이 작품은 프로젝트 SAF2017-82736-C2-1-R 장관 드 시엔시아 e 이노바시온에서 MTM-F, Fundación Universidad Francisco de Vitoria에서 JS에 의해 재정적으로 지원되었다.

참고: 동물 실험은 국가 및 기관 생물 윤리 위원회에 의해 승인되었습니다.
1. ihOEG (Ts12 및 Ts14) 문화
참고: 이 절차는 조직 배양 생물 안전 캐비닛의 멸균 조건하에서 수행됩니다.
<ol><li>표 1에제공된 50mL ME10 OEG 배양 배지를 준비한다.</li>
	<li>표 1에제공된 대로 DMEM/F12-FBS 5mL을 15mL 원문 튜브에 준비합니다.</li>
	<li>깨끗한 수조에서 37°C에서 두 미디어를 15분 동안 데우습니다.</li>
	<li>깨끗한 수조에서 Ts12 및 Ts14 세포바이알을 37°C에서 해동합니다.</li>
	<li>2단계에서 제조된 DMEM/F12-FBS 배양 배지에 세포를 다시 일시 중지하고 추가합니다.</li>
	<li>200 x g에서5 분 동안 원심 분리기.</li>
	<li>상체를 갈망합니다.</li>
	<li>ME10 배지 의 500 μL을 추가하고 펠릿을 다시 중단합니다.</li>
	<li>ME10의 3mL로 p60 세포 배양 접시를 준비하고 셀룰러 서스펜션을 드롭 와이즈로 추가합니다.</li>
	<li>플레이트 에 걸쳐 균일하게 세포를 배포하려면 이동합니다.</li>
	<li>배양 세포는 37°C에서 5% CO2로한다. 참고: 합류에 도달한 후, 적어도 다른 구절은 공동 문화를 위한 세포를 최적화하기 위하여 행해져야 합니다. 90%의 합류가 공동 문화를 위해 커버립에 시드하기 전에 필요합니다. Confluent p-60은 Ts14의 경우 7 x 105의 평균 셀 수와 Ts12 세포주에 대해 2.5 x 106의 평균 셀 번호를 가지고있다. Ts12 및 Ts14 세포주마다 2-3일마다 통과되어야 합니다.</li>
</ol>2. 분석용 ihOEG(Ts12 및 Ts14) 준비
참고: 이 단계는 RGN 해부 및 공동 문화 전에 24h를 수행해야 합니다.
<ol><li>12mm Ø 커버립을 10 μg/mL 폴리 L-리신(PLL)으로 1시간 동안 처리합니다. 참고: 커버립은 PLL 솔루션에서 하룻밤 동안 방치할 수 있습니다.</li>
	<li>커버립을 1x 인산완충식염(PBS)으로 세 번 씻습니다.</li>
	<li>p60 세포 배양 접시로부터 Ts12 및 Ts14 ihOEG 세포를 분리한다.<ol>
<li>DMEM/F12-FBS 배양 배지(표1)의4mL을 15mL 원문 튜브에 추가합니다. 깨끗한 수조에서 37 °C에서 따뜻하게합니다.</li>
		<li>접시에서 배지를 제거하고 1x PBS-EDTA1mL로 세포를 세척합니다.</li>
		<li>OEG 세포에 트립신-EDTA 1mL을 추가하고 37°C에서 3-5분, 5% CO2에서3-5분 동안배양한다.
</li>
		<li>p1000 파이펫으로 세포를 수집하고 3.1 단계에서 준비된 매체로 전송합니다.</li>
		<li>200 x g에서5 분 동안 원심 분리기.</li>
		<li>상체를 갈망합니다.</li>
		<li>ME10 배지 1mL을 추가하고 펠릿을 다시 중단합니다.</li>
		<li>혈종계에서 셀 수를 계산합니다.</li>
	</ol>
</li>
	<li>종자 80,000 Ts14 세포 또는 100,000 Ts12 세포 /잘 ME10 배지의 500 μL에서 24 웰 플레이트에서 커버립에.</li>
	<li>배양 세포는 37°C에서 5%CO2,24h.</li>
</ol>3. 망막 조직 해부
참고: 2개월 된 수컷 위스타 쥐는 RGN 소스로 사용됩니다. 24웰 세포 접시의 20 개의 우물에 대한 두 개의 망막 (한 마리의 쥐). 사용하기 전에 자동 클클레이브 수술 재료. 파팡 해리 키트는 상업적으로 구입(재료의 표). 재구성에 대한 공급자의 지침을 따릅니다. 5mM 스톡에서 D,L-2-아미노-5-인포노발레산(APV)을 재구성하고 알리쿼트준비한다.
<ol><li>분석 당일, 다음 미디어를 준비한다.<ol>
<li>차가운 EBSS (파아펜 해리 키트의 바이알 1)의 5mL와 p60 세포 배양 접시를 준비합니다.</li>
		<li>p60 세포 배양 접시를 파팔 해리 키트의 재구성된 바이알 2(papain)와 50 μL의 APV로 준비한다.  그런 다음 250 μL의 상반신 3(DNase plus 5 μL)를 추가합니다.</li>
		<li>멸균 튜브 믹스 2.7 mL의 유리병 1과 300 μL 의 유리병 4 (알부민-ovomucoid 프로테아제 억제제). 바이알 3(DNase)의 150 μL과 30 μL의 APV를 추가합니다.</li>
		<li>표 1에제공된 바와 같이 신경분사-B27 배지(NB-B27)의 20mL를 준비한다.</li>
	</ol>
</li>
	<li>CO2로질식하여 쥐를 희생하십시오.</li>
	<li>기요틴으로 참수하여 머리를 제거하십시오. 100mm 페트리 접시에 넣고 머리에 에탄올 70%로 스프레이한 후 라미나르 플로우 후드에 넣습니다.</li>
	<li>쥐의 수염을 가위로 잘라 서 눈 조작을 방해하지 않도록 합니다.</li>
	<li>메스로 눈을 가로질러 절개를 할 수 있을 만큼 안구를 꺼내서 포셉으로 시신경을 잡아냅니다.</li>
	<li>렌즈와 유리체 유머를 제거하고 망막 (오렌지 와 같은 조직)을 꺼내고 눈의 나머지 층은 내부에 머물러 있습니다 (안료 상피 층을 포함).</li>
	<li>3.1.1 단계에서 제조된 p60 세포 배양 접시에 망막을 놓습니다.</li>
	<li>3.1.2 단계에서 제조된 p60 세포 배양 접시에 망막을 옮기고 메스로 메스를 대략 적인 크기의 작은 조각으로 자르고 1mm.</li>
	<li>15mL 플라스틱 튜브로 옮김합니다.</li>
	<li>조직을 30분 동안 배양하고, 가습된 인큐베이터에서 5% CO2미만의 37°C에서, 10분마다 교반한다.</li>
	<li>유리 파스퇴르 파이펫으로 위아래로 피펫을 하여 세포 덩어리를 분리합니다.</li>
	<li>5 분 동안 200 x g에서 세포 현탁액을 원심 분리합니다.</li>
	<li>수퍼나탄을 버리고 파아픔을 비활성화하고, 3.1.3 단계에서 준비된 용액의 세포 펠릿을 재보중단한다. (2 눈에 1.5 mL).</li>
	<li>이 셀 서스펜션을 재구성된 바이알 4의 5mL로 조심스럽게 파이프화합니다.</li>
	<li>원심 분리기 200 x g에서 5 분 동안.</li>
	<li>원심분리하는 동안, OEG 24 웰 셀 플레이트(이전에 2단계에서 제조됨)에서 ME-10 배지를 완전히 제거하고 잘 당 NB-B27 배지의 500 μL로 대체한다.</li>
	<li>상체를 버리고 NB-B27 배지의 2mL에서 세포를 다시 중단한다.</li>
	<li>m24 플레이트의 웰당 망막 셀 서스펜션 100 μL을 PLL 처리 또는 OEG 단층 커버립에 플레이트.</li>
	<li>NB-B27 배지에서 96h에 대해 5%CO2로 37°C에서 배양을 유지한다.</li>
</ol>4. 면역 염색
<ol><li>96h 후, 배양 배지(600 μL)(PFA 최종 농도 2%)에 1x PBS에서 4%의 파라포름데히드(PFA)의 동일한 부피를 첨가하여 10분 동안 세포를 고정한다.</li>
	<li>24-멀티웰 플레이트에서 미디어와 PFA를 제거하고 다시 한 번 1배 PBS에 4% 파라포름알데히드(PFA)의 500 μL을 추가합니다. 10 분 동안 인큐베이션하십시오.</li>
	<li>픽서를 버리고 1x PBS로 3번 5분 동안 세척하십시오.</li>
	<li>PBS(PBS-TS)에서 0.1% 트리톤 X-100/1% FBS를 30~40분 동안 차단합니다.</li>
	<li>다음과 같이 PBS-TS 완충제에서 1차 항체를 준비하십시오: SMI31 (MAP1B 및 NF-H 단백질에 대하여) 단클론 항체 (1:500). 514 (MAP2A 및 B 단백질인식) 토끼 폴리클론 항혈제 (1:400).</li>
	<li>1 차적인 항체를 균원에 추가하고 4°C에서 하룻밤 동안 배양합니다.</li>
	<li>다음날, 항체를 버리고 커버립을 1x PBS로 3번, 5분 동안 씻는다.</li>
	<li>다음과 같이 PBS-TS 버퍼에서 이차 항체를 준비하십시오: SMI-31의 경우, 항 마우스 알렉사 플루어 488 (1:500). 514, 안티 토끼 알렉사-594 (1:500).</li>
	<li>1 h, RT에서, 어둠 속에서 대응하는 형광 이차 항체를 가진 세포를 배양한다.</li>
	<li>커버립을 1x PBS로 3회, 어둠 속에서 5분 동안 씻습니다.</li>
	<li>마지막으로, 장착 매체(재료의 표)와커버립을 마운트하고 4 ° C에서 유지합니다. 참고: 필요할 때마다 DAPI(4,6-디아미드노-2-페닐린돌)를 가진 형광 핵 염색이 수행될 수 있다. 장착하기 전에, DAPI (1x PBS에서 10 μg /mL)와 어둠 속에서 10 분 동안 세포를 배양한다. 커버립을 1x PBS로 3번 세척하고 마지막으로 마운팅 매체로 커버립을 장착합니다.</li>
</ol>5. 축축한 재생 정량화
참고: 샘플은 상피 성화 현미경의 40배 목표에 따라 정량화됩니다. 최소 30장의 사진을 무작위 필드에서 촬영해야 하며, 적어도 200개의 뉴런이 있는 각 치료에 대해 정량화되어야 합니다. 각 실험은 최소 3회 반복되어야 합니다.
<ol><li>RGNs의 총 인구에 비해 축축 (SMI31 양성 중성염)을 가진 뉴런의 백분율을 정량화 (MAP2A/B 514 뉴런 바디및 원원수의 양성 면역 염색으로 확인).</li>
	<li>축 축 재생 지수 또는 평균 축 산 길이(μm/뉴런)를 정량화합니다. 이 매개변수는 모든 확인된 축축의 길이(μm)의 합으로 정의되며, 각축을 제시했는지 여부에 관계없이 계산된 뉴런의 총 수로 나뉜다. 축 사 길이 이미지 소프트웨어 ImageJ (NIH-미국)의 플러그인 NeuronJ를 사용 하 여 결정 됩니다.</li>
	<li>적절한 소프트웨어를 사용하여 평균, 표준 편차 및 통계적 유의를 계산합니다.</li>
</ol>

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A., Phelps, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+olfactory+ensheathing+glia+enhance+axon+outgrowth+on+a+myelin+substrate+in+vitro.">Mouse olfactory ensheathing glia enhance axon outgrowth on a myelin substrate in vitro.</a> Experimental Neurology. 216 (1), 95-104 (2009).</li><li>Witheford, M., Westendorf, K., Roskams, A. 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저자는 공개 할 것이 없습니다.

CNS 상해 부위에서 OEG 이식은 구성적인 프로 신경 재생특성7,8,9로인해 CNS 부상에 대한 유망한 치료법으로 간주됩니다. 그러나, 조직원에 따라 후각점막(OM-OEG)과 후각전(OB-OEG)-또는 기증자의 나이에 따라, 이러한 용량26,31,33,36에상당...

성인 중추 신경계 (CNS) 뉴런은 부상이나 질병 후 재생 능력이 제한되어 있습니다. CNS 재생을 촉진하는 일반적인 전략은 줄기 세포, 슈완 세포, 성상세포 또는 후각 내성 신경교(OEG) 세포1,2,3,4,5와같은 축축성장을 유도하는 세포 유형의 부상부위에서이식된다.
OEG는 신경 문장6에서 파생되고 후각 점막과 후각 전구에서 찾습니다. 성인에서, 후각 감각 뉴런은 환경 노출의 결과로 정기적으로 죽고 새로 분화 된 뉴런으로 대체됩니다. OEG는 이러한 새로운 후각 축축을 둘러싸고 안내하여 후각 전구에 들어가 CNS7에서목표를 가진 새로운 시냅스를확립합니다. 이러한 생리적 특성으로 인해 OEG는 척수 나 시신경 손상과 같은 CNS 부상 의 모델에 사용되어 왔으며 신경 생성 및 신경 보호 특성은8,9,10,11로입증되었습니다. 세포외 매트릭스 프로테아제 생산 또는 신경영양 및 축삭 성장 인자12,13,14의분비를 포함한 이러한 세포의 재생 특성에 대한 여러 가지 요인으로 확인되었습니다.
1차 OEG 세포를 확장하는 기술적 한계를 감안할 때, 우리는 이전에 균일 한 OEG의 무제한 공급을 제공하는 가역 불멸의 인간 OEG (ihOEG) 클론 라인을 설립하고 특성화했습니다. 이러한 ihOEG 세포는 부검에서 얻은 후각 전구에서 제조된 1차 배양에서 유래한다. 그(것)들은 텔로머라아제 촉매 서브유닛 (TERT)과 종양균 Bmi-1의 전도에 의해 불멸되고 SV40 바이러스 큰 T 항원15,16,17,18로변형되었다. 이들 ihOEG 세포주 중 2개는 Ts14로, 이는 본래 배양과 Ts12의 재생 능력을 유지하며, 이러한 실험에서 낮은 재생 제어로 사용되는 낮은 재생성라인(18)이다.
신경 손상 후 축 하 재생을 육성 하는 OEG 용량을 평가 하기 위해, 여러 시험 관 모델이 구현 되었습니다. 이러한 모델에서, OEG는 서로 다른 뉴런 기원과 중성염 형성 및 신장의 배양에 적용되며, 이는 글리아교 공동 문화에 대한 반응으로 분석된다. 이러한 뉴런 소스의 예로는 신생아 쥐 피질뉴런(19)이있으며, 피질조직(20)에서쥐 배아 뉴런에 수행된 스크래치 상처, 쥐 망막 이병21,쥐 시상하또는 해마 산후 뉴런22,23,산후 쥐 등쪽 뿌리 신경절뉴런(24), 산후 마우스 코르티코피타피통뉴런(25,인간 NT2 뉴런26)또는 사후 뇌피질 뉴런이 반응성 성상세포 흉터와 같은 배양(27)을 한다.
그러나 이러한 모델에서는 재생 분석이 부상당한 성인 뉴런에 없는 본질적인 가소성을 가진 배아 또는 산후 뉴런에 의존합니다. 이러한 단점을 극복하기 위해, 우리는 원래 Wigley등28,29,30,31에 의해 개발된 것을 기반으로 성인 망막 신경절 뉴런(RGNs)과 OEG 라인의 공동 배양에서 성인 축축산 재생 모델을 제시하고 우리 그룹12,13,14, 15,15, 16,16, 17, 18, 18, 18, 18, 18, 17, 18, 18, 18, 18, 18, 18, 15, 16, 18,18, 18, 18, 18, 18, 18, 18, 18, 17, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 17, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 17, 18, 18, 18, 18,1 간략하게, 망막 조직은 성인 쥐에서 추출하고 papain로 소화됩니다. 망막 세포 현탁액은 폴리리신 처리 커버립 또는 Ts14 및 Ts12 단층층상에 도금됩니다. 배양은 축산(MAP1B 및 NF-H 단백질)34 및 소마토덴드리틱(MAP2A 및 B)35마커를 고정하기 전에 96시간 동안 유지된다. 축사 재생은 축축을 가진 뉴런의 백분율로 정량화되며, RGNs및 축축 재생 지수의 총 인구에 대하여 뉴런당 평균 축축수 길이로 계산된다. 이 프로토콜은 ihOEG의 신경 재생 잠재력을 평가하는 데 유용할 뿐만 아니라 OEG 또는 다른 신경교 세포의 다른 소스로 확장 될 수 있습니다.

<table>
	<tbody>
		<tr>
			<td>antibody 514</td>
			<td>Reference 34</td>
			<td></td>
			<td>Rabbit polyclonal antiserum, which recognizes MAP2A and B.</td>
		</tr>
		<tr>
			<td>antibody SMI-31</td>
			<td>BioLegend</td>
			<td>801601</td>
			<td>Monoclonal antibody against MAP1B and NF-H proteins</td>
		</tr>
		<tr>
			<td>anti-mouse Alexa Fluor 488 antibody</td>
			<td>ThermoFisher</td>
			<td>A-21202</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rabbit Alexa Fluor 594 antibody</td>
			<td>ThermoFisher</td>
			<td>A-21207</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>D,L-2-amino-5-phosphonovaleric acid</td>
			<td>Sigma</td>
			<td>283967</td>
			<td>NMDA receptor inhibitor</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>Nuclei fluorescent stain</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>11573397</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr>
			<td>FBS-Hyclone</td>
			<td>Fisher Scientific</td>
			<td>16291082</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr>
			<td>Fluoromount</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td>Mounting medium</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health (NIH-USA)</td>
			<td></td>
			<td>Image software</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Lonza</td>
			<td>BE17-605F</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>Neuronal cells culture medium</td>
		</tr>
		<tr>
			<td>Papain Dissociation System</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LK003150</td>
			<td>For use in neural cell isolation</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS-EDTA</td>
			<td>Lonza</td>
			<td>H3BE02-017F</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin/Amphotericin B</td>
			<td>Lonza</td>
			<td>17-745E</td>
			<td>Bacteriostatic and bactericidal</td>
		</tr>
		<tr>
			<td>Pituitary extract</td>
			<td>Gibco</td>
			<td>13028014</td>
			<td>Bovine pituitary extract</td>
		</tr>
		<tr>
			<td>Poly -L- lysine (PLL)</td>
			<td>Sigma</td>
			<td>A-003-M</td>
			<td></td>
		</tr>
	</tbody>
</table>

coculture of axotomized rat retinal ganglion neurons with olfactory ensheathing glia, as an in vitro model of adult axonal regeneration

후각 내시경 신경교 (OEG) 세포는 후각 점막에서 후각 전구의 후각 신경 층 (ONL)으로 모든 방법을 국소화된다. 성인 생활 내내, 그들은 새로 생성 된 후각 뉴런의 축 하 성장에 대 한 열쇠, 라미나 프로 프리 테리토리에서 ONL. 그들의 프로 재생 속성으로 인해, 이 세포는 척수 또는 시신경 상해 모형에 있는 축축재생을 육성하기 위하여 이용되었습니다.
우리는 신경 손상 후 OEG 신경 재생 용량을 분석하고 측정하기 위해 체외 모델을 제시합니다. 본 모델에서, 가역적으로 불멸의 인간 OEG(ihOEG)는 단층으로 배양되고, 망막은 성인 쥐로부터 추출되고 망막 신경절 뉴런(RGN)은 OEG 단층상에 공동 배양된다. 96h 후, RGNs의 축산 및 소종 절인 마커는 축축및 평균 축산 길이/뉴런을 가진 RG의 수에 의해 정량화된다.
이 프로토콜은 배아 또는 산후 뉴런에 의존하는 다른 체외 분석에 비해 장점이 있으며 성인 조직에서 OEG 신경 재생 특성을 평가합니다. 또한 ihOEG의 신경 재생 잠재력을 평가하는 데 유용할 뿐만 아니라 OEG 또는 다른 신경교 세포의 다른 소스로 확장 될 수 있습니다.

이 프로토콜에서, 우리는 신경 손상 후 OEG 신경 재생 용량을 분석하는 체외 모델을 제시한다. 도 1에도시된 바와 같이, OEG 소스는 부검15,17,18에서얻은 후각 전구로부터 제조된 1차 배양으로부터 유래한 가역불멸인간 OEG 클론 세포주-Ts12-이다. 망막 조직은 성인 쥐로부터 추출되고, 소화되고, 망막 신경절 ?...

Watch this Scientific Journal Video about Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration at JoVE.com

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

우리는 신경 손상 후 후각 내성 glia (OEG) 신경 재생 능력을 평가하는 체외 모델을 제시한다. 그것은 RGN 축상 및 somatodritic 마커를 분석해서, OEG 단층 및 축 변성의 후속 연구 결과에 축축한 성인 망막 신경절 신경절 뉴런 (RGN)의 공동 문화를 기반으로합니다.

축 축 하 재생의 시험관 내 모델로, 후각 천시 글리아와 축 축 절 망막 신경의 공동 문화

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

coculture-axotomized-rat-retinal-ganglion-neurons-with-olfactory

Research

JoVE Journal

Neuroscience

3.8K 조회수.  Universidad Francisco de Vitoria. 우리는 신경 손상 후 후각 내성 glia (OEG) 신경 재생 능력을 평가하는 체외 모델을 제시한다. 그것은 RGN 축상 및 somatodritic 마커를 분석해서, OEG 단층 및 축 변성의 후속 연구 결과에 축축한 성인 망막 신경절 신경절 뉴런 (RGN)의 공동 문화를 기반으로합니다.

비디오: Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

차 쥐 두피 뉴런과 Glia의 Bilaminar의 공동 문화

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

연구

신경과학

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

만성에 대한 성인 태어나 강한 뉴런의 선택 바이러스성 전달 생체내에 Optogenetic의 자극

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

교양 성인 지느러미 루트 신경절의 신경 세포와 축삭 재생의 유전 연구

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

말초 신경 손상 후 기능 회복을 평가하기 위해 후각 Ensheathing 세포의 이식

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

<em&gt; 체외</em성인 마우스 Myenteric 신경총에서 분리 된 장내 뉴런과 교세포의&gt; 준비

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

형광 염료와 성인 Zebrafish의 망막 신경절 세포의 퇴행성 라벨링

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

모델로 후각 시스템은 축삭 성장 패턴 및 형태를 연구하는<em&gt; 생체</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

간접 신경-성상 세포 공동 배양 분석 일 :<em&gt; 체외</em&gt; 신경 세포 - glia 상호 작용의 상세한 조사를위한 셋업

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

M&amp;#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

레이저 유도 된 망막 변성 및 재생 Zebrafish 모델 뮐러 명과 셀 활성화

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

성인 마우스 지 루트 신경 절의 Electroporation Vivo에서 조사 포유류 축 삭 재생:

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...