This work was supported by the Australian Research Council Centre of Excellence for Integrative Brain Function (ARC Centre Grant CE140100007), an NHMRC project grant (#1086643). Prof. George Paxinos is supported by a Senior Principal Research Fellow NHMRC grant (#1043626).

윤리 문 : 동물 과목을 포함하는 모든 절차 (승인 ACEC 번호 14 / 94A입니다) 뉴 사우스 웨일즈 대학의 동물 관리 및 윤리위원회 (ACEC)의 지침을 따르십시오. 투명 마우스 척수 1. 준비 <ol><li> 아이스 콜드 하이드로 겔 용액의 조제 <ol><li> 16 % 파라 포름 알데히드 용액의 조제 (PFA) <ol><li> 70 ml의 미리 예열 증류수 (50-55 °에 C)에 16g 파라 포름 알데히드 분말을 추가하고 파라 포름 알데히드가 용해 될 때까지 가열 자석 교반기에서 교반한다. 참고 :이 솔루션은 이상 55 ° C 열 및 파라 포름 알데히드는 독성이 알고 있어야하는 것을 허용하지 않습니다. </li><li> 실린더에 파라 포름 알데히드 용액을 전송하고 100 ㎖에 증류수를 추가합니다. 4 ℃의 냉장고를 사용하여 파라 포름 알데히드 용액을 냉각. </li></ol></li><li> 증류수 26.25 mL에 125 mg의 VA-044 개시제를 용해 4 용액을 냉각C의 냉장고 °. </li><li> 차가운 5 ㎖ 아크릴 아미드 수용액 (40 %) 1.25 mL의 비스 용액 (2 %) (주의 : 비스 아크릴 아미드 솔루션은 유전 적 결함이나 암을 유발할 수 있고, 독성), 5 ㎖의 10 배 PBS, 12.5 ml의 16 % PFA 용액 및 얼음 VA-044의 개시제 용액. </li><li> 얼음 위의 솔루션을 섞는다. 주 : 전량을 50 ㎖이다. </li></ol></li><li> 마우스 척수의 관류 및 컬렉션 <ol><li> 0.9 % 생리 식염수에 희석 케타민 (80 ㎎ / ㎏) 및 xyzaline (5 ㎎ / ㎏)의 복강 내 주사로 마우스를 마취. 마우스의 투여 량은 생쥐의 경우보다 더 낮을 수있다. </li><li> 마우스가 다리의 피부에 핀치에 응답하지 않습니다 일단 흄 후드에 적합한 플라스틱 표면에, (접착 테이프가 효과가있다) 떨어져 몸에서 마우스의 사지를 고정합니다. </li><li> 가슴 위에 마우스 피부를 잘라 후 뼈 가슴은 가위로 가슴을 노출. </li><li> 25 G 바늘 토륨에 부착하여, 연동 펌프를 사용하여튜브의 E 단부는 심장의 좌심실로 니들을 삽입 약 10 ㎖ / 분의 속도를 피 종료점을 만든 다음 40 ml의 0.9 % 생리 식염수로 세정을 시작 우심방 자르지. </li><li> 완료되면, 35 ml의 차가운 얼음 하이드로 겔 용액으로 마우스를 관류. 참고 : 10 ML의 속도로 관류 / 분 신경 조직의 부종을 방지 할 수 있습니다. </li><li> 블레이드와 뒷면에 피부를 절개하고 척추 옆 근육을 제거합니다. 일측에 척추 아치를 절단하고, 다른 쪽에게 튀기는 후 미세 시저와 척주에서 척수를 취할. </li></ol></li><li> 마우스 척수 지우기 <ol><li> 밤새 4 ℃에서 하이드로 겔 용액을 15 ml의에서 척수를 놓습니다. </li><li> 겔 용액 10 ㎖를 제거하고 5 ㎖ 튜브에 척수 세그먼트 용액의 나머지를 붓는다. 그것이 완전히 찰 때까지 겔 용액 5 ㎖ 튜브를 채우기. </li><li> 5 ㎖ 튜브의 맨 위에 파라 필름의 작은 조각을 스트레칭 후 튜브 목 파라 필름 랩. 참고 : 파라 필름 접촉을 하이드로 겔 솔루션을 확인하고 하이드로 겔 용액과 파라 필름 사이에 거품이 없습니다. </li><li> 37 년 5 ML 튜브를 넣어 &#160; ° C 오븐에서 밤새. 이 겔하게까지 하이드로 겔 솔루션을 관찰한다. </li><li> 오븐에서 5 ML 튜브를 타고 (예 : 삽 1 ML의 피펫 팁 등) 스패너와 튜브에서 젤을 제거합니다. 겔에 거친 조직을 고집하여 척수 조직에서 젤을 제거하고 (겔 조직에 충실하고, 따라서 조직에 의해 제거됩니다) 떨어진 거친 조직을. </li><li> 척수 조직으로부터 겔을 제거 후, ​​쉐이커상에서 24 시간 동안 1X PBS (PH 7.4)와 척수 4 회 반복한다. </li><li> 3.85 g의 우레아 및 385 g의 N, N, N &#39;, N&#39;- 테트라 키스 (2- 히드 록시 프로필) ethylenediam 용해 CUBIC 클리어 용액을 준비5.38 용액에 오프라인은 증류수. 주 : 뜨거운 교반기가 사용됩니다. 분명히하고 실온까지 냉각 후, 용액에 2.31 g의 폴리에틸렌 글리콜 모노 메틸 -p- isooctylphenyl 에테르 / 트리톤 X-100을 추가한다. 참고 :이 세 가지 화학 물질 10 g을 하나의 마우스입니다. </li><li> 면도날 2-3 mm 길이 세그먼트로 coronally 마우스 척수를 잘라 위의 CUBIC 청소 용액 5 ㎖로했습니다. 37 ℃ 오븐에서 진탕 놓고 &#160; 3 일간 C를 °. </li><li> 3 일 후 새로운 하나에 청소 솔루션을 변경합니다. 주 : 상기 용액은 사용 전에 제조한다. </li><li> 2 ~ 3 일 후에 CUBIC 청소 솔루션을 새로 고침 후, 글꼴 8 문자 용지에 조직의 투명성을 확인합니다. 이 투명한 경우, 글자 삭제 조직을 통해 알 수있다. (0.1 % 트리톤-X100)는 조직 4 회 (6 시간마다)을 씻어 청소 용액을 제거하고 PBST의 4를 가하여. </li></ol></li></ol><ol><li> 37 ° C 오븐에서 진탕 기에서 3 일 : 차 항체 용액에서 척수 세그먼트를 품어 (PBST 100 항 세로토닌은 토끼에서 제기, 1 희석). </li><li> 항체 용액을 제거하고 4 PBST를 가하여 (0.1 % 트리톤 X100)는 37 ° C의 오븐에서 척수 세그먼트 4 회 (매 6 시간)을 씻어. </li><li> 37 ° C 오븐에서 진탕 기에서 3 일간 조직을 배양 : PBST 용액을 제거하고 이차 항체 솔루션을 추가 (PBST 100 594 복합 염소 항 - 토끼 IgG의를 1 희석). </li><li> 이차 항체 용액을 제거하고 37 ° C 오븐에서 통에 하루 척수 세그먼트 4 번 씻어 (6 시간마다) PBST 4를 가하여. 다음날 조직 이미징을위한 준비가되어 있습니다. </li></ol> 3. 이미지 <ol><li> PBST 용액을 제거하고, 심지어 조직의 굴절률을 85 % 글리세롤 (4)를 가하여. </li><l난&gt; 조직의 선명도를 확인합니다. 그것은 약 1 시간 분명되면 멀티 포토 형광 현미경 유지 프레임 맞는 크고 얇은 유리 슬라이드 상 조직을 넣어. </li><li> 척수 조직 옆에 85 % 글리세롤을 몇 방울을 넣어 22 X 50mm 유리 커버 슬립이 그것을 커버 슬립. 참고 : 척수의 방향은 이미지가 어떻게 보이는지를 결정한다. </li><li> 다중 광자 형광 현미경의 유지 프레임에 유리 슬라이드를 넣고 빛 경로로 조직을 이동합니다. </li><li> 헬륨 네온 레이저 라인 594 nm의 선택 및 라이브 화상에 양 신호의 밝기를 검사하여 최적의 레벨로 레이저의 세기를 조절한다. </li><li> (물, NA 0.7) 20X 대물 렌즈를 이용하여 척수 조직의 스캔 영역을 선택하고, Z 스택을 만들 준비하고,이 3 μm로 설정된 각 단계의 깊이. </li><li> 20X 미만의 objec Z 스택의 상단부터 하단까지의 조직을 검사적인하고 (오일 중, NA 1.4)를 63X 대물 각각 (스텝 크기는 1 ㎛였다). 3D 소프트웨어 (28)를 이용하여 3 차원 영상을 재구성한다. </li></ol>

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bringing+CLARITY+to+gray+matter+atrophy.">Bringing CLARITY to gray matter atrophy.</a> NeuroImage. 101, 625-632 (2014).</li><li>Ando, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inside+Alzheimer+brain+with+CLARITY:+senile+plaques,+neurofibrillary+tangles+and+axons+in+3-D.">Inside Alzheimer brain with CLARITY: senile plaques, neurofibrillary tangles and axons in 3-D.</a> Acta Neuropathol. 128 (3), 457-459 (2014).</li><li>Zhang, H., Rinaman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+CLARITY+for+visualizing+immunofluorescence+labeling+in+the+developing+rat+brain.">Simplified CLARITY for visualizing immunofluorescence labeling in the developing rat brain.</a> Brain Struct Funct. , (2015).</li><li>Lee, H., Park, J. H., Seo, I., Park, S. H., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+application+of+the+electrophoretic+tissue+clearing+technology,+CLARITY,+to+intact+solid+organs+including+brain,+pancreas,+liver,+kidney,+lung,+and+intestine.">Improved application of the electrophoretic tissue clearing technology, CLARITY, to intact solid organs including brain, pancreas, liver, kidney, lung, and intestine.</a> BMC Dev Biol. 14, 781 (2015).</li><li>Yang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+phenotyping+within+transparent+intact+tissue+through+whole-body+clearing.">Single-cell phenotyping within transparent intact tissue through whole-body clearing.</a> Cell. 158 (4), 945-958 (2014).</li><li>Rosset, A., Spadola, L., Ratib, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OsiriX:+an+open-source+software+for+navigating+in+multidimensional+DICOM+images.">OsiriX: an open-source software for navigating in multidimensional DICOM images.</a> J Digit Imaging. 17, 205-216 (2004).</li></ol>

The authors have nothing to disclose.

이 프로토콜은 결합 된 선명도와 CUBIC 기술로 마우스 척수의 이미지 세로토닌 섬유하는 방법을 보여줍니다 설명했다. 이 청 등. 14 토 메르 외. (15)에 의해 개발 된 수동적 지우기 프로토콜에 비해 빨리 소거 과정을 소개하고 척수 조직이 아니라 클리어시 하이드로 겔에 의해 지원 될 수있다. 청 등. (14)와 토 메르 등. (15)?...

Supraspinal projections are responsible for the modulation of diverse behaviors such as pain perception. One of the projections carrying nociceptive information contains serotoninergic fibers, which originate from the hindbrain raphe and adjacent reticular nuclei1,2. Physiological and pharmacological studies have demonstrated an increased release of serotonin in the dorsal horn of the spinal cord after electrical stimulation of the raphe nuclei in the hindbrain3-5. In the rat and opossum, serotonergic raphespinal fibers have dense terminals, not only in the dorsal horn6-8, but also in the intermediate zone7,9,10, the ventral horn7,11, and even lamina 1012,13. There are no similar studies in the mouse. The present study aimed to map the termination pattern of serotonergic fibers arising from the hindbrain raphe nuclei and their adjacent reticular nuclei in the mouse spinal cord using the recently published CLARITY14 method and its modification - CUBIC15. 
Conventional fluorescence or peroxidase immunohistochemistry of the spinal cord clearly shows the distribution of serotonergic fibers in the gray matter of the spinal cord in 30-40 &#181;m thick cross-sections. However, this approach does not show the continuity of the serotonergic fiber tracts in the white matter and their collaterals in the gray matter. Although the 3D reconstruction of histological sections has advanced our knowledge of fiber tracts, it remains a challenge for histologists and anatomists to follow a single tract due to small distortions in the tissue caused by cutting. To circumvent this obstacle a number of researchers have developed various protocols for making the whole tissue structure transparent, and collecting an image of unaltered tissue in a single video file17-21. So far, the clear, lipid-exchanged, acrylamide-hybridized rigid, imaging/ immunostaining compatible, tissue hydrogel (CLARITY) technique, developed by Deisseroth's group14,15, as well as CUBIC, developed by Susaki et al16 are the most successful. Since the publication of the protocols, many researchers have started using these techniques to investigate various aspects of biological tissues, including, not only the brain22-25, but also the heart, kidneys, intestine, and the lungs26,27. 
By fixing the mouse spinal cord with the hydrogel solution (CLARITY) and clearing with the CUBIC reagents (which is a much faster method than that described by the original CLARITY protocol14,15), a spinal cord tissue block of 2-3 mm long was cleared within two weeks and immunofluorescence staining for serotonin completed in eight days. With just a combination of chemical agents, conventional immunohistochemistry can be used to create an image of individual fiber tracts in a 3D video file in approximately one month.

<table border="0" cellpadding="0" cellspacing="0" width="2081">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:495px;">Photoinitiator VA044</td>
			<td style="width:495px;">Wako</td>
			<td style="width:284px;">va-044/225-02111</td>
			<td style="width:807px;">http://www.wako-chem.co.jp/specialty/waterazo/VA-044.htm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40% acrylamide solution</td>
			<td>Bio Rad</td>
			<td>161-0140</td>
			<td>http://www.bio-rad.com/en-au/sku/161-0140-40-acrylamide-solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2% Bis Solution</td>
			<td>Bio Rad</td>
			<td>161-0142</td>
			<td>http://www.bio-rad.com/en-au/sku/161-0142-2-bis-solution?parentCategoryGUID=5e7a4f31-879c-4d63-ba0b-82556a0ccf1d</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paraformaldehyde</td>
			<td>Sigma</td>
			<td align="right">158127</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sial/158127?lang=en&#38;region=AU</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">urea</td>
			<td>Merck Millipore</td>
			<td align="right">66612</td>
			<td>http://www.merckmillipore.com/AU/en/product/Urea---CAS-57-13-6---Calbiochem,EMD_BIO-66612</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N,N&#8217;,N&#8217;-tetrakis (2-hydroxypropyl) ethylenediamine</td>
			<td>Merck Millipore</td>
			<td align="right">821940</td>
			<td>http://www.merckmillipore.com/AU/en/product/Ethylenediamine-N,N,N&#39;,N&#39;-tetra-2-propanol,MDA_CHEM-821940</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton-X 100</td>
			<td>Merck Millipore</td>
			<td align="right">648462</td>
			<td>http://www.merckmillipore.com/AU/en/product/TRITON&#174;-X-100-Detergent---CAS-9002-93-1---Calbiochem,EMD_BIO-648462</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/s0389?lang=en&#38;region=AU</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,2&#8217;,2&#8217;&#8217;- nitrilotriethanol</td>
			<td>Merck Millipore</td>
			<td align="right">137002</td>
			<td>http://www.merckmillipore.com/AU/en/product/Triethanolamine-(Trolamine),MDA_CHEM-137022</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">serotonin antibody</td>
			<td>Merck Millipore</td>
			<td>AB938</td>
			<td>http://www.merckmillipore.com/AU/en/product/Anti-Serotonin-Antibody,MM_NF-AB938</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 594 conjugate</td>
			<td>Life Technologies</td>
			<td>&#160;A-11012</td>
			<td>https://www.lifetechnologies.com/order/genome-database/antibody/Rabbit-IgG-H-L-Secondary-Antibody-Polyclonal/A-11012</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">multi-photon microscope</td>
			<td>Leica</td>
			<td>Leica TCS SP5 MP STED</td>
			<td>http://www.leica-microsystems.com/products/confocal-microscopes/details/product/leica-tcs-sp5-mp/</td>
		</tr>
	</tbody>
</table>

imaging serotonergic fibers in the mouse spinal cord using the clarity/cubic technique

척수 긴 하강 섬유 운동 통증 지각, 및 다른 동작을 위해 필수적이다. 이러한 섬유 시스템의 대부분의 척수 광섬유 종단 패턴 철저 종 조사되지 않았다. 척수 돌출 세로토닌 섬유, 조직 학적 섹션에 쥐 주머니쥐 연구되었으며 기능적 의미는 척수에서의 광섬유 종단 패턴에 기초하여 도출되었다. 명확성과 CUBIC 기술 개발과 함께,이 광 시스템 및 세로토닌 척 주상 경로의 알려지지 않은 특징을 밝힐 가능성 척수에서의 분포를 조사하는 것이 가능하다. 여기서는 결합 선명도 CUBIC 기술을 사용하여 마우스 척수 세로토닌 섬유 영상화 상세한 프로토콜을 제공한다. 방법은 COMBIN와 조직 겔 용액 설명과 마우스의 관류를 포함시약을 삭제의 ATION. 척수 조직 바로 아래 2 주 클리어하고, 세로토닌에 대한 후속 면역 염색 미만 십일 년에 완성되었다. 다중 광자 형광 현미경으로 조직을 검사하고, 3D 이미지 Osirix 소프트웨어를 사용하여 재구성 하였다.

이 섹션은 명확성과 CUBIC 프로토콜의 조합을 사용하여 투명 마우스 척수 세로토닌 항체 염색의 결과를 나타낸다. 우리는 (또한 비디오 (1) 그림 1 참조) 세로토닌 섬유는 복부 뿔의 복부 부분에서 우위와 척수의 모든 라미에 존재하는 것으로 나타났다. 제어 조직은 긍정적 인 섬유 (결과가 표시되지 않은)하지 않았다. 복부 호른에서 조밀 세로토닌 ?...

Watch this Scientific Journal Video about Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique at JoVE.com

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

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-serotonergic-fibers-mouse-spinal-cord-using-claritycubic

Research

JoVE Journal

Neuroscience

10.4K 조회수.  Neuroscience Research Australia. 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.

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

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

척수 전기 생리학 II : 세포 흡입 전극 제작

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

연구

신경과학

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 ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

애벌레 Zebrafish의 척수 절개

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

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 ...

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.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

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 ...

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 ...

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 ...

Diffusion Tensor Magnetic Resonance Imaging in Chronic Spinal Cord Compression

Chronic spinal cord compression is the most common cause of spinal cord impairment in patients with nontraumatic spinal cord damage. Conventional magnetic resonance imaging (MRI) plays an important role in both confirming the diagnosis and evaluating the degree of compression. However, the anatomical detail provided by conventional MRI is not sufficient to accurately estimate neuronal damage and/or assess the possibility of neuronal recovery in chronic spinal cord compression patients. In contrast, diffusion tensor imaging (DTI) can provide quantitative results according to the detection of water molecule diffusion in tissues. In the present study, we develop a methodological framework to illustrate the application of DTI in chronic spinal cord compression disease. DTI fractional anisotropy (FA), apparent diffusion coefficients (ADCs), and eigenvector values are useful for visualizing microstructural pathological changes in the spinal cord. Decreased FA and increases in ADCs and eigenvector values were observed in chronic spinal cord compression patients compared to healthy controls. DTI could help surgeons understand spinal cord injury severity and provide important information regarding prognosis and neural functional recovery. In conclusion, this protocol provides a sensitive, detailed, and noninvasive tool to evaluate spinal cord compression.

Here, we present a protocol for the application of diffusion tensor imaging parameters to evaluate spinal cord compression.

확산 텐서 만성 척수 압축에서 자기 공명 이미징

Chronic spinal cord compression is the most common cause of spinal cord impairment in patients with nontraumatic spinal cord damage. Conventional magnetic ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

쥐와 마우스 두뇌에 신경 줄기 세포의 동맥 내 납품: 대뇌 허혈에 응용 프로그램

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...