This work was supported in part by National Institutes of Health (NIH) Grants R01 GM-41514 (to M.D.C.), R39 GM-048071 (to I.P.), and R01 NS-074987 (to T.E.L.) and the National Multiple Sclerosis Society (NMSS) Collaborative Center Research Award CA1058-A-8 (to C.M.W., T.E.L. and M.D.C.), NMSS Grant RG4925, NIH Training Grant T32-AI-060573 (to M.L.G.), NMSS Postdoctoral Fellowship FG 1960-A-1 (to J.G.W.), and funding from the George E. Hewitt Foundation for Medical Research (M.P.M.).

참고 : 윤리 문 : 동물 처리를위한 프로토콜은 캘리포니아, 얼바인, 프로토콜 # 2010년부터 2943년까지 대학의 기관 동물 관리 및 사용위원회 (IACUC)에 의해 승인되었다. 척수 1. 제거 <ol><li> 챔버를 안락사에 ~ 100 % 액체 이소 플루 란, USP 적신 종이 타월을 놓고 위에 마른 종이 타월을 배치합니다. 마우스가 이소 플루 란 접촉되지 않도록 마른 종이 타월 위에 챔버에 마우스를 놓고 챔버가 포함되어 있는지 확인합니다. 마우스가 안락사 될 수 있도록 호흡 중단 후, 1 분 정도 기다립니다. </li><li> 마우스를 안락사 보장하기 위해 목의 척추 절개를 수행합니다. 이 척수에 손상을 줄 수 있습니다 자궁 경부 전위를 수행하지 마십시오. </li><li> 젖은 모발, 70 % 에탄올로 마우스 스프레이. </li><li> (약 자궁 얇은 판 1에서 척추를 노출 C1를 마우스의 뒷면에서 머리와 피부를 제거하는 날카로운 미세 가위를 사용하여) 성례의 얇은 판 4 (S4)에. </li><li> # 10 메스 블레이드를 사용하여, 근육과 지방에서 분리하는 척주의 왼쪽과 오른쪽을 절개. 이 척수의 제거가 훨씬 쉽게 만들 것입니다. </li><li> 옵션 : 곡선 루어 rongeurs 추가 살을 멀리 퍼하는 데 사용할 수 있습니다. </li><li> 톱니 모양의 Graefe 씨 집게와 척수 척추의 상단을 누른 상태에서 척수를 만지지 않도록 조심하면서, 최대 C1에 노출 된 척추에 곡선면 티타늄 곡선 Vannas 가위를 삽입합니다. </li><li> 모든 길 오른쪽에있는 티타늄 곡선 Vannas 가위를 밀어 하나의 작은 상처를합니다. 작은 크런치 소리가 들리지와 가위가 척추를 잘라으로 생각해야한다. 코드의 맨 왼쪽에이 컷을 반복합니다. 너무 빨리 이동하거나이 가위 코드의 손상 위험하므로 절단 너무 큰 확인하지 않도록주의하십시오. </li><li> 하나의 얇은 판은 척추의 측면이 절단되면 집게로 해제 할 수 있어야합니다. R보유 척수 척추를 뒤로 당겨 계속 S4까지 각각의 얇은 판에 대해이 단계를 EPEAT. 참고 :이 이식 사이트로 삭감되면 이식 사이트 위의 척추 가능성이 붙지 않습니다. 바로 이식 사이트 아래에 시작하는 절차를 진행합니다. </li><li> 옵션 : 이식 사이트이며, 측정 및 ~ 이식 사이트에 20mm 주동이의 꼬리 잘라 곳을보​​고 아래 척추를하다. 참고 : 더 일반적으로 15mm보다 더 멀리 이동하지 않는 이식의 NPC로 필요하지 않습니다 NPC의 이식. 척수의 필요한 양은 이식 세포 유형의 실험 및 이동 특성에 의존 할 것이다. 이식 사이트에 등거리 주동이의 꼬리 컷을 만들기 이미징 동안 더 쉽게 위치 할 수있는 이식 사이트를 가능하게 할 것이다. </li><li> # 11 블레이드와 메스를 사용하여 조심스럽게 오른쪽에있는 신경을 절감하고 주동이의 끝에서 시작하여 척수의 복부 측면의 왼쪽. 이 w병이보다 용이하게 복부 척추로부터 제거 될 수 있도록 척수. 참고 : 빨리 완료는 (1.7-1.11 단계), 척수가 건조하지 않습니다. 그러나, 1X PBS 촉촉한 유지하는 코드로 투여 될 수있다. </li><li> 마우스 전환 등 척수가 아래로 향 마우스를 저장할 수 있습니다. 조심스럽게 폐쇄 톱니 모양의 Graefe 씨의 집게를 사용하여 척수를 껍질. 척수 새김없이 신중 척수 손상 단일 편에서 해제 될 수 있도록 나머지 신경절을 잘랐다. </li><li> 척수는 RPMI-1640에 있는지 확인하고 2P 현미경에 전송 얼음에 놓습니다. </li></ol> 이미징 척수 2. 준비 <ol><li> 아가로 오스 척수를 포함하기 <ol><li> 촬영하기 전에 얼음에 냉장 RPMI-1640에 고립 된 척수를 유지합니다. </li><li> 아가 로스 (저 겔화 온도)을 칭량하고 1X PBS 5 ml의 5 % 용액을 제조 하였다. AG를 용해 15 초 동안 5 % 아가로 오스 용액을 마이크로파일어나 37 ° C로 냉각 솔루션을 보자. </li><li> 복부면이 위를 향하도록 파라 필름 한 장에 배치하여 척수를 준비합니다. </li><li> 피펫 척수의 복부 측면에 걸쳐 5 % 아가로 오스 솔루션의 약 5 ml의. 아가 로스를 실온으로 냉각시켜 고화하자. 전체 응고 약 5 분 소요됩니다. </li><li> 22mm 사각형 커버 슬립에 조직 접착제의 가벼운 코트를 적용합니다. </li><li> 파라 필름에 복부 측면을 배치, 임베디드 척수 전환. 몸 윗면은 지금 직면한다. 등쪽에 커버 슬립을 준수하고, 접착제를 응고 RMPI의 아가로 오스 포함 된 척수 / 커버 슬립 준비 잠수함. 과잉 면도날을 사용하여 아가로 오스를 응고 제거합니다. </li></ol></li><li> 현미경 무대에서 척수를 장착 <ol><li> 커버 슬립의 바닥에 바셀린을 적용합니다. 이것은 재관류 동안 제제를 안정화한다. </li><li> 척수 제제를 놓고25X 찍기 목표를 향해 위로 향하도록 복부 측면과 현미경 무대에 잘 이미징. 맞춤형 이미징 아니라 약 20mm, 깊이 50mm 길고, 곳곳에서 50mm입니다. </li><li> 이미지 산소 예열과 준비 (37 ° C), superfusing 동안 혈청이없는 (95 : 이산화탄소 carbogen : 5 산소) RPMI-1640 배지를. <ol><li> 전 따뜻한 물을 욕조에 37 ° C에 미디어 그것은 미디어 병에 튜브를 삽입하여 튜브 펌프에 연결합니다. </li><li> 챔버에 튜브의 연결에서 37 ° C 히터 장치에서 미디어 온도​​를 유지합니다. Superfuse 미디어 우물가를 통해 3 ㎖ / 튜브 펌프 (그림 1B)에 연결된 튜브를 사용하여 분. </li></ol></li></ol></li></ol> 복부 척수 3. 2P 이미징 <ol><li> 현미경 설정 <ol><li> 900 nm의 조정 사파이어 레이저 : 카멜레온 울트라 티를 사용하여 Thy1-EYFP 척수의 이미지를 획득. 에서의 레이저 파워를 감쇄&lt;5 % 표본은 최소 광독성 (16)을 보장합니다. 촬상 안정을 보장하기 위해 드리프트 및 조직 및 세포 손상을 방지하기 위해 단일 ​​인라인 히터 용액을 이용하여 일정한 37 ℃에서 산소 - 관류 RPMI-1640을 환류시켜두면 온도를 유지한다. </li><li> eGFP는 및 EYFP 형광 신호를 분리하기 위해, 의도적으로 시인성을 향상시키기 위해 녹색 발광을 분할, 세 개의 채널로 2P 발광을 분리하는 일련의 520 nm의 단일 에지 이색 및 560 nm의 단일 에지 이색 성 빔 스플리터를 배치했다. 참고 :이 채널은 파란색 - 녹색 (발광 &lt;520 ㎚), (520-560 nm의) 녹색 - 노란색과 빨간색 (발광&gt; 560 ㎚)라고합니다. 광전자 배증 관은 각 채널의 광을 방출 감지합니다. </li></ol></li><li> 관심의 위치 이미징 영역 <ol><li> 접안 렌즈와 시야 광원을 이용하여, 기준점을 설정하는 척수의 복부 가장자리에 침지 대물 초점. </li><li> 반드시 주변 광원을 끄고 자상 있는지 확인레이저 셔터를 열어 2P 여기에 가려움. </li><li> 가능하면 관심 분야에 대한 조직을 검색 할 때, 최종 이미지를 획득 할 때보다 낮은 해상도 설정, 디지털 줌없이 높은 볼륨, 높은 스캔 속도를 사용합니다. 관심 영역에 대한 조직 검색이 시스템의 전형적인 설정은 다음과 같습니다 해상도 : 256 픽셀; 볼륨 : X = Y 600 μm의 = 600 μm의, Z = 0-300 μm의. </li><li> 척수의 배쪽 가장자리 근처 EYFP 축삭을 준수하십시오. 콜라겐 (파란색)에서 두 번째 고조파 신호는 척수 조직의 가장자리에 밝은 될 것입니다. EYFP 축삭 그냥 콜라겐에서 2 차 고조파 신호에 지느러미 찾아 EYFP 신호는 녹색 - 노란색 채널에 보여집니다. </li><li> 척수 준비 (14)의 길이 방향의 중앙에 이식 사이트를 찾습니다. eGFP는-NPC 이식 다음 마우스 일일를 들어, 깊은 조직으로 z 평면에서 빔 경로를 중심으로 이식 사이트를 찾습니다. 이식 사이트 위스콘신LL 동물 사이에 다소 차이가 있지만, 일반적으로 복부 가장자리에서 ~ 200 μm의 거리에 있습니다. 1 일 후 이식 후 쥐의 복부 및 측면 가장자리에 가까운, 백색질에 eGFP는-의 NPC의 클러스터를 관찰합니다. </li></ol></li><li> 최종 이미지를 획득 <ol><li> 512 픽셀의 이미지 해상도를 취득; 볼륨 : X = 270 Y μm의 = 212 μm의, 및 z = 100 μm의 사용 Slidebook 6 소프트웨어. 2.5 μm의 단위로 순차적 초점면을 인수하여 Z-스택을 컴파일합니다. </li><li> 척수에있는 모든 마이그레이션 객체의 신속한 정량을 보장하기 위해 오프셋 적절한 인터레이스와 양방향 검사를 수행합니다. 척수는 약 1 프레임 / 초입니다 내에서 이상적인 획득 프레임 속도가 휴대 전화 속도를 결정하는 것을 확인합니다. 주 : 이미징 설정은 포착 프레임 율, 해상도 및 촬상 촬상 볼륨으로 개별 사용자 선호도로 변경 될 수있다. 큰 이미지 볼륨은 일반적으로 GIV에서 획득을 더 오래 걸립니다욕실 해상도. </li><li> 이러한 비트 평면 Imaris 7.7 같은 영상 분석 소프트웨어를 자르기, 매끄러운 및 의사 색조 Slidebook 이미지 파일을 분석. Slidebook와 Imaris의 자동화 된 프로세스를 사용하여 연속적인 영상 볼륨을 빗질하여 최종 시간 경과 비디오를 생성합니다. </li></ol></li></ol>

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The authors have no competing interests.

실시간은 그대로 조직의 2P 영상은 탈수 초 마우스 척수에 이식 다음 NPC의 반응 속도 및 상호 작용을 조사하기 위해 필요합니다. 생체 내에 2P 촬상 일반적 살아있는 쥐 척수의 등쪽에 휴대 동성을 결정하는 데 사용되며, 탈수 초성 질환 17-19에서 척수 탈수 초를 연구하기 위해 사용되었다. 이식의 NPC가 2P 현미경을 사용하여 현장에서 이미지에 너무 깊이 자리 잡고 복부 백질로 이동하기 때?...

실험자가 면역 뇌척수염 (EAE) 및 neuroadapted 마우스 형 간염 바이러스 (MHV)과 두개 내 감염을 포함하여 탈수 초화의 마우스 모델은, 분자 경로 및 질병과 관련된 세포의 상호 작용을 연구하는 훌륭한 도구입니다. 그들은 주도하고 FDA의 효과는 주로자가 면역 및 염증 일의 중단을 대상으로, 제약 요법을 승인 지원했다. 내생 재유 수화가 실패하지만 일단, 현재 승인 된 치료는 효과적으로 중추 신경계의 탈수 초 병변을 복구하지 않습니다. 따라서, 질병의이 단계에서 복구에 초점을 맞춘 치료는 만성 증상과 삶의 질 향상 등의 개선을 위해 중요하다. 최근, 신경 전구 세포 (NPC들)은 염증과 탈수 초화의 영역을 대상으로 잠재적 인 재생 치료 양상으로 최전선에왔다. 몇몇 연구 endogeno을 유도하는 능력의 NPC를 강조우리는 재유 수화 및 재유 수화 2-8에 직접 참여한다. NPC의 재유 수화가 직접 관여되어 있기 때문에, 다음과 같은 내인성 세포 이식 그들의 동력학 및 상호 작용을 이해하는 것이 필수적이다. 이식 후, NPC가 다음 rostrally 및 이식 사이트 5,9에 꼬리 쪽 상대 백질 손상의 영역에 배쪽으로 이동한다. 이주의 반응 속도는 환경 단서에 대한 응답에 차이가; 비 손상된 척수에 이식 NPC가 손상된 척수 6에 이식 NPC들보다 더 큰 속도를 가지고있다. 철새 기간이 지나면 그대로 척수 6 손상된 척수 상대적으로 더 높은 비율로, NPC가 광범위하게 확산 옮겼다. 마지막으로, NPC의 대부분은 희소 돌기 아교 세포로 분화 및 직접 재유 수화의 4,6,9을 시작합니다. 탈수 초 병변은 복잡하고 여러 단계에서 세포의 다양한 인구를 포함 할 수 있습니다ctivati​​on. 예를 들어, 활성화 된 다발성 경화증 (MS) 병변은 활성화 T 세포, M1의 미세 아교 세포 및 M1 식세포하지만 만성 자동 MS 병변은 주로 약간의 염증 세포 (10-13)와 반응성 성상 세포로 구성 될 수의 현저한 인구를 포함 할 수있다. 때문에 이펙터 세포의 다양성, 탈수 초화의 마우스 모델에서 이광자 (2P)는 촬상 병변 내의 로컬 셀룰러 상호 작용의 이해를 돕기 위해 매우 유용한 도구이다. MS 많은 널리 사용 MS 연구 모델에서, 병변의 대부분 의한 병변의 깊이와 척수 높은 지질 함량 척수, 생체 내에의 2P 촬상 접근 할 수없는 영역의 복부 측에 위치한다. 복부 척수를 따라 병변 내에서 이러한 문제와 연구 세포 - 세포 상호 작용을 회피하기 위해 우리는 생체 2P 이미징 준비 6 간단한을 개발했다. 이 연구는 보여 이전의 방법 간행물, 최대 다음MHV 유도 (14)의 탈수 초화 JHMV 균주 다음 마우스의 척수에 증강 된 녹색 형광성 단백질 (EGFP) 발현의 NPC 이식 절차. 5 주 된 쥐 JHMV에 감염 14 일 후 감염에 흉부 레벨 9에 eGFP는-NPC가 이식된다. 여기에 제시된 프로토콜은 생체 아가로 오스 준비를하기 위해선, 척수를 추출, 향상된 노란색 형광 단백질 (EYFP) 발현 축삭과 이미지 이식 eGFP는-NPC 상호 작용하는 방법에 대한 자세한 단계를 제공합니다. 뉴런 특이 Thy1 EYFP 프로모터하에 발현 된 마우스는이 절차 (15)에 사용 하였다. 만 축삭의 일부는 개별 축삭을 이미징 유용하게 EYFP을 표현한다. 여기에서 우리는 칠일 이식 후에서 제거 척수를 보여; 그러나, 척수는 식후 어느 시점에서 추출 될 수있다. 우리가 손상된 축색의 NPC와 상호 작용을 표시하는 동안, 우리는 프로토콜 형광 마커 유전자와 조합하여 사용할 수있다다른 세포 유형 마우스 척수 걸쳐 일어나는 세포 상호 작용의 다수를 알아보고자 하였다.

<table border="0" cellpadding="0" cellspacing="0" width="858">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Name of Reagent/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:381px;">Isoflurane, USP</td>
			<td style="width:175px;">Piramal Critical Care, Inc</td>
			<td style="width:136px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Fine scissors</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td style="width:136px;">14060-09</td>
			<td>sharp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">scalpel blade #10</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td style="width:136px;">10010-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">scalpel handle</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td style="width:136px;">10003-12</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Luer rongeurs</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td style="width:136px;">16001-15</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Graefe forceps</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td style="width:136px;">11052-10</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Vannas scissors</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td style="width:136px;">15615-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">scalpel blade #11</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td style="width:136px;">10011-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">RPMI-1640</td>
			<td style="width:175px;">Gibco</td>
			<td style="width:136px;">12-115F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">agarose, low gelling temperature</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:136px;">A9414-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Parafilm</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:136px;">13-374-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Vetbond (tissue adhesive)</td>
			<td style="width:175px;">3M</td>
			<td style="width:136px;">1469SB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">22 mm square cover slip</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:136px;">12-547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">25x dipping objective, 1.1 NA</td>
			<td style="width:175px;">Nikon</td>
			<td style="width:136px;">CFI Apo LWD 25XW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Single inline solution heater</td>
			<td style="width:175px;">Warner Instruments</td>
			<td style="width:136px;">64-0102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">520 nm single-edge dichroic beam splitter</td>
			<td style="width:175px;">Semrock</td>
			<td style="width:136px;">FF520-Di02-25x36</td>
			<td>Brightline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">560 nm single-edge dichroic beam splitter</td>
			<td style="width:175px;">Semrock</td>
			<td style="width:136px;">FF560-FDi01-25x36</td>
			<td>Brightline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">photomultiplier tubes</td>
			<td style="width:175px;">Hamamatsu</td>
			<td style="width:136px;">R928</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">C/L variable-speed tubing pump</td>
			<td style="width:175px;">Masterflex</td>
			<td style="width:136px;">77122-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">digital thermometer</td>
			<td style="width:175px;">Comar Instruments</td>
			<td style="width:136px;">3501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Chameleon Ultra Ti:Sapphire laser&#160;</td>
			<td style="width:175px;">Coherent</td>
			<td style="width:136px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Slidebook 6 software</td>
			<td style="width:175px;">3i</td>
			<td style="width:136px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Imaris 7.7 software</td>
			<td style="width:175px;">Bitplane</td>
			<td style="width:136px;">N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

two-photon imaging of cellular dynamics in the mouse spinal cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

<html> 외식 척수 촬상 프로토콜 척수 내의 형광을 시각화하는데 이용 될 수 있지만, 우리는 대표적인 결과 EYFP-축삭-eGFP는 NPC와 상호 작용을 보여준다. 첫째, 우리는 그림 1A에 포함 된 복부 척수 준비를 보여줍니다. 다음으로, 우리는 그림 1B에서 2P 현미경 설정 및 주요 구성 요소를 보여줍니다. (2) 복부 척수 내에서 단일 Z-스택에 eGFP는 및 EYFP 형광을 보여?...

Watch this Scientific Journal Video about Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord at JoVE.com

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

마우스 척수 이미징을위한 새로운 생체 준비. 이 프로토콜은 척수 걸쳐 라이브 세포 상호 작용의 두 광자 이미징을 허용한다.

마우스 척수의 세포 역학의 두 광자 이미징

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

two-photon-imaging-of-cellular-dynamics-in-the-mouse-spinal-cord

Research

JoVE Journal

Neuroscience

10.1K 조회수.  University of California, Irvine.  마우스 척수 이미징을위한 새로운 생체 준비. 이 프로토콜은 척수 걸쳐 라이브 세포 상호 작용의 두 광자 이미징을 허용한다. 

비디오: Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

두 광자 현미경을 사용하여 마우스 척수의 생체내 이미징에

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

연구

신경과학

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

두피 뉴런에서 생체 내 두 - 광자의 영상은 경험에 의존 분자 변경

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

종의 척추 상공 회의소를 주입하는 절차<em&gt; 생체</em&gt; 마우스 척수의 영상

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Diffusion Imaging in the Rat Cervical Spinal Cord

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a diagnostic and prognostic tool in cases of disease or injury. Diffusion weighted imaging (DWI), is sensitive to the thermal motion of water molecules and allows for inferences of tissue microstructure. This report describes a protocol to acquire and analyze DWI of the rat cervical spinal cord on a small-bore animal system. It demonstrates an imaging setup for the live anesthetized animal and recommends a DWI acquisition protocol for high-quality imaging, which includes stabilization of the cord and control of respiratory motion. Measurements with diffusion weighting along different directions and magnitudes (b-values) are used. Finally, several mathematical models of the resulting signal are used to derive maps of the diffusion processes within the spinal cord tissue that provide insight into the normal cord and can be used to monitor injury or disease processes noninvasively.

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

쥐 자궁 경부 척수에 확산 이미징

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

동시에 두 광자<em&gt; 생체</em&gt; 마우스 Retrosplenial 코어 텍스에서 시냅틱 입력의 이미징 및 시냅스 대상

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

CLARITY를 사용하여 마우스 척수에서 세로토닌 섬유 이미징 / CUBIC 기술

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Thy1를 사용 하 여 기본 Somatosensory 피 질에서 신경 활동을 이미징-GCaMP6s 유전자 변형 쥐

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

에 비보 듀오 컬러 이미징 혈관 역학 Contusive 척수 상해를 따르는 방법

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

두뇌 조각에서 신경 모 수석에서 2 광자 칼슘 이미지

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

마우스에 있는 Laminectomy 및 척수 창 이식

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...