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다진 최
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1 KISEP Original Article 대한간질학회지 26;1(1):3-1 Kainic Acid 유발마우스간질모델에서 에의한 Caspase 의존성세포고사억제 신하영 조양제 조경주 김현우 김현정 김경환 이병인 허경 연세대학교의과대학신경과학교실, 뇌연구소 Inhibits Caspase-Dependent Cell Death Pathway and is Neuroprotective against Hippocampal Damage after Kainic Acid-Induced Seizure in Mice Ha Young Shin, M.D., Yang-Je Cho, M.D., Kyoung-Joo Cho, Hyun-Woo Kim, Hyun-Jung Kim, Gyung Whan Kim, M.D., Ph.D., Byung In Lee, M.D. and Kyoung Heo, M.D. Department of Neurology, Yonsei University College of Medicine, Brain Research Institute, Seoul, Korea Purpose: Despite current acceptance of its neuroprotective property, whether the minocycline affords neuroprotection or how it protects neurons against seizures in the animal model of epilepsy is not clear. This prompts us to investigate whether minocycline is neuroprotective against kainic acid (KA)-induced seizure in mice through inhibition of caspase-dependent mitochondrial apoptotic pathways. Methods: Adult male ICR mice were subjected to seizures by intrahippocampal KA injection with treatment of vehicle or minocycline. For cell death analysis, histological analysis using cresyl-violet staining, TdT-mediated dutp-biotin nick end labeling (TUNEL), and histone-associated DNA fragmentation analysis were performed. Evaluation of cytochrome c, cleaved caspase-3, and caspase-3 activity were also performed. Results: Hippocampal neuronal death was evident by cresyl violet staining, TUNEL, and cell death assay in vehicle-treated mice after KA injection; however, there was significant reduction of cell death in the minocycline-treated group. Significant decrease of both cytosolic translocation of cytochrome c and subsequent activation of caspase-3 after treatment of minocycline were demonstrated by Western blot analysis, immunohistochemical staining, and caspase-3 activity assay. Conclusion: This study suggests that minocycline may be neuroprotective against hippocampal damage after KA-induced seizure through inhibition of caspase-dependent cell death pathways. (J Korean Epilep Soc 26;1(1):3-1) KEY WORDS: Seizure Apoptosis Caspase Cytochrome c. 서 론 은 tetracycline 계항생제의일종으로류마치스관절염이나, 여드름등의치료제로서임상적안전성이확립된제제이다. 1 또한 minocycline 은경구투여 Received 2 May 26 Accepted 3 May 26 Corresponding author: Kyoung Heo, M.D., Department of Neurology, Yonsei University College of Medicine, 134 Sinchondong, Seodaemun-gu, Seoul , Korea Kheo@yumc.yonsei.ac.kr This work was supported by Yonsei University Research Fund of 25 ( ). 가가능하고, 생체이용률 (bioavailability) 이높은특성이있으며, 특히작은분자량 (495 kda) 과높은지질친화성으로인해뇌혈관장벽을효과적으로통과한다는장점이있다. 2 최근, Yrjanheikki 등 3 에의해백서전대뇌허혈모델에서 minocycline이신경보호 (neuroprotective) 효과가있음이보고된이후, 뇌경색, 3,4 Huntington s disease, 5,6 근위축성축삭경화증, 7 다발성경화증 8 및파킨슨병 9 과같은여러실험동물모델에서 minocycline 의신경보호효과가보고되었다. 아직까지 minocycline 의신경보호기전은명확하진않으나, 소교세포 (microglia) 의활성화억제를통한염증반응억제, matrix metalloproteinase의억 대한간질학회지 26;1(1):3-1 3

2 KA 유발간질모델에서 의세포보호효과 제, 미토콘드리아에서의 cytochrome c 유리억제와 caspase-1, caspase-3 등의활성억제를통한 caspase 의존성세포고사경로억제등이그기전으로제시된바있다. 1,3,5,7 이렇듯여러신경계질환의동물모델에서 minocycline 의신경보호효과가입증되었지만, 간질동물모델에서의 minocycline 의세포보호효과에대해서는아직연구가이루어지지않은상태이다. 따라서본실험에서는측두엽간질모델의하나인 kainic acid (KA) 유발마우스간질모델 1,11 에서 minocycline 이신경세포사멸을감소시킬수있는지를알아보고, 그기전으로 minocycline 이 caspase 의존성세포고사경로를억제하는지를살펴보고자하였다. 방법 실험동물모든실험동물들은연세대학교의과대학동물실험윤리위원회의지침에따라사용되었다. 수컷 ICR (Institute of Cancer Research) 마우스 (3개월, 체중 35~4 g, Samtako, 경기도, 대한민국 ) 를이용하였다. 한우리 (cage) 당 5마리로사육하였고, 실내온도는 22±.5 로유지하였으며, 12시간간격으로낮과밤의주기를두었다. 물과먹이는자유롭게접근하도록하였다. Kainic acid 유발간질모델과 minocycline 투여실험동물들을 2.5% isoflurane과질소와산소혼합기체 (7%/3%) 로마취시켰다. 수술도중가열받침 (heating pad) 과가열전등 (heating lamp) 으로정상체온을유지시켰다. 동물의머리를정위장치 (stereotaxic frame) 에고정시킨후, 두피의정중선을절개하여두개골을노출시킨다음, bregma 의위치를확인하였다. 우측해마위치 (anteroposterior (AP)=-2. mm; mediolateral (ML)=1.7 mm; dorsoventral (DV)=-1.8 mm) 에 32 gauge Hamilton syringe (Hamilton, Reno, NV, USA) 를사용하여 KA (.5 μg/. 5μl in.9% saline; Sigma, St. Louis, MO, USA) 를 micropump를통해 1분에걸쳐투여하였다. 주사바늘제거후나타날수있는 KA의역류를막기위하여 KA 투여후주사바늘을 1 분간더그자리에위치시켰다. 생리식염수에용해시킨 minocycline hydrochloride (45 mg/kg; Sigma) 는 KA 처치 12시간전에복강내로주사하였고, KA 처치 3분후에 minocycline (9 mg/kg) 을다시주사하였다. 3 처리군은 KA 처 치후, minocycline과같은양 (volume) 의.9% saline 을같은시간대에복강내로주사하였다. 정상대조군은 KA나 vehicle 을처리하지않은마우스를대상으로하였다. KA 처치후 1시간동안 plexiglass chamber에서마우스의행동을관찰하여 KA를처치한모든마우스에서발작 (seizure) 이나타나는것을확인하였고, 발작지속시간을일정하게유지하기위해 KA 처치 6분뒤에 diazepam (2.5 ml/kg; Roche, Paris, France) 을복강내로주사하여발작을정지시켰다. Racine stage 5이상의지속적인전신발작을보인마우스만을실험에포함시켰다. 조직절편을이용한분석절편제작 Urethane 을이용하여실험동물을희생시킨뒤 1 U/ml heparin이든.9% saline과 3.7% formaldehyde를차례로심장을통하여관류시키고, 뇌를적출하여 3.7% formaldehyde에고정하였으며, 그후 3% sucrose에저장하였다. 잘게부순드라이아이스속에서뇌조직을급냉시키고, 동결절편기 (microtome cryostat) 를이용하여 2 μm 두께로관상절편을만든후, 슬라이드에올렸다. Cresyl violet 염색제작된절편을 2~3 시간이상건조시킨후, 자일렌에 3분간 2회담가두었다. 그뒤 1%, 9%, 7% 에탄올에차례대로 1분 3초씩담근후증류수에 3분간 2회함수과정을거쳤다. 함수과정을마친절편을 cresyl violet (Sigma) 염색액에 8분간담가두었고, 이후탈수과정으로증류수와 7%, 9%, 1% 에탄올에순서대로넣었다가자일렌으로세척하였다. 커버글라스를덮고퍼마운트로봉입한후, 광학현미경으로관찰하였다. 반정량적 (semi-quantitative) 분석을위해대조군 (KA -untreated), vehicle 투여군및 minocycline 투여군각각의마우스배측해마에서 3 μm 간격의연속된 4개의절편을이용하였으며, CA1 과 CA3 부위에서형태학적으로손상받은신경세포수를세어이를비교하였다. 12 DNA 분절 (fragmentation) 현상검출 DNA 분절현상을확인하기위하여, TdT-mediated dutp-biotin nick end labeling (TUNEL) 염색을시행하였다. 위에기술한방법으로준비한조직절편을 37 의어두운방에서 1시간동안 TUNEL reaction mixture (Roche Diagnostics GmbH, Penzberg, Germany) 4 대한간질학회지 26;1(1):3-1

3 신하영 조양제 조경주등 5 μl와반응시켰다. 이절편을조심스럽게세척을한뒤, 핵대조염색을위해실온에서 2분간 propidium iodine (PI; Sigma).5 g/ml 와반응시켰다. 절편을세척한후 Vectashield mounting medium (Vector laboratories, Burlingame, CA, USA) 으로봉입하였고, LSM 51 confocal laser scanning microscopy (Carl Zeiss, Thornwood, NY, USA) 을이용하여관찰하였다. 면역조직화학염색 (Immunohistochemical staining) Cytochrome c에대한면역조직화학검사를위해준비한조직절편을 PBS로세척을하고일차항체로반응시켰다. Cytochrome c에대한일차항체로는 rabbit anticytochrome c polyclonal antibody (1:1; Santa Cruz Biotechnology, Santa Cruz, CA, USA) 를사용하였다. 절편을일차항체로 4 에서 8시간정도반응시킨후, 이차항체인 FITC-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) 를실온에서 1시간동안반응시켜면역조직화학검사를시행하였다. PI를이용해서핵대조염색후, confocal laser scanning microscope (Carl Zeiss) 을이용하여관찰하였다. Western blot analysis KA 주입후 1시간, 4시간, 그리고 24시간뒤에실험동물을희생시키고뇌조직을적출하였다. 곧바로 KA를주입한쪽의해마를적출하여잘게부순드라이아이스로급냉시키고 -8 에보관하였다. 각조직을 4 에서 lysis buffer (5 mm KH 2 PO 4,.1 mm EDTA, ph 7.8) 로균질화 (homogenize) 하였다. 이시료를미토콘드리아분획, 세포질분획, 세포핵분획으로나누기위하여원심분리를하였다. 시료를 4, 75 g에서 1분간원심분리한후, 이 pellet 을핵분획으로사용하였다. 이후, 다시상층액을모아서 4, 1, g에서 2분간원심분리한후, 여기서얻어진 pellet 을미토콘드리아분획으로이용하였다. 다시남겨진상층액을 1, g로 6분간원심분리를하였고그상층액을세포질분획으로사용하였다. Cytochrome c의세포질내전위 (cytosolic translocation) 를확인하기위해미토콘드리아분획과세포질분획에서 cytochrome c에대한 Western blot 을시행하였으며, cleaved caspase-3의발현정도를확인하기위해서전세포분획 (whole cell fraction) 을사용하여 Western blot 을시행하였다. 동량의단백질을같은부피의 2X sample buffer (125 mm Tris/HCl, 2% SDS, 1% glycerin, 1 mm DDT, and.2% bromphenol blue, ph 6.9) 에넣고 5분간가열하였다. SDS-polyacrylamide gels (7.5%) 에서전기영동한후, PVDF 막 (Amersham Biosciences, Piscataway, NJ, USA) 으로옮겼다. 이 PVDF 막을.1% Tween 2이포함된 TBS (5 mm Tris/HCl, 14 mm NaCl, ph 7.3) 로세척하고 5% skim milk 로 4 에서 8시간이상 blocking 하였다. 일차항체로 rabbit anti-cytochrome c polyclonal antibody (1:2; Santa Cruz Biotechnology) 와 rabbit anti-cleaved caspase-3 polyclonal antibody (1:5; Cell Signaling Technologies, Beverly, MA, USA) 를사용하여 37 에서 1시간동안반응을시켰다. 이후, 5차례.1% Tween 2이포함된 TBS (5 mm Tris/HCl, 14 mm NaCl, ph 7.3) 로세척하고 horseradish peroxidase (1:5 in TBS plus 5% milk) 가결합된이차항체로실온에서 6분간반응시킨후 ECL plus kit (Amersham International, Buckinghamshire, England) 으로감광시켜확인하였다. LAS-1 plus (Fuji Film Co., Tokyo, Japan) 를이용하여이미지를얻었고, TINA 2. (Raytest GmbH, Straubenhardt, Germany) 소프트웨어를사용하여분석하였다. Caspase inhibitor 처리 Pan-caspase inhibitor인 N-benzyloxycarbonylval-ala-asp-fluoromethyl ketone (z-vad.fmk; Sigma) 2 μl를 Hamilton syringe (Hamilton) 를사용하여뇌실 (AP=.2 mm; ML=1. mm; DV=-3.1 mm) 에 KA 처치 3 분후에주입하였다. 대조군은같은양의.25% DMSO가포함된 PBS을주입하였다. Caspase-3 활성측정 Caspase-3 의활성을측정하기위해서, 활성화된 caspase-3 에의해기질의 N-acetyl-Asp-Glu-Val-Asp AFC (DEVD-AFC) 절단부위를특이적으로인식하여 ELISA 방법으로생물학적발광을내는 Caspase-3 activity assay kit, fluorometric (Calbiochem, Darmstadt, Germany) 을사용하였다. 세포질표본은 Western blot 분석방법대로준비하였다. 세포질표본의단백질을정량하여, 같은양의시료에 kit 와함께제공된 assay buffer (2 mm HEPES, ph 7.4, 5 mm NaCl,.2 mm EDTA, 4 mm dithiothreitol) 및 caspase-3 substrate conjugate 을넣고 37 에서 6분동안반응시킨후형광 (excitation/emission: 4/55 nm) 을측정하였다. 대한간질학회지 26;1(1):3-1 5

KA 유발간질모델에서 의세포보호효과 Cell death assay DNA 분절정량을위해세포질내의 histone-associated DNA 분절을감지하는 cell death detection PLUS kit (Roche Diagnostics

KA를주입하고 24시간뒤에 KA를주입한쪽의해마를적출하여 4 에서조직의 7배부피의용해완충액 (lysis buffer; 5 mm KH 2 PO 4,.1 mm EDTA, ph 7.

단백질을정량한뒤같은양의시료를이용하여 cell death detection PLUS kit (Roche Diagnostics) 에서제공된방법에따라 ELISA 를시행하였다. 통계학적분석통계자료들은평균 ± 표준편차 (mean±sd) 로표시하였다.

결과 조직학적분석을통한 KA에의한발작후신경세포사멸과 minocycline 의효과 을투여한군에서는, KA 처치 24시간후 cresyl violet 염색에서해마세포사멸이뚜렷이관찰되었 다 (Figure 1A).

또한정량적분석을통해, minocycline 투여군에서는 vehicle 투여군에비해의미있게많은 CA1과 CA3 pyramidal 세포들이생존함을관찰할수있었다 (percentage of surviving cells against control:

1)(figure 1B). KA에의한발작후 cytochrome c 의세포질내전위와 minocycline의효과 KA 처치후 1시간과 4시간째에 cytochrome c의세포질내전이를관찰하였다.

KA유발발작후 vehicle 을투여한군에서는, 세포질분획에서도강한 cytochrome c 항체반응이관찰되어 cytochrome c 의세포질내전이를확인할수있었다.

13; vehicle-treated 1 h, 2.11±2.2; vehicle-treated 4 h, 23.77±1.72; minocycline-treated 1 h, 14.33±1.54 ; minocycline-treated 4 h, 16.

Histological analysis of mice hippocampus at 24 h after KA treatment.

4 KA 유발간질모델에서 의세포보호효과 Cell death assay DNA 분절정량을위해세포질내의 histone-associated DNA 분절을감지하는 cell death detection PLUS kit (Roche Diagnostics GmbH, Penzberg, Germany) 을이용하였으며, 13 Saito 등 14 이기술하였던방법을약간변경하여시행하였다. KA를주입하고 24시간뒤에 KA를주입한쪽의해마를적출하여 4 에서조직의 7배부피의용해완충액 (lysis buffer; 5 mm KH 2 PO 4,.1 mm EDTA, ph 7.8) 으로균질화 (homogenize) 시켰고, 4, 75 g에서 1분간원심분리하여상층액을얻었다. 이상층액을 1, g에서 2분간원심분리하여얻은상층액을다시 4, 1, g에서 6분간원심분리를하였다. 원심분리후얻어진상층액을세포질분획으로사용하였다. 단백질을정량한뒤같은양의시료를이용하여 cell death detection PLUS kit (Roche Diagnostics) 에서제공된방법에따라 ELISA 를시행하였다. 통계학적분석통계자료들은평균 ± 표준편차 (mean±sd) 로표시하였다. 두군간의통계비교는 unpaired t-test (StatView, SAS Institute, Inc., Cary, NC, USA) 로시행하였으며, p<.5 를통계적으로유의한것으로판정하였다. 결과 조직학적분석을통한 KA에의한발작후신경세포사멸과 minocycline 의효과 을투여한군에서는, KA 처치 24시간후 cresyl violet 염색에서해마세포사멸이뚜렷이관찰되었 다 (Figure 1A). 하지만, minocycline을투여한마우스해마에서는이같은신경세포손상소견이 vehicle 투여군에비해상대적으로감소함이관찰되었다. 또한정량적분석을통해, minocycline 투여군에서는 vehicle 투여군에비해의미있게많은 CA1과 CA3 pyramidal 세포들이생존함을관찰할수있었다 (percentage of surviving cells against control: minocycline-ca1, 32.47±4.56; vehicle-ca1, 2.87±.64 ; minocycline-ca3, 44.8 ±2.13; vehicle-ca3, 11.99±2.1 ; t-test, p<.1, p<.1)(figure 1B). KA에의한발작후 cytochrome c 의세포질내전위와 minocycline의효과 KA 처치후 1시간과 4시간째에 cytochrome c의세포질내전이를관찰하였다. Western blot 분석에서, KA 를처치하지않은정상대조군에서는세포질분획에서는 11 kda의 cytochrome c 항체반응이거의나타나지않았고, 미토콘드리아분획에서만 cytochrome c 항체반응이강하게일어났다. KA유발발작후 vehicle 을투여한군에서는, 세포질분획에서도강한 cytochrome c 항체반응이관찰되어 cytochrome c 의세포질내전이를확인할수있었다. 하지만, minocycline 을투여한군에서는세포질분획의 cytochrome c의항체반응은 vehicle을투여한군에비하여의미있게감소하였다 (Optical density (OD): control, 1.37±.13; vehicle-treated 1 h, 2.11±2.2; vehicle-treated 4 h, 23.77±1.72; minocycline-treated 1 h, 14.33±1.54 ; minocycline-treated 4 h, 16.32±.7 ; t-test, p<.1, p<.1) (Figure 2A). 5 Control Surviving cells (% of control) CA1 CA3 A B Hippocampal subfields Figure 1. Histological analysis of mice hippocampus at 24 h after KA treatment. A: In normal control, there was no apparent hippocampal cell death. Profound neuronal loss and nuclear fragmentation were noted in the CA1 and CA3 subfield in the vehicle-treated mice at 24 h after kainic acid injection. Neuronal cell death in the same subfields was prominently reduced by minocycline treatment. Bar=1 μm in left panel and 5 μm in middle and right panel. B: Surviving cells in both CA1 and CA3 were significantly increased in minocycline-treated mice at 24 h after KA treatment, compared to vehicle-treated mice. 6 대한간질학회지 26;1(1):3-1

신하영 조양제 조경주등 Cytochrome c에대한면역조직화학염색을 KA 처치후 4시간째에시행하였다. KA를처치하지않은정상대조군에비하여, KA유발발작후 vehicle을투여한군에서해마신경세포질내에 cytochrome c에대한면역반응 (green color) 이뚜렷이증가되었다.

KA유발발작후 vehicle을투여한군에서는많은 TUNEL 양성세포들이관찰되었으며, CA1, CA3 부근에서많이관찰되었다. 하지만, minocycline 을투여한군에서는이러한 TUNEL 양성세포가 vehicle 투여군에비해의미있게감소하였다 (TUNEL-positive cells: vehicle-ca1, 3538±265.

KA 처치후 4시간, 24시간째에 cleaved caspase-3 에대한 Western blot 분석을시행하였으며, 17 kda과 19 kda에해당하는부분에서 cleaved caspase-3에대한면역반응이두개의띠로관찰되었다.

43; vehicle-treated, 4 h, 57.48±3.16; 24 h, 85.39 ±4.98; minocycline-treated, 4 h, 34.91±3.98 ; 24 h, 56.54±4.16 ; t-test, p<.1, p<.1)(figure 3B).

5 신하영 조양제 조경주등 Cytochrome c에대한면역조직화학염색을 KA 처치후 4시간째에시행하였다. KA를처치하지않은정상대조군에비하여, KA유발발작후 vehicle을투여한군에서해마신경세포질내에 cytochrome c에대한면역반응 (green color) 이뚜렷이증가되었다. 이에비해, minocycline을처치한군은세포질내 cytochrome c에대한면역반응이 vehicle 을처치한군에비하여감소됨을관찰할수있었다 (Figure 2B). KA에의한발작후 caspase 의존성세포고사와 minocycline의효과 KA를처치하지않은정상대조군의해마에서는 TU- NEL 양성세포들이관찰되지않았다. KA유발발작후 vehicle을투여한군에서는많은 TUNEL 양성세포들이관찰되었으며, CA1, CA3 부근에서많이관찰되었다. 하지만, minocycline 을투여한군에서는이러한 TUNEL 양성세포가 vehicle 투여군에비해의미있게감소하였다 (TUNEL-positive cells: vehicle-ca1, 3538±265.89/ mm 2 ; minocycline-ca1, 1586±46.54/mm 2 ; vehicle -CA3, 2542±14.87/mm 2 ; minocycline-ca3, 178 ±161.39/mm 2 ; t-test, p<.1, p<.1)(figure 3A). KA 처치후 4시간, 24시간째에 cleaved caspase-3 에대한 Western blot 분석을시행하였으며, 17 kda과 19 kda에해당하는부분에서 cleaved caspase-3에대한면역반응이두개의띠로관찰되었다. KA를처치하지않은정상대조군에비해, KA유발발작후 vehicle을투여한군에서는 cleaved caspase-3의발현양이뚜렷이증가함을알수있었다. 이에반해 minocycline 을투여한군에서는 vehicle 투여군에비해 cleaved caspase-3의발현이유의하게감소됨을관찰하였다 (OD: control, 7.64 ±.43; vehicle-treated, 4 h, 57.48±3.16; 24 h, ±4.98; minocycline-treated, 4 h, 34.91±3.98 ; 24 h, 56.54±4.16 ; t-test, p<.1, p<.1)(figure 3B). KA유발발작후 24시간째에서 KA처치후 vehicle 을투여한군, KA처치후 caspase 억제제인 z-vad.fmk 을투여한군, 그리고 KA처치후 minocycline 을투여한 Mitochondrial COX-IV Cytosolic Ctr 1h 4h 1h 4h Relative optical density A Beta-actin Ctr 1h 4h B Figure 2. Reduction of cytosolic release of cytochrome c by minocycline. A: In western blot analysis, the amount of cytosolic cytochrome c was significantly reduced in minocycline-treated mice at 1 h and 4 h after KA injection, compared to the vehicletreated mice. B: In immunohistochemistry of cytochrome c (green color), the increased immunoreactivity of cytosolic cytochrome c of vehicle-treated mice was evident at 4 h after KA injection, compared to control. In contrast, minocycline-treated mice showed reduction of cytochrome c immunoreactivity in cytosol, compared to vehicle-treated mice. Bar=5 μm. Ctr, control; COX, cytochrome oxidase. COX-IV and beta-actin; internal controls of each mitochondrial and cytosolic subfractions. Samples are from five independent studies. 대한간질학회지 26;1(1):3-1 7

KA 유발간질모델에서 의세포보호효과 4, CAl TUNEL-positive cells/mm 2 3, 2, 1, A CA3 CA1 CA3 Hippocampal subfields 1 Cleaved caspase-3

4 Relative functional unit (RFU) 4 3 2 1 NS Relative optical density 1.2 1..8.6.4.2 C z-vad.fmk Caspase 3 activity. z-vad.fmk DNA fragmentation Figure 3.

A: Immunofluorescent TUNEL staining and its semi-quantitative analysis showed that TUNEL-positive cells in both CA1 and CA3

B: In Western blot analysis, the amount of cleaved caspase-3 was significantly reduced in minocycline-treated mice, compared

C: Comparison of caspase-3 activity and histone-associated DNA fragmentation assay among the normal control, z-vad.

군에서 caspase-3 activity assay를시행하였는데, vehicle 투여군에비해 z-vad.

69 ; minocycline, 41.65±3.34 ; t-test, p<.1). 하지만 z-vad.fmk 투여군과 minocycline 투여군사이에는 caspase-3 활성도의차이가없었다 (p=.96)(figure 3C).

6 KA 유발간질모델에서 의세포보호효과 4, CAl TUNEL-positive cells/mm 2 3, 2, 1, A CA3 CA1 CA3 Hippocampal subfields 1 Cleaved caspase-3 Beta-actin Ctr 4h 24h 4h 24h Relative optical density B Ctr 4h 24h Time 1.4 Relative functional unit (RFU) NS Relative optical density C z-vad.fmk Caspase 3 activity. z-vad.fmk DNA fragmentation Figure 3. Reduction of caspase-dependent apoptotic cell death by minocycline after KA-induced seizure. A: Immunofluorescent TUNEL staining and its semi-quantitative analysis showed that TUNEL-positive cells in both CA1 and CA3 were notably reduced in minocycline-treated mice after KA injection, compared to the vehicle-treated mice. Bar=5 μm. B: In Western blot analysis, the amount of cleaved caspase-3 was significantly reduced in minocycline-treated mice, compared to vehicle-treated mice. Samples are from five independent studies. Ctr, control. C: Comparison of caspase-3 activity and histone-associated DNA fragmentation assay among the normal control, z-vad.fmk- and minocycline-treated mice at 24 h after KA injection. Samples are from five independent studies. 군에서 caspase-3 activity assay를시행하였는데, vehicle 투여군에비해 z-vad.fmk 투여군과 minocycline 투여군에서 caspase-3 활성도가의미있게감소하였다 (OD: control, 22.76±5.66; vehicle, 57.27±6.99; z-vad.fmk, 37.59±1.69 ; minocycline, 41.65±3.34 ; t-test, p<.1). 하지만 z-vad.fmk 투여군과 minocycline 투여군사이에는 caspase-3 활성도의차이가없었다 (p=.96)(figure 3C). 역시위와같은조건에서, cell death detection kit을이용하여 histone-associated DNA 분절을비교하였는데, vehicle 투여군에비해 z-vad.fmk 투여군과 minocycline 투여군에서 DNA 분절이의미있게감소하였다 (OD: control,.18±.2; vehicle, 1.23±.1; z-vad.fmk,.87±.6; minocycline,.56±.11; t-test, p<.1). 하지만, z-vad.fmk 투여군과 minocycline 투여군에서 caspase-3 활성의차이가없었음에도불구하고, mino- 8 대한간질학회지 26;1(1):3-1

7 신하영 조양제 조경주등 cycline 투여군에서 z-vad.fmk 투여군에비해 DNA 분절양이통계적으로의미있게감소하였다 ( p<.1) (Figure 3C). 고찰 저자들은이번실험을통해, KA에의해유발된경련발작으로인한세포사멸에서 minocycline 이신경보호효과를지니고있음을보여주었으며, 이러한 minocycline 의신경보호효과기전의하나로 caspase 의존성세포고사억제가중요한역할을한다는것을제시하였다. 본실험의결과를요약하면다음과같다. 첫째, minocycline 투여로인해 KA 유발발작후나타나는해마세포사멸과 DNA 분절이감소하였다 (Figure 1 and 3A). 둘째, minocycline 의투여로인해 caspase 의존성세포고사기전인 cytochrome c의세포질내전위와 (Figure 2), caspase-3 활성도가감소함을확인하였다 (Figure 3B and C). 병적인 KA의증가는흥분성신경세포독성 (excitotoxic injury) 을유발하는것으로잘알려져있으며, 또한 KA 투여에의한경련발작으로인해, 해마등변연계신경손상및세포고사가유발되는것으로잘알려져있다. 1,11,15-18 본실험에서도 KA 처치로유발된경련발작후, 조직학적으로해마손상이뚜렷이관찰되었으며 (Figure 1), TUNEL 염색, 그리고 histone-associated DNA 분절정량을통해 KA 투여에의한경련발작으로인해서세포고사가유발됨을확인할수있었다 (Figure 3A and C). 또한본실험결과는 minocycline 의투여가 KA에의한경련발작후발생하는해마세포사멸을감소시킴을보여주고있는데 (Figure 1, 3A and 3C), 이는일차신경배양 (primary neuronal culture) 이나백서선조체에 NMDA 를투여하여, 흥분성세포독성에대한 minocycline 의신경세포보호효과를보여준기존실험들의결과와잘부합한다. 19,2 또한, 이러한결과는다른여러뇌질환동물모델에서나타난 minocycline 의신경세포보호효과와도잘부합하여본실험결과를뒷받침하고있다. 5,9,21-23 저자들은또한이번실험을통해 KA에의한경련발작후나타나는뇌신경손상에대한 minocycline 의신경세포보호기전의하나로, minocycline 이미토콘드리아에서세포질로의 cytochrome c 유리와그에따른 caspase- 3 활성화를억제함을밝혔다 (Figure 2, 3B and 3C). 이한결과들역시 minocycline 이 caspase 의존성세포고사를억제함으로써, 신경보호효과를나타냄을보여준이 전실험들의결과 5-7,24-26 와잘부합하여우리의결과를뒷받침한다. 최근연구에따르면, minocycline 이 caspase 비의존성경로또한억제함으로써세포보호효과를나타내는것으로보고되었다. 6 저자들은 pan-caspase 억제제인 z- VAD.fmk 를사용하여정상대조군, caspase 억제군, 그리고 minocycline 투여군에서의 caspase-3 활성도와 DNA 분절현상과의관계를조사하였다 (Figure 3C). Caspase 억제군과 minocycline 투여군모두비슷한정도의 caspase-3 활성억제를보인반면, 그러나세포고사와연관된 DNA 분절의양은 minocycline 투여군에서 caspase 억제군에비해의미있게감소하였다 (Figure 3C). 이는 minocycline이 caspase 의존성경로만을억제할뿐만아니라다른기전즉, caspase 비의존성경로의억제를통해세포보호에일정부분역할을할것이라는가능성을제시한다. 향후, minocycline 의 caspase 비의존성경로억제에대한연구가이루어져야할것으로생각된다. 결론적으로, 이번연구를통해저자들은 KA에의해유발된경련발작으로인한해마의신경세포손상이 minocycline 투여에의해감소함을나타내었다. 은 caspase 의존성세포고사경로를억제하였으며, 이는 minocycline 의신경세포보호기전의하나로생각된다. 이러한소견은향후간질발작에의한뇌신경세포보호및간질발생의억제치료에있어하나의유망한치료전략으로서의 minocycline 의가능성을제시한다. REFERENCES 1. Yong VW, Wells J, Giuliani F, Casha S, Power C, Metz LM. The promise of minocycline in neurology. Lancet Neurol 24;3: Macdonald H, Kelly RG, Allen ES, Noble JF, Kanegis LA. Pharmacokinetic studies on minocycline in man. Clin Pharmacol Ther 1973;14: Yrjanheikki J, Keinanen R, Pellikka M, Hokfelt T, Koistinaho J. Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia. Proc Natl Acad Sci USA 1998;95:15, Arvin KL, Han BH, Du Y, Lin SZ, Paul SM, Holtzman DM. markedly protects the neonatal brain against hypoxicischemic injury. Ann Neurol 22;52: Chen M, Ona VO, Li M, et al. inhibits caspase-1 and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington disease. Nat Med 2;6: Wang X, Zhu S, Drozda M, et al. inhibits caspaseindependent and -dependent mitochondrial cell death pathways in models of Huntington s disease. Proc Natl Acad Sci USA 23; 1: Zhu S, Stavrovskaya IG, Drozda M, et al. inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice. Nature 22;417: Popovic N, Schubart A, Goetz BD, Zhang SC, Linington C, Duncan ID. Inhibition of autoimmune encephalomyelitis by a tetra- 대한간질학회지 26;1(1):3-1 9

8 KA 유발간질모델에서 의세포보호효과 cycline. Ann Neurol 22;51: Du Y, Ma Z, Lin S, et al. prevents nigrostriatal dopaminergic neurodegeneration in the MPTP model of Parkinson s disease. Proc Natl Acad Sci USA 21;98:14, Bouilleret V, Ridoux V, Depaulis A, Marescaux C, Nehlig A, Le Gal La Salle G. Recurrent seizures and hippocampal sclerosis following intrahippocampal kainate injection in adult mice: electroencephalography, histopathology and synaptic reorganization similar to mesial temporal lobe epilepsy. Neuroscience 1999;89: Suzuki F, Heinrich C, Boehrer A, et al. Glutamate receptor antagonists and benzodiazepine inhibit the progression of granule cell dispersion in a mouse model of mesial temporal lobe epilepsy. Epilepsia 25;46: Narasimhan P, Sugawara T, Liu J, Hayashi T, Noshita N, Chan PH. Overexpression of human copper/zinc-superoxide dismutase in transgenic animals attenuates the reduction of apurinic/apyrimidinic endonuclease expression in neurons after in vitro ischemia and after transient global cerebral ischemia. J Neurochem 25;93: Bonfoco E, Krainc D, Ankarcrona M, Nicotera P, Lipton SA. Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures. Proc Natl Acad Sci USA 1995;92: Saito A, Hayashi T, Okuno S, Nishi T, Chan PH. Oxidative stress is associated with XIAP and Smac/DIABLO signaling pathways in mouse brains after transient focal cerebral ischemia. Stroke 24; 35: Ben-Ari Y. Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy. Neuroscience 1985;14: Suzuki F, Junier MP, Guilhem D, Sorensen JC, Onteniente B. Morphogenetic effect of kainate on adult hippocampal neurons associated with a prolonged expression of brain-derived neurotrophic factor. Neuroscience 1995;64: Ben-Ari Y, Cossart R. Kainate, a double agent that generates seizures: two decades of progress. Trends Neurosci 2;23: Bengzon J, Kokaia Z, Elmer E, Nanobashvili A, Kokaia M, Lindvall O. Apoptosis and proliferation of dentate gyrus neurons after single and intermittent limbic seizures. Proc Natl Acad Sci USA 1997;94:1, Tikka T, Fiebich BL, Goldsteins G, Keinanen R, Koistinaho J., a tetracycline derivative, is neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia. J Neurosci 21;21: Goni-Allo B, Ramos M, Jordan J, Aguirre N. In vivo studies on the protective role of minocycline against excitotoxicity caused by malonate or N-methyl-d-aspartate. Exp Neurol 25;191: Yrjanheikki J, Tikka T, Keinanen R, Goldsteins G, Chan PH, Koistinaho J. A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window. Proc Natl Acad Sci USA 1999;96:13, He Y, Appel S, Le W. inhibits microglial activation and protects nigral cells after 6-hydroxydopamine injection into mouse striatum. Brain Res 21;99: Kriz J, Nguyen MD, Julien JP. slows disease progression in a mouse model of amyotrophic lateral sclerosis. Neurobiol Dis 22;1: Scarabelli TM, Stephanou A, Pasini E, et al. inhibits caspase activation and reactivation, increases the ratio of XIAP to smac/diablo, and reduces the mitochondrial leakage of cytochrome C and smac/diablo. J Am Coll Cardiol 24;43: Teng YD, Choi H, Onario RC, et al. inhibits contusion-triggered mitochondrial cytochrome c release and mitigates functional deficits after spinal cord injury. Proc Natl Acad Sci USA 24;11: Wang J, Wei Q, Wang CY, Hill WD, Hess DC, Dong Z. up-regulates Bcl-2 and protects against cell death in mitochondria. J Biol Chem 24;279:19, 대한간질학회지 26;1(1):3-1

신하영 이두재 조경주등 Kainate 유발간질모델 - - 크레질바이올렛 (cresyl violet) 염색 Kainate 유발간질모델에서의뇌파검사 대상과방법 산소음이온라디칼 (superoxide anion radical, O.- 2 ) 검출 in situ- - 대한간질학

신하영 이두재 조경주등 Kainate 유발간질모델 - - 크레질바이올렛 (cresyl violet) 염색 Kainate 유발간질모델에서의뇌파검사 대상과방법 산소음이온라디칼 (superoxide anion radical, O.- 2 ) 검출 in situ- - 대한간질학 KISEP Original Article 대한간질학회지 2004;8(2):108 8(2):108-115 115 Kainate 유발간질모델에서 Apurinic/Apyrimidinic Endonuclease 의빠른소실과이에따른세포고사 신하영 이두재 조경주 김미애 이용현 허경 김경환 이병인 Rapid Loss of Apurinic/Apyrimidinic Endonuclease