이 작품은 호주 국립 보건 의학 연구위원회, 헌터 의학 연구소, 뉴캐슬 대학에서 연구 기금에 의해 지원되었다.

모든 절차는 NSW 동물 연구 법, NSW 동물 연구 규정 및 대한 연습의 호주 코드를 포함하여 관련 지침 및 규정에 따라뿐만 아니라 뉴캐슬 대학의 동물 관리 및 윤리위원회에서 승인을 수행 관리 및 과학적인 목적을위한 동물의 사용. 1. 뇌 조직의 해부 <ol><li> 잘린 십이주 된 수컷 쥐를 희생. 쥐도 근사하고 잘린 희생 할 수있다, 또는 최초의 이소 플루 란 (5 % 유도, 1.5 % 유지) 70 % N 2 30 %의 O 2 마취 할 수 있고, 그 목을 베. </li><li> 가능한 빨리 두개골에서 뇌를 제거 (이상적으로,이보다 2 분에 수행해야합니다). </li><li> 프리 핸드의 해부에 의해 피질을 제거합니다. 마이하도록 절개 가능한 뇌 적게 손상, 가능한 최단 시간 내에 수행되어야슬라이스의 무결성을 ntain. </li><li> 잔류 피부를 제거하기 위해, 기타 퍼, 혈액,,, 37 ° C 크렙스 완충액 (118 mM 염화나트륨, 4.7 mM의 염화칼륨, 1.3 mM의 염화칼슘의 소량 (3-5 ml)에 뇌를 담가 1.2 ㎜의 인산이 수소 칼륨, 1.2 mM의 황산 마그네슘, 25 mM의 나트륨, 탄산 수​​소, 11.7 mM의 글루코오스, 0.03 mM의 디 나트륨 에틸렌 디아민 테트라 아세트산 (EDTA)을 95 % 산소 / 5 % 이산화탄소 (carbogen)의 pH 7.4)로 평형화. </li></ol> 2. Microslices의 준비 <ol><li> 여과지의 두 조각을 배치하고 따뜻한 크렙스 버퍼를 살짝 묻혀있다하는 맥일 웨인 헬기의 무대에서 뇌를 놓습니다. 두뇌와 따뜻한 크렙스 버퍼에 적신 여과지를 유지하지만 너무 젖은 뇌가 미끄러지지 않을 수 있습니다 종이의 표면에 액체가 무료입니다. </li><li> 250 μm의 코로나 섹션으로 머리를 조각. 영양소, 산소 및 노폐물 th로일반적으로 혈액 공급이 절연 신경 조직에서 조직을 둘러싸는 유체 교환하여 교환에서, 슬라이스의 사이즈는 적절한 산소 및 영양소 교환이 발생할 수 있도록하는 두께로 400 μM이어야한다. </li><li> 맥일 웨인 단계 90 ° 돌립니다. </li><li> 250 μm의 시상 조각으로 머리를 조각. 여기서, 이러한 뇌 섹션 (가변 길이의 250 μM × 250 μm의 슬라이스 조직의 두께에 따라) microslices로 지칭된다. </li><li> (사용 크렙스 버퍼의 양이 조직의 양에 따라 달라집니다) 15 ㎖를 37 ° C 크렙스 버퍼 둥근 바닥 튜브에 microslices를 놓습니다. </li></ol> 3. Microslices의 평형 <ol><li> 부드럽게 개별 microslices가 일시 중단 될 때까지 교반 한 후 그 중력에서 해결 할 수 있습니다. </li><li> 조심스럽게 인해 일시 중단 된 세포 파편에 흐린 될 상층 액을 제거하고 신선한 37 ° C 10 mL를 넣고크렙스는 튜브에 버퍼. </li><li> 파편의 대부분 제거 될 것이다,이 세척 절차를 네 번 반복합니다. </li><li> 이 후, 부드러운 흔들림 / 반전으로 37 ° C에서 microslices을 평형 및 크렙스는 총 1 시간 동안 매 15 분을 버퍼로 변경합니다. </li><li> 이 때, 2 ML 신선한 37 ° C 크렙스 버퍼를 추가하고 평평한 바닥 폴리스티렌 15-20 microslices (microslice 현탁액 150 μL)를 전송하여 만들 수 있습니다 부드럽게 가장자리 (과 여분의 넓은 구멍 피펫 팁을 사용하여 5 ㎖ 튜브 P1000 팁의 끝에서 1 ~ 2 mm 절삭). 각 microslice가 산소 분위기에서 몇 밀리미터보다 더되지 않도록, 단층의 튜브의 기부에 균일 microslices 스프레드. </li></ol> 4. 흥분 독성 자극 <ol><li> 37 ° C에서 microslices을 품어, 지속적으로 위치하는 가스 라인을 사용하여 150 μL의 microslice 서스펜션의 표면에 carbogen 전달다만 microslice 서스펜션 (기계 혼란을 피하기 위해)의 표면 위에 에드. </li><li> (: 최종 농도 2.5 mM의 글루타민산 염 + 50 μM 글리신 흥분 독성 자극) 또는 단독으로 150 μL 크렙스 버퍼 (nonstimulated를 제어) 150 ㎕의 5 mM의 글루타민산 염 + 100 크렙스 버퍼 μM 글리신과 microslices을 처리합니다. 다양한 자극이 단계에서 사용될 수있다 (를 포함 하나 이에 국한되지 않는 AMPA, NMDA + 글리신, 탈분극의 KCl, 및 산소 / 글루코스 부족). </li><li> 여러 번 후 자극 (0, 30, 60, 90, 120, 300 초)에서 배양 용액을 제거하고 얼음처럼 차가운 균질화 버퍼 (30 MM 트리스, 산도 7.4, 1 ㎜의 에틸렌 글리콜의 300 μL로 교체 디아민 테트라 아세트산, 4 mM의 EDTA, 100 μM 몰리브덴 산 암모늄, 5 mM의 나트륨 피로 포스페이트, 25 mM의 불소화 나트륨, 1 mM의 나트륨 오르토 바나 데이트, 1 밀리미터 phenylmethanesulfonylfluoride 완전 프로테아제 억제제 칵테일). </li><li> 유리 테플론 다운스 균질화를 사용하여 얼음에 microslices 균질화(20 스트로크, 700 RPM). </li><li> 등 -80 ° C 및 다양한 신호 분자, 죽음 분자에 보관 균질 서양 얼룩에 의해 측정 할 수있다. </li></ol>

<ol>
	<li>Lo, E. H., Dalkara, T., Moskowitz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms,+challenges+and+opportunities+in+stroke.">Mechanisms, challenges and opportunities in stroke.</a> Nat. Rev. Neurosci. 4, 399-415 (2003).</li><li>Wang, Y., Qin, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+cellular+mechanisms+of+excitotoxic+neuronal+death.">Molecular and cellular mechanisms of excitotoxic neuronal death.</a> Apoptosis. 15, 1382-1402 (2010).</li><li>Vogt Weisenhorn, D. M., Roback, L. J., Kwon, J. H., Wainer, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+of+cAMP/PKA+and+MAPK+signaling+in+neuronal+cells+is+dependent+on+developmental+stage.">Coupling of cAMP/PKA and MAPK signaling in neuronal cells is dependent on developmental stage.</a> Exp. Neurol. 169 (1), 44-55 (2001).</li><li>Kharlamov, E., Cagnoli, C. M., Atabay, C., Ikonomovic, S., Grayson, D. R., Manev, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposite+effect+of+protein+synthesis+inhibitors+on+potassium+deficiency-induced+apoptotic+cell+death+in+immature+and+mature+neuronal+cultures.">Opposite effect of protein synthesis inhibitors on potassium deficiency-induced apoptotic cell death in immature and mature neuronal cultures.</a> J. Neurochem. 65 (3), 1395-1398 (1995).</li><li>Kristensen, B. W., Noraberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+excitotoxic+profiles+of+ATPA,+AMPA,+KA+and+NMDA+in+organotypic+hippocampal+slice+cultures.">Comparison of excitotoxic profiles of ATPA, AMPA, KA and NMDA in organotypic hippocampal slice cultures.</a> Brain Res. 917 (1), 21-44 (2001).</li><li>Lecrux, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneously+hypertensive+rats+are+highly+vulnerable+to+AMPA-induced+brain+lesions.">Spontaneously hypertensive rats are highly vulnerable to AMPA-induced brain lesions.</a> Stroke. 38, 3007-3015 (2007).</li><li>Sattler, R., Charlton, M. P., Hafner, M., Tymianski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+influx+pathways,+not+calcium+load,+determine+neuronal+vulnerability+to+calcium+neurotoxicity.">Distinct influx pathways, not calcium load, determine neuronal vulnerability to calcium neurotoxicity.</a> J. Neurochem. 71, 2349-2364 (1998).</li><li>Morrison, B. 3. r. d., Saatman, K. E., Meaney, D. F., McIntosh, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+central+nervous+system+models+of+mechanically+induced+trauma:+a+review.">In vitro central nervous system models of mechanically induced trauma: a review.</a> J. Neurotrauma. 15, 911-928 (1998).</li><li>McIlwain, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+Neurochemistry.">Practical Neurochemistry.</a> , (1975).</li><li>Skelding, K. A., Spratt, N. J., Fluechter, L., Dickson, P. W., Rostas, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha+CaMKII+is+differentially+regulated+in+brain+regions+that+exhibit+differing+sensitivities+to+ischemia+and+excitotoxicity.">alpha CaMKII is differentially regulated in brain regions that exhibit differing sensitivities to ischemia and excitotoxicity.</a> J. Cereb. Blood Flow Metab. 32 (12), 2181-2192 (2012).</li><li>Kavanagh, J. M., Dodd, P. R., Rostas, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3H]MK-801+binding+in+immature+and+mature+chicken+forebrain.">3H]MK-801 binding in immature and mature chicken forebrain.</a> Neurosci. Lett. 134 (1), 83-87 (1991).</li><li>Kavanagh, J. M., Bunn, S. J., Boyd, T. L., Rostas, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+in+glutamate+receptor+stimulated+inositol+phospholipid+metabolism+and+45Ca(2+)-accumulation+in+posthatch+chicken+forebrain.">Developmental changes in glutamate receptor stimulated inositol phospholipid metabolism and 45Ca(2+)-accumulation in posthatch chicken forebrain.</a> Neurosci. Lett. 194 (3), 161-164 (1995).</li><li>Sharps, E. S., McCarl, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+performance+liquid+chromatographic+method+to+measure+32P+incorporation+into+phosphorylated+metabolites+in+cultured+cells.">A high performance liquid chromatographic method to measure 32P incorporation into phosphorylated metabolites in cultured cells.</a> Anal. Biochem. 124, 421-424 (1982).</li><li>Bradbury, D. A., Simmons, T. D., Slater, K. J., Crouch, S. P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurment+of+the+ADP:ATP+ratio+in+human+leukaemic+cell+lines+can+be+used+as+an+indicator+of+cell+viability,+necrosis+and+apoptosis.">Measurment of the ADP:ATP ratio in human leukaemic cell lines can be used as an indicator of cell viability, necrosis and apoptosis.</a> J. Immunol. Methods. 240 (1-2), 1-2 (2000).</li><li>Rodnight, R., McIlwain, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+in+tissue+metabolism:+3.+Study+of+tissue+fragments+with+little+or+no+added+aqueous+phase,+and+in+oils.">Techniques in tissue metabolism: 3. Study of tissue fragments with little or no added aqueous phase, and in oils.</a> Biochem. J. 57, 649-661 (1954).</li><li>Shelanski, M. L., Gaskin, F., Cantor, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+assembly+in+the+absence+of+added+nucleotides.">Microtubule assembly in the absence of added nucleotides.</a> Proc. Natl. Acad. Sci. U.S.A. 70 (3), 765-768 (1973).</li></ol>

저자는이 논문에서 수행 된 작업의에 대한 관심의 충돌이 없다는 것을 선언합니다.

여기서, 본래 성숙한 뇌 조직에서 흥분 독성 및 허혈 - 매개 세포 사멸에 관여하는 분자 메커니즘을 조사하는 데 사용될 수 microslices의 생성을위한 시험 관내 기술이 설명된다. 이 기술은 실행 가능한 조직 (1-3 피규어), 즉 큰의 Organotypic 조각 15 대사와 유사하다을 생산하고 있습니다. 또한,이 microslice 모델은 밀접하게 생체 내 10 흥분 독성 매개 뇌 손상 다?...

뇌졸중의 가장 일반적인 형태는 대뇌 혈관 폐색 될 때 발생하는 허혈성 뇌졸중이다. 혈액의 흐름 중단 한 결과 조직의 국소 빈혈은 세포막의 탈분극 확산, 흥분성 신경 전달 물질의 방출을 일으키고, 세포 죽음의 활성화에 이르게 세포 내 칼슘의 지속적인 상승은 1 경로. 이 과정은 &#39;흥분 독성 (excitotoxicity)&#39;라고하고, 스트로크 2 등의 병리의 다양한 의해 생성 된 신경 세포의 죽음에 관련된 일반적인 경로입니다되었습니다. 흥분 독성 및 다른 신경 세포 죽음의 계곡에 관련된 신호 전달 경로의 억제는 뇌졸중 다음 신경 세포의 손상을 제한 할 수있는 매력적인 방법입니다. 동물 전체 시스템을 사용할 때 흥분 독성 (excitotoxicity)과 허혈성 뇌졸중에 관련된 정확한 분자 메커니즘을 확인하는 것은 어려울 수 있습니다. 같은 차 배아와 &#39;신경과 같은&#39;(예를 들어 neuroblasto로엄마와 선암 불후의 선)는 세포 배양 시스템은 자주 사용된다. 이러한 모델의 주요 이점들은, 상대적으로 비용 효과적인 쉽게 조작 할 수 있으며 세포 사멸이 용이하고 정량 측정 할 수 있다는있다. 그러나, 신호 전달 경로는 배양 3,4를 사용 조건 및 미성숙 신경 세포에 의해 변경하고 선이 다른 수용체를 표현하고 뇌 5-8 성숙에 비해 신호 분자 수 불멸 할 수 있습니다. (공 배양 시스템이 사용되는 경우, 또는 2 개)의 손상 뇌 조직이 서로 상호 작용하는 다양한 종류의 세포를 함유하는 이종 반면 또한, 배양 된 신경 세포는 단지 하나의 세포 유형의 검사를 허용한다. Organotypic 슬라이스 배양 시스템 (뇌 조직의 얇은 절편)도 사용되며, 이들이 생체 내에서 발견되는 이러한 모델은 이종의 세포 집단의 연구를 허용한다. 이 기술을 사용하는 경우에는, 조직의 제한된 양 슬라이스는, 각각의 동물로부터 수득 할 수있다한 불후의 세포주로 배양 할, 장기 문화 매체는 슬라이스의 경로와 수용체 신호에 변화가 발생할 수 없습니다. 성숙한 두뇌의 Organotypic 조각을 생성하는 데 사용할 수있는 반면, 미성숙 뇌에서 슬라이스 문화에 더 많은 의무, 그리고 더 일반적으로 사용된다. 신경 신호 전달 경로를 검사 할 수있는 사용하기 쉬운 그대로 성숙한 뇌, 모방 또는 표현 모델을 개발하기 위해 필요하다. 여기서, 흥분 독성 또는 허혈성 모욕 다음 세포사에 관여 분자 메커니즘을 규명하는 데 사용할 수있는 손상 신경 조직을 포함하는 생체 외 기법을 설명한다. 이 기술은, 실험을 수행하는 데 필요한 동물의 수를 감소 재현이며, 더 큰 슬라이스의 Organotypic 대사 유사한 방식으로 동작 가능한 조직을 생성한다. 또한, 신경 회로, 셀룰러 상호 작용 및 시냅스 콜로라도mpartment 부분적으로 그대로 유지됩니다. 사용되는 생리 버퍼는 셀 &#39;봉인&#39;에 막, 그리고 수 세포가 원래의 막 저항 9를 복구 할 수 있습니다. 이 뇌 조각 모델을 충실하게 흥분 독성 매개 뇌 손상 10 다음 관찰 된 반응을 모방 할 수있다, 뇌졸중에 관련된 분자 메커니즘을 검사하는 데 사용할 수 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Guillotine</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Used to decapitate animal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical equipment</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Forceps, scissors, tweezers, etc, for brain removal and dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">McIlwain chopper</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Used to generate 100-400 &#956;m brain sections.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>McIlwain choppers are manufactured/distributed by a range of companies including Mickle Engineering, Harvard Apparatus, Campden Instruments and Ted Pella.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Round bottom plastic tubes</td>
			<td style="width:71px;">Greiner</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water bath</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>For keeping tissue at 37 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aerating apparatus</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Used to keep microslices in a humidified, oxygenated environment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Flat bottomed polystyrene tubes</td>
			<td style="width:71px;">Nunc</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dounce Homogenizer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

excitotoxic stimulation of brain microslices as an in vitro model of stroke

전체 동물 체제를 사용하는 경우 허혈성 뇌졸중과 같은 신경 병리학 적 조건에 관련된 분자 메커니즘을, 검사, 어려울 수 있습니다. 예컨대, 기본 또는 &#39;신경 조의&#39;세포 배양 시스템은 일반적으로 이용된다. 이러한 시스템은 작동 비교적 용이하며, (예컨대 세포사와 같은) 다양한 기능적 결과를 쉽게 정량화 될 수있는 유용한 모델 시스템 (예 흥분 독성 - 매개 세포 사멸 경로 등) 배양 된 미숙 뉴런의 조사 결과와 경로이지​​만 반드시 성숙한 뇌에서 관찰 된 것과 같은, 또는 손상 조직에 없습니다. 따라서, 성숙한 신경 조직의 세포 메커니즘을 조사 할 수있는 모델을 개발할 필요가있다. 우리는 신경 조직 손상의 분자 경로의 다양성을 조사하기 위해 사용될 수있는 시험 관내 기술을 개발했다. 설명 된 기술은 본 명세서 쥐 대뇌 피질 조직을 활용하지만,이 기술은 조직을 사용하도록 구성 될 수있다또는 뇌 영역 (예를 들어, 해마, 선조체, 등) (예 : 마우스, 토끼, 기니 돼지, 닭 등) 종의 다양 한에서. 또한, 자극 / 치료의 다양한 (예를 들어, 흥분 독성, 억제제의 투여 등)을 사용할 수있다. 결론적으로, 본원에 기재된 뇌 슬라이스 모델 흥분 독성 매개 성 뇌 손상에 관련된 분자 메커니즘의 다양성을 조사 할 수있다.

이 절차를 사용하여 생성 Microslices가 실용적이며, 종의 다양성 (예를 들어, 래트, 마우스, 닭) microslices을 생성하기 위하여 사용될 수있다. 생존의 세 가지 독립적 인 조치를 활용 한 : 호흡 속도 (그림 1), 아데닌 뉴클레오티드 비율 (그림 2), 및 조직의 칼륨 함량 (그림 3). 이러한 방법을 사용하면, microslices 적어도 2 시간 후 생성을 위해 실행 가능한 남아 있다고 ...

Watch this Scientific Journal Video about Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke at JoVE.com

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

우리는 흥분 독성 매개 성 뇌 손상에 관련된 분자 메커니즘을 조사하는 데 사용될 수있는 뇌 슬라이스 모델을 개발 하였다. 이 기술은 실행 가능한 성숙한 뇌 조직을 생성하고, 부분적으로 손상 신경 회로, 세포 상호 작용과 시냅스 구획을 유지하면서, 실험에 필요한 동물의 수를 줄일 수 있습니다.

같은 뇌 Microslices의 흥분 독성 자극<em&gt; 체외</em&gt; 스트로크의 모델

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

excitotoxic-stimulation-brain-microslices-as-an-vitro-model

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JoVE Journal

Medicine

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

8.4K 조회수.  The University of Newcastle.  우리는 흥분 독성 매개 성 뇌 손상에 관련된 분자 메커니즘을 조사하는 데 사용될 수있는 뇌 슬라이스 모델을 개발 하였다. 이 기술은 실행 가능한 성숙한 뇌 조직을 생성하고, 부분적으로 손상 신경 회로, 세포 상호 작용과 시냅스 구획을 유지하면서, 실험에 필요한 동물의 수를 줄일 수 있습니다. 

비디오: Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

초점 대뇌 국소 빈혈에 조기 허혈성 공차를 유도하기 위해 적절한 전처리 자극으로 양측 경동맥 동맥 폐색

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

연구

의학

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

지주막 하 출혈의 생쥐 모델

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

로 뇌 미세 혈관 내피 세포 (CEnd를)에 기지개<em&gt; 체외</em혈액 뇌 장벽의&gt; 외상성 뇌 손상 모델

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

설정 - 업<em&gt; 체외</em쥐 뇌 혈관 벽의&gt; 모델 (BBB)​​ : BBB 불 침투성 및 수용체 매개 운송에 초점

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

같은 산소 포도당 박탈과 재산 소화<em&gt; 체외</em&gt; 허혈 - 재관류 손상 모델 공부 뇌 혈관 장벽 부전

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

A Multimodal Imaging- and Stimulation-based Method of Evaluating Connectivity-related Brain Excitability in Patients with Epilepsy

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations over time in the blood-oxygenation level dependent signal. rs-fcMRI has been applied extensively to identify abnormalities in brain connectivity in different neurologic and psychiatric diseases. However, the relationship among rs-fcMRI connectivity abnormalities, brain electrophysiology and disease state is unknown, in part because the causal significance of alterations in functional connectivity in disease pathophysiology has not been established. Transcranial Magnetic Stimulation (TMS) is a technique that uses electromagnetic induction to noninvasively produce focal changes in cortical activity. When combined with electroencephalography (EEG), TMS can be used to assess the brain's response to external perturbations. Here we provide a protocol for combining rs-fcMRI, TMS and EEG to assess the physiologic significance of alterations in functional connectivity in patients with neuropsychiatric disease. We provide representative results from a previously published study in which rs-fcMRI was used to identify regions with abnormal connectivity in patients with epilepsy due to a malformation of cortical development, periventricular nodular heterotopia (PNH). Stimulation in patients with epilepsy resulted in abnormal TMS-evoked EEG activity relative to stimulation of the same sites in matched healthy control patients, with an abnormal increase in the late component of the TMS-evoked potential, consistent with cortical hyperexcitability. This abnormality was specific to regions with abnormal resting-state functional connectivity. Electrical source analysis in a subject with previously recorded seizures demonstrated that the origin of the abnormal TMS-evoked activity co-localized with the seizure-onset zone, suggesting the presence of an epileptogenic circuit. These results demonstrate how rs-fcMRI, TMS and EEG can be utilized together to identify and understand the physiological significance of abnormal brain connectivity in human diseases.

Resting-state functional-connectivity MRI has identified abnormalities in patients with a wide range of neuropsychiatric disorders, including epilepsy due to malformations of cortical development. Transcranial Magnetic Stimulation in combination with EEG can demonstrate that patients with epilepsy have cortical hyperexcitability in regions with abnormal connectivity.

간질 환자의 뇌 흥분을 연결 관련 평가하는 멀티 모달 Imaging- 및 자극 기반 방법

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

신생아 쥐의 달팽이관 Explant 기법으로<em&gt; 체외에서</em&gt; 청력 연구의 스크리닝 도구

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

생체 외에서 혈액-뇌 장벽 돼지 뇌 내 피 세포를 기반으로 하는 모델의 설립에 대 한 방법 개선

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Vagus Nerve Stimulation As an Adjunctive Neurostimulation Tool in Treatment-resistant Depression

Vagus nerve stimulation (VNS) is an approved neurostimulation therapy. The purpose of the method is to treat patients with therapy-resistant depression (TRD). VNS exhibits antidepressive and stabilizing effects. This method is particularly useful as a long-term treatment, in which up to two-thirds of patients respond. The vagus nerve stimulator is positioned on the left vagus nerve during a surgical procedure and is activated telemetrically by a wand connected to a handheld computerized device. The treating physician can perform various adjustments of the vagus nerve stimulator during in-office visits (e.g., by modifying stimulation intensity or stimulation frequency) to achieve maximum therapeutic effects with low side effects. Set-up of the device usually takes several months. Typical side effects include wound infection, temporary salivation, coughing, paralysis of the vocal cords, bradycardia, or even asystole. The patient can stop the VNS by placing a magnet over the generator. The current protocol describes delivery of the specific stimulation tool and methods for adjusting the tuning parameters to achieve the best remission rates in patients with TRD.

Vagus nerve stimulation (VNS) has been shown to be effective as an adjunct treatment for treatment-resistant depression (TRD). VNS leads to antidepressive and antisuicidal effects and quality of life improvements. This protocol offers a step-by-step guide to managing and adjusting a vagus nerve stimulator for efficient treatment of TRD.

미주 신경 자극 치료 내성 우울증에 대체 통제 도구로

Vagus nerve stimulation (VNS) is an approved neurostimulation therapy. The purpose of the method is to treat patients with therapy-resistant depression ...

Updated Technique for Reliable, Easy, and Tolerated Transcranial Electrical Stimulation Including Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a noninvasive method of neuromodulation using low-intensity direct electrical currents. This method of brain stimulation presents several potential advantages compared to other techniques, as it is noninvasive, cost-effective, broadly deployable, and well-tolerated provided proper equipment and protocols are administered. Even though tDCS is apparently simple to perform, correct administration of the tDCS session, especially the electrode positioning and preparation, is vital for ensuring reproducibility and tolerability. The electrode positioning and preparation steps are traditionally also the most time consuming and error-prone. To address these challenges, modern tDCS techniques, using fixed-position headgear and pre-assembled sponge electrodes, reduce complexity and setup time while also ensuring that the electrodes are consistently placed as intended. These modern tDCS methods present advantages for research, clinic, and remote-supervised (at home) settings. This article provides a comprehensive step-by-step guide for administering a tDCS session using fixed-position headgear and pre-assembled sponge electrodes. This guide demonstrates tDCS using commonly applied montages intended for motor cortex and dorsolateral prefrontal cortex (DLPFC) stimulation. As described, selection of the head size and montage-specific headgear automates electrode positioning. Fully assembled pre-saturated snap-electrodes are simply affixed to the set position snap-connectors on the headgear. The modern tDCS method is shown to reduce setup time and reduce errors for both novice and expert operators. The methods outlined in this article can be adapted to different applications of tDCS as well as other forms of transcranial electrical stimulation (tES) such as transcranial alternating current stimulation (tACS) and transcranial random noise stimulation (tRNS). However, since tES is application specific, as appropriate, any methods recipe is customized to accommodate subject, indication, environment, and outcome specific features.

When administering transcranial direct current stimulation (tDCS), reproducible electrode preparation and placement are vital for a tolerated and effective session. The purpose of this article is to demonstrate updated modern setup procedures for the administration of tDCS and related transcranial electrical stimulation techniques, such as transcranial alternating current stimulation (tACS).

경두개 직접 전류 자극을 포함한 신뢰할 수 있고 쉬우며 용인된 경두개 전기 자극을 위한 기술 업데이트

Transcranial direct current stimulation (tDCS) is a noninvasive method of neuromodulation using low-intensity direct electrical currents. This method of ...