduces orofacial nociception and Fos expression in the TNC, and its antinociceptive action on orofacial pain may be associated with the inhibition of p-p38 MAPK in the microglia.

• Biomedical Science Letters
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• v.25 no.2
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• pp.177-184
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• 2019

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease (PD). LRRK2 contains a functional kinase and GTPase domains. A pathogenic G2019S mutation that is the most prevalent among the LRRK2 mutations and is also found in sporadic cases, increases its kinase activity. Therefore, identification of LRRK2 kinase substrates and the development of kinase inhibitors are under intensive investigation to find PD therapeutics. Several recent studies have suggested members of Rab proteins, a branch of the GTPase superfamily, as LRRK2 kinase substrates. Rab proteins are key regulators of cellular vesicle trafficking. Among more than 60 members of human Rab proteins, Rab3, Rab5, Rab8, Rab10, Rab12, Rab29, Rab35, and Rab43 have been identified as LRRK2 kinase substrates. However, most studies have used human embryonic kidney (HEK) 293T cells overexpressing LRRK2/Rab proteins or murine embryonic fibroblast (MEF) cells which are not relevant to PD, rather than neuronal cells. In this study, we tested whether Rab proteins are phosphorylated by LRRK2 in astroglia in addition to neurons. Among the various Rab substrates, we tested phosphorylation of Rab10, because of the commercial availability and credibility of the phospho-Rab10 (pRab10) antibody, in combination with a specific LRRK2 kinase inhibitor. Based on the results of specific LRRK2 kinase inhibitor treatment, we concluded that LRRK2 phosphorylates Rab10 in the tested brain cells such as primary neurons, astrocytes and BV2 microglial cells.

• Journal of Oriental Neuropsychiatry
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• v.32 no.2
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• pp.111-127
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• 2021

Objectives: To review and analyze clinical and preclinical evidence of effectiveness, safety, and underlying mechanisms of yokukansan (YKS), a herbal medicine, in alleviating aggression. Methods: Classical records on YKS were searched in the Korean Traditional Medicine Knowledge Database (KTMKD). By searching five electronic databases, prospective clinical studies and preclinical studies of YKS for alleviating aggression/agitation published up to March 30, 2021 were included. Results: Only two classical records on YKS were found from the KTMKD. A total of 11 clinical studies and 15 preclinical studies were found from the five electronic databases. Among 11 clinical studies, seven enrolled patients with dementia and four enrolled patients with other neuropsychiatric disorders. Most clinical studies reported significant improvement in one or more outcomes related to aggression in the YKS group after treatment. Among 15 preclinical studies, all studies except two reported a significant decrease in aggression/agitation-related behavior of YKS or yokukansankachimpihange. Suggested underlying mechanisms of YKS or yokukansankachimpihange for aggression/agitation in these studies included regulation of serotonin receptor, amelioration of abnormal glucocorticoid level related to the hypothalamic-pituitary-adrenal axis, regulation of orexin secretion, amelioration of degeneration in brain cells including glia cells, and suppression of excessive glutamatergic or dopaminergic activity. Conclusions: There were some clinical and preclinical evidence supporting the effectiveness and safety of YKS for alleviating aggression. Given that aggression is the most frequent and destructive symptoms of behavioral and psychological symptoms of dementia, applicability of YKS as a herbal medicine should be further investigated in future high-quality research.

• Journal of Life Science
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• v.17 no.2 s.82
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• pp.198-203
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• 2007

DNA transfection is a powerful tool for studying gene functions. The $Ca^{2+}$-phosphate precipitation remains one of the most popular and cost-effective transfection techniques. Mature neurons are more resistant to transfection than young ones and most other cell types, and easy to die if microenvironment changes. Here, we report a transfection protocol for mature neurons. The critical modifications are inclusion of glial cells in culture and careful control of $Ca^{2+}$-phosphate precipitation under microscope. Cerebral glial cells were grown until ${\sim}70-80%$ confluence in DMEM/10% horse serum, which was thereafter replaced with serum-free Neurobasal/Ara-C, and 319 hippocampal neurons were plated onto the glial layer Formation of fine $DNA/Ca^{2+}$-phosphate precipitates was induced using Clontech $CalPhos^{TM}$ Mammalian Transfection Kit, and the size ($0.5-1\;{\mu}m$ in diameter) and density(about 10 particles/$100\;{\mu}m^2$) were carefully controlled by the time of incubation in the medium. This modified protocol can be reliably applied for transfection of mature neurons that are maintained longer than two weeks in vitro, resulting in 10-15 healthy transfected neurons per a well of 24-well plates. The efficacy of the protocol was verified by punctate expression of $pEGFP-CaMKII{\alpha}$, a synaptic protein, and diffuse expression of pDsRed2. Our protocol provides a reliable method for transfection of mature neurons in vitro.

• International Journal of Oral Biology
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• v.39 no.1
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• pp.49-56
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• 2014

We investigated the role of central P2X receptors in inflammatory pain transmission in the orofacial area in rats. Experiments were carried out using male Sprague-Dawley rats weighing 230-280g. Complete Freund's adjuvant (CFA, $40{\mu}L$) was applied subcutaneously to the vibrissa pad to produce inflammatory pain. The intracisternal administration of iso-PPADS tetrasodium salt, a non-selective P2X receptor antagonist, A317491 sodium salt hydrate, a $P2X_{2/3}$ receptor antagonist, 5-BDBD, a $P2X_4$ receptor antagonist, or A438079 hydrochloride, a $P2X_7$ receptor antagonist, was performed 5 days after CFA injection. Subcutaneous injections of CFA produced increases in thermal hypersensitivity. Intracisternal injections of iso-PPADS ($25{\mu}g$) or A438079 (25 or $50{\mu}g$) produced significant anti-hyperalgesic effects against thermal stimuli compared to the vehicle group. A317491 or 5-BDBD did not affect the head withdrawal latency times in rats showing an inflammatory response. Subcutaneous injections of CFA resulted in the up-regulation of OX-42, a microglia marker, and GFAP, an astrocyte marker, in the medullary dorsal horn. The intracisternal administration of A438079 reduced the numbers of activated microglia and astrocytes in the medullary dorsal horn. These results suggest that a blockade of the central $P2X_7$ receptor produces antinociceptive effects, mediated by inhibition of glial cell function in the medullary dorsal horn. These data also indicate that central $P2X_7$ receptors are potential targets for future therapeutic approaches to inflammatory pain in the orofacial area.

• 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.

• Toxicological Research
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• v.11 no.2
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• pp.315-327
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• 1995

Diverse neurotoxic insults result in proliferation and hypertrophy of astrocytes, a subtype of glia in central nervous system. The hallmark of this response, often terms "reactive gliosis", is the enhanced expression of the major intermediate filament protein of castrocytes, glial fibrillary acidic protein (GFAP). These changes in the astrocytes suggest that GFAP may be a useful biochemical indicator of neurotoxicity. To investigate this possibility, we administered intra-peritoneally prototype nerotoxicants, metharnphetamine (MAP, 5 mg/kg), cocaine (30 mg/kg), N-buthyl benzenesulfonamide (NBBS, 300 mg/kg) and trimethytin (TMT, 8 mg/kg) to Wistar Rats and then assessed the effects of these agents on content of GFAP, which were determined by Sandwish ELISA and evaluated with neurotoxic symptoms, and quantitative changes of imrnunoreactivity of GFAP by light microscopic image analysis in specific regions. We found that assay of GFAP revealed time- and region-dependant patterns of neurotoxicity. The GFAP immunoreactivity of rat brain was increased in substantia nigra and hippocampus by MAP, NBBS and TMT; in roedial septal nucleus and nucleus accurnbens, it was also increased by RrBBS. Sandwich ELISA showed that GFAP levels of cerebrum in all groups on days 3 and 7 and that of brainstem(including cerebellum) in MAP, NBBS groups on day 1 and 3 were increased. A review of the background, design and results of these experiments are presented in this paper. Our findings indicate that GFAP is a sensitive and specific biomarker of neurotoxicity.otoxicity.

• Parasites, Hosts and Diseases
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• v.52 no.6
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• pp.613-619
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• 2014

Neospora caninum (Apicomplexa; Sarcocystidae) is a protozoan that causes abortion in cattle, horses, sheep, and dogs as well as neurological and dermatological diseases in dogs. In the central nervous system of dogs infected with N. caninum, cysts were detected that exhibited gliosis and meningitis. Flavonoids are polyphenolic compounds that exhibit antibacterial, antiparasitic, antifungal, and antiviral properties. In this study, we investigated the effects of flavonoids in a well-established in vitro model of N. caninum infection in glial cell cultures. Glial cells were treated individually with 10 different flavonoids, and a subset of cultures was also infected with the NC-1 strain of N. caninum. All of the flavonoids tested induced an increase in the metabolism of glial cells and many of them increased nitrite levels in cultures infected with NC-1 compared to controls and uninfected cultures. Among the flavonoids tested, 3',4'-dihydroxyflavone, 3',4',5,7-tetrahydroxyflavone (luteolin), and 3,3',4',5,6-pentahydroxyflavone (quercetin), also inhibited parasitophorous vacuole formation. Taken together, our findings show that flavonoids modulate glial cell responses, increase NO secretion, and interfere with N. caninum infection and proliferation.