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The Journal of Internal Korean Medicine > Volume 25(3); 2004 > Article
The Journal of Internal Korean Medicine 2004;25(3): 461-472.
뇌해마의 장기양 조직배양을 이용한 한약물의 뇌신경세포손상 보호효능 연구
이원철1,3, 정혁상2, 손낙원3
1동국대학교 한의과대학 심계내과학교실
2경희대학교 한의과대학 해부학교실
3경희대학교 동서의학대학원 신경과학교실
Neuroprotective Effects of Medicinal Herbs in Organotypic Hippocampal Slice Cultures
Won-Chul Lee1,3, Hyuk-Sang Jung2, Nak-Won Sohn3
1Department of Internal Medicine, College of Oriental Medicine, Dongguk University
2Department of Anatomy, College of Oriental Medicine, Kyung Hee University
3Department of Neuroscience, Graduate School of East-West Medical Science, Kyung Hee University
Correspondence  Won-Chul Lee ,Tel: 02-3416-9791, Fax: 02-3416-9790, Email: noosa@naver.com
Received: July 7, 2004,   Accepted: July 28, 2004,   Published online: September 30, 2004.
ABSTRACT
Objectives:
For the screening of neuroprotective effects of medicinal herbs, the complex system of animal models suffer some disadvantages in controlling critical parameters such as blood pressure and body temperature. Additionally, application of drugs to the appropriate brain area sometimes is difficult, due to poor permeability though the blood brain barrier, and so potential protective effects might be masked.

Methods :
Organotypic hippocampal slice culture (OHSC) method has the advantages of being relatively easy to prepare and of maintaining the general structure, including tissue integrity and the connections between cells. Drugs can easily be applied and neuronal damage can easily be quantified by using tissues and culture media. This study demonstrates neuroprotective effects of Puerariae radix (葛根, PR), Salviae miltiorrhizae radix (丹蔘, SR), Rhei rhizoma (大黃, RR), and Bupleuri radix (柴胡, BR). These were screenedand compared to MK-801, antagonist of NMDA receptors, by using OHSC of 1 week-old Sprague-Dawley rats. Oxygen/glucose deprivation (OGD) were conducted in an anaerobic chamber (85% N2, 10% CO2 and 5% H2) in a deoxygenated glucose-free medium for 60 minutes. Water extracts of each herbs were treated to culture media with 5 μg/㎖ for 48 hours.

Results:
Neuronal cell death in the cultures was monitored by densitometric measurements of the cellular uptake of propidium iodide (PI). PI fluorescence images were obtained at 48 hours after the OGD and medicinal herb treatment. Also TUNEL-positive cells in the CA1 and DG regions and LDH concentrations in culture media were measured at 48 hours after the OGD. According to measured data, MK-801, PR, SR and BR demonstrated significant neuroprotective effect against excessive neuronal cell death and apoptosis induced by the OGD insult. Especially, PR revealed similar neuroprotective effect to MK-801 and RR demonstrated weak neuroprotective effect.

Conclusions:
These results suggest that OHSC can be a suitable method for screening of neuroprotective effects of medicinal herbs. (This work was supported by the research program of Dongguk University and Grant 01-PJ9-PG1-01CO03-0003 from Ministry of Health & Welfare.)
Key words: Puerariae radix(葛根, PR), Salviae miltiorrhizae radix(丹蔘, SR), Rhei rhizoma(大黃, RR), Bupleuri radix(柴胡, BR), oxygen-glucose deprivation(OGD), organotypic hippocampal slice cultures(OHSC)
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