Name: coculture of axotomized rat retinal ganglion neurons with olfactory ensheathing glia, as an in vitro model of adult axonal regeneration
Uploaded: 2020-11-02T15:00:00.000+00:00
Duration: 7 min 57 s
Description: 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

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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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

Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is very poor 1. The failure of axon regeneration is a combined result of several factors including the hostile glial cell environment, inhibitory myelin related molecules and decreased intrinsic neuron regenerative capacity 2. Astrocytes are the most predominant glial cell type in central nervous system and play important role in axon functions under physiology and pathology conditions 3. Contrast to the homologous oligodendrocytes, astrocytes are a heterogeneous cell population composed by different astrocyte subpopulations with diverse morphologies and gene expression 4. The functional significance of this heterogeneity, such as their influences on axon growth, is largely unknown.
To study the glial cell, especially the function of astrocyte heterogeneity in neuron behavior, we established a new method by co-culturing high purified dorsal root ganglia neurons with glial cells obtained from the rat cortex. By this technique, we were able to directly compare neuron adhesion and axon growth on different astrocytes subpopulations under the same condition.
In this report, we give the detailed protocol of this method for astrocytes isolation and culture, dorsal root ganglia neurons isolation and purification, and the co-culture of DRG neurons with astrocytes. This method could also be extended to other brain regions to study cellular or regional specific interaction between neurons and glial cells.

In this protocol, we described a new method to study the influence of glial cell heterogeneity on axon growth with an in vitro co-culture system. Rat cortical glial cells were cultured to confluence and cocultured with highly purified rat dorsal root ganglia neurons. Different glial cell influence on neurons adhesion and axon growth was compared directly in the same culture. This method provides a new way to directly study the glial cell heterogeneity influence on neuron adhesion and axon growth.

새 Coculture 방법을 사용 액슨 성장에 대한 연구 Glial 세포 이질 영향

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is ...

연구

신경과학

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

포유류의 CNS에서 시신경의 절개 후 실험에 대한 노는 방법

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

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 ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

병아리 배아에서 분리 및 해리 감각 뉴런의 문화

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

등쪽 뿌리 신경절 사출 및 지느러미 루트 감각 축삭의 재생을위한 모델로 부상 크러시

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

주변에 Neuroregeneration 중앙 신 경계를 연구 하 고 대 한 Vivo에서 초파리 부상 모델

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

의학

Sheath-Preserving Optic Nerve Transection in Rats to Assess Axon Regeneration and Interventions Targeting the Retinal Ganglion Cell Axon

Retinal ganglion cell (RGC) axons converge at the optic nerve head to convey visual information from the retina to the brain. Pathologies such as glaucoma, trauma, and ischemic optic neuropathies injure RGC axons, disrupt transmission of visual stimuli, and cause vision loss. Animal models simulating RGC axon injury include optic nerve crush and transection paradigms. Each of these models has inherent advantages and disadvantages. An optic nerve crush is generally less severe than a transection and can be used to assay axon regeneration across the lesion site. However, differences in crush force and duration can affect tissue responses, resulting in variable reproducibility and lesion completeness. With optic nerve transection, there is a severe and reproducible injury that completely lesions all axons. However, transecting the optic nerve dramatically alters the blood brain barrier by violating the optic nerve sheath, exposing the optic nerve to the peripheral environment. Moreover, regeneration beyond a transection site cannot be assessed without reapposing the cut nerve ends. Furthermore, distinct degenerative changes and cellular pathways are activated by either a crush or transection injury.
The method described here incorporates the advantages of both optic nerve crush and transection models while mitigating the disadvantages. Hydrostatic pressure delivered into the optic nerve by microinjection completely transects the optic nerve while maintaining the integrity of the optic nerve sheath. The transected optic nerve ends are reapposed to allow for axon regeneration assays. A potential limitation of this method is the inability to visualize the complete transection, a potential source of variability. However, visual confirmation that the visible portion of the optic nerve has been transected is indicative of a complete optic nerve transection with 90-95% success. This method could be applied to assess axon regeneration promoting strategies in a transection model or investigate interventions that target the axonal compartments.

This protocol describes an optic nerve transection that preserves the optic nerve sheath in rats. Hydrostatic pressure from microinjections into the optic nerve generate a complete transection, allowing for sutureless reapposition of the transected optic nerve ends and direct targeting of the axonal compartment in a transection model.

Retinal ganglion cell (RGC) axons converge at the optic nerve head to convey visual information from the retina to the brain. Pathologies such as ...

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.

In the coculture system of dorsal root ganglia and Schwann cells, myelination of the peripheral nervous system can be studied. This model provides experimental opportunities to observe and quantify peripheral myelination and to study the effects of compounds of interest on the myelin sheath.

쥐 등쪽 뿌리 신경절 외식편과 슈반 세포의 공동 배양에서 말초 축삭의 시험관 내 수초화

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, ...

Coculturing of Retinal Ganglion Neurons with Olfactory Ensheathing Glia

Source: Portela-Lomba, M., et al. <a href="https://app.jove.com/v/61863">Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration.</a> J. Vis. Exp. (2020)
This video demonstrates the procedure to isolate retinal ganglion neurons (RGNs) from a rat&#39;s retina and coculture them with olfactory ensheathing glia (OEG) cells. This method allows for studying the neuro-regenerative potential of OEGs after neural injury.

망막 신경절 뉴런과 후각 외피 신경교세포의 공동 배양

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Co-culture and Interaction Studies

Co-culturing of a Dorsal Root Ganglion Explant with Schwann Cells for Neuron Myelination

Source: Blusch, A. et al., <a href="https://app.jove.com/v/64768">In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells.</a> J. Vis. Exp. (2023)
This video demonstrates the coculturing model of a dorsal root ganglion explant and Schwann cells. In coculture conditions, ascorbic acid promotes the differentiation of Schwann cells into myelinating forms. Myelinating Schwann cells wrap the axons multiple times to create a myelin sheath, which is essential for nerve function.

Neuron Myelination을 위한 Schwann Cells와 Dorsal Root Ganglion Explant의 공동 배양

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Schwann cell culture

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