Name: laminectomy and spinal cord window implantation in the mouse
Uploaded: 2019-10-23T12:30:00
Description: Watch this Scientific Journal Video about Laminectomy and Spinal Cord Window Implantation in the Mouse at JoVE.com

S.E. Lutz는 그랜트 KL2TR002002 및 일리노이 대학 시카고 의과 대학 창업 기금에 따라 국립 번역 과학, 국립 보건원, 국립 연구소의 지원을 받고 있습니다. 사이먼 알포드는 RO1 MH084874에 의해 지원됩니다. 이 콘텐츠는 전적으로 저자의 책임이며 반드시 NIH의 공식 견해를 나타내는 것은 아닙니다. 저자는 TG eGFP에 대한 컬럼비아 대학 의료 센터의 신경학과에서 Dritan Agalliu 감사합니다: 클라우딘-5 마우스, 과학적 토론, 수술 프로토콜 및 이미징 응용 프로그램의 개발에 대한 통찰력. 저자는 캘리포니아 대학의 신경 생물학 및 행동학과의 Sunil P. Gandhi에게 감사하고, 어바인은 입체 적 장치 및 동물 온도 컨트롤러의 첫 번째 프로토 타입을 설계하고, 수술 프로토콜에 대한 논의, 2 광자 현미경 검사법에서 훈련. 저자는 또한 수술 입체 현미경을 사용자 정의에 도움을 스티브 피켄스 (W. Nuhsbaum, Inc.) 감사, 론 리핀스키 (고래 제조) 입체 부품을 가공.

모든 실험은 일리노이 대학, 시카고 기관 동물 관리 및 사용 위원회 프로토콜을 따릅니다. 이것은 터미널 절차입니다.
1. 시약 준비
<ol><li>인공 대뇌 척수액 (aCSF)을 준비하여 125 mM NaCl, 5 mM KCl, 10 mM 포도당, 10 mM HEPES, 2 mM MgCl2·6H2O, ddH2O. 멸균 필터에서 2mM CaCl2/2H 2O를 포함하고 개별 사용 별표에서 동결합니다. 사용 전에 수조에서 aCSF를 39?...

<ol>
	<li>Weinger, J. G., 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=Two-photon+imaging+of+cellular+dynamics+in+the+mouse+spinal+cord.">Two-photon imaging of cellular dynamics in the mouse spinal cord.</a> Journal of Visualized Experiments. (96), (2015).</li><li>Davalos, D., Akassoglou, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+the+mouse+spinal+cord+using+two-photon+microscopy.">In vivo imaging of the mouse spinal cord using two-photon microscopy.</a> Journal of Visualized Experiments. (59), (2012).</li><li>Farrar, M. J., Schaffer, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+procedure+for+implanting+a+spinal+chamber+for+longitudinal+in+vivo+imaging+of+the+mouse+spinal+cord.">A procedure for implanting a spinal chamber for longitudinal in vivo imaging of the mouse spinal cord.</a> Journal of Visualized Experiments. (94), (2014).</li><li>Fenrich, K. K., Weber, P., Rougon, G., Debarbieux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implanting+glass+spinal+cord+windows+in+adult+mice+with+experimental+autoimmune+encephalomyelitis.">Implanting glass spinal cord windows in adult mice with experimental autoimmune encephalomyelitis.</a> Journal of Visualized Experiments. (82), e50826 (2013).</li><li>Figley, S. A., 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=A+spinal+cord+window+chamber+model+for+in+vivo+longitudinal+multimodal+optical+and+acoustic+imaging+in+a+murine+model.">A spinal cord window chamber model for in vivo longitudinal multimodal optical and acoustic imaging in a murine model.</a> PLOS ONE. 8 (3), e58081 (2013).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nature Methods. 2 (12), 932-940 (2005).</li><li>Liebner, S., 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=Functional+morphology+of+the+blood-brain+barrier+in+health+and+disease.">Functional morphology of the blood-brain barrier in health and disease.</a> Acta Neuropathologica. 135 (3), 311-336 (2018).</li><li>Shen, L., Weber, C. R., Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tight+junction+protein+complex+undergoes+rapid+and+continuous+molecular+remodeling+at+steady+state.">The tight junction protein complex undergoes rapid and continuous molecular remodeling at steady state.</a> Journal of Cell Biology. 181 (4), 683-695 (2008).</li><li>Knowland, 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=Stepwise+recruitment+of+transcellular+and+paracellular+pathways+underlies+blood-brain+barrier+breakdown+in+stroke.">Stepwise recruitment of transcellular and paracellular pathways underlies blood-brain barrier breakdown in stroke.</a> Neuron. 82, 1-15 (2014).</li><li>Lutz, S. E., 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=Caveolin1+Is+Required+for+Th1+Cell+Infiltration,+but+Not+Tight+Junction+Remodeling,+at+the+Blood-Brain+Barrier+in+Autoimmune+Neuroinflammation.">Caveolin1 Is Required for Th1 Cell Infiltration, but Not Tight Junction Remodeling, at the Blood-Brain Barrier in Autoimmune Neuroinflammation.</a> Cell Reports. 21 (8), 2104-2117 (2017).</li><li>Harrison, M., 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=Vertebral+landmarks+for+the+identification+of+spinal+cord+segments+in+the+mouse.">Vertebral landmarks for the identification of spinal cord segments in the mouse.</a> Neuroimage. 68, 22-29 (2013).</li><li>Cupido, A., Catalin, B., Steffens, H., Kirchhoff, F., Bakota, L., Brandt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laser Scanning Microscopy and Quantitative Image Analysis of Neuronal Tissue. , 37-50 (2014).</li><li>Sekiguchi, K. J., 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=Imaging+large-scale+cellular+activity+in+spinal+cord+of+freely+behaving+mice.">Imaging large-scale cellular activity in spinal cord of freely behaving mice.</a> Nature Communications. 7, 11450 (2016).</li><li>Nadrigny, F., Le Meur, K., Schomburg, E. D., Safavi-Abbasi, S., Dibaj, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser-scanning+microscopy+for+single+and+repetitive+imaging+of+dorsal+and+lateral+spinal+white+matter+in+vivo.">Two-photon laser-scanning microscopy for single and repetitive imaging of dorsal and lateral spinal white matter in vivo.</a> Physiological Research. 66 (3), 531-537 (2017).</li><li>Adelsperger, A. R., Bigiarelli-Nogas, K. J., Toore, I., Goergen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+Low-flow+Digital+Anesthesia+System+for+Mice+and+Rats.">Use of a Low-flow Digital Anesthesia System for Mice and Rats.</a> Journal of Visualized Experiments. (115), (2016).</li><li>Damen, F. W., Adelsperger, A. R., Wilson, K. E., Goergen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Traditional+and+Integrated+Digital+Anesthetic+Vaporizers.">Comparison of Traditional and Integrated Digital Anesthetic Vaporizers.</a> Journal of the American Association for Laboratory Animal Science. 54 (6), 756-762 (2015).</li><li>Miyamoto, 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=Selective+COX-2+inhibitor+celecoxib+prevents+experimental+autoimmune+encephalomyelitis+through+COX-2-independent+pathway.">Selective COX-2 inhibitor celecoxib prevents experimental autoimmune encephalomyelitis through COX-2-independent pathway.</a> Brain. 129 (Pt 8), 1984-1992 (2006).</li><li>Muthian, G., 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=COX-2+inhibitors+modulate+IL-12+signaling+through+JAK-STAT+pathway+leading+to+Th1+response+in+experimental+allergic+encephalomyelitis.">COX-2 inhibitors modulate IL-12 signaling through JAK-STAT pathway leading to Th1 response in experimental allergic encephalomyelitis.</a> Journal of Clinical Immunology. 26 (1), 73-85 (2006).</li></ol>

여기에 설명된 방법은 유리 창을 통해 마우스에서 척수의 안정적인 이미징을 허용합니다. 이 방법은 형광 BBB 단단한 접합 단백질을 발현하는 형질전환 eGFP:Claudin5+/- 마우스에서 BBB 리모델링을 평가하기 위하여 적용되었습니다, 그러나 척수에 있는 어떤 형광성 단백질 또는 세포의 연구 결과를 위해 동등하게 잘 적용될 수 있었습니다.
라민 절제술과 척수 안정화를위한 여러 ?...

형광성 단백질을 발현하는 형질전환 동물 모델은 분과내 현미경 검사법과 결합될 때 생물학 및 병리생리학을 해결하기 위한 강력한 플랫폼을 제공합니다. 이러한 기술을 척수에 적용하려면 척수를 이미징을 위해 특수 프로토콜이 필요합니다. 그러한 전략 중 하나는 라민 절제술과 척수 창 이식을 수행하는 것입니다. 현미경 검사법을 위한 이상적인 laminectomy 프로토콜의 주요 특징은 네이티브 조직 구조 및 기능의 보존, 화상 진찰 필드의 안정성, 빠른 처리 시간 및 결과의 재현성을 포함합니다. 특별한 도전은 호흡과 심장 박동에 의해 유도된 운동에 대하여 화상 진찰 필드를 안정시키는 것입니다. 여러 ex vivo 및 생체 내 전략은 이러한 목표를 달성하기 위해보고되었다1,2,3,4,5. 대부분의 생체 내 방법은 척추2,4의 측면을 클램핑하는 것을 포함하고 종종 수술 중 안정성을 위해 경질 금속 장치3,4를 이식하고 다운스트림 이미징 애플리케이션. 척추를 고정하면 잠재적으로 혈류를 손상시키고 혈액 -뇌 장벽 (BBB) 단백질 리모델링을 유도 할 수 있습니다.
이 방법의 목적은 프로토콜의 침략을 최소화하고 결과를 개선하면서 살아있는 마우스에서 광학 이미징에 사용할 수있는 그대로 척수를 만드는 것입니다. 우리는 여전히 강력한 기계적 안정성을 달성 최소 침습 타원형 플라스틱 3D 인쇄 백 플레이트와 결합 한 단일 laminectomy 및 커버 유리 이식 절차를 설명합니다. 백플레이트는 치과 시멘트가있는 전방 및 후방 척추 척추에 직접 부착됩니다. 백플레이트에는 금속 암을 통해 현미경 스테이지에 단단히 부착되는 나사 구멍이 있는 측면 확장 암이 장착되어 있습니다. 이것은 효과적으로 현미경 단계에 온전한 전방 및 후방 척추를 고정하고, 그렇지 않으면 호흡과 심장 박동에 의해 소개될 운동 아티팩트에 기계적 저항을 제공합니다. 이 방법은 흉부 수준 12에서 단일 척추의 하구 절제술에 최적화되어 생체 내 이미징 중 안정성을 위한 대체 전략에 사용되는 클램프를 생략합니다. 절차는 마우스 당 대략 30 분 취하는, 급속합니다.
이 프로토콜은 BBB의 질병 기전을 연구하는데 사용될 수 있다. BBB는 중추 신경계 (CNS)에 대한 매우 선택적 환경을 제공하는 내피 세포, 혈관 평활근, pericytes 및 성상 세포 발 과정으로 구성된 동적 미세 혈관 시스템입니다. 대표적인 데이터는 향상된 녹색 형광 단백질(eGFP):클라우딘-5, BBB 단단한 접합 단백질을 발현하도록 설계된 형질전환 마우스에서 이 프로토콜의 적용을 묘사한다. 제공된 백플레이트 인쇄 파일은 대체 응용 프로그램에 맞게 사용자 지정할 수도 있습니다.

<table>
	<tbody>
		<tr>
			<td>3D printer</td>
			<td>Raise3D</td>
			<td>Pro2</td>
			<td>For printing backplates</td>
		</tr>
		<tr>
			<td>PLA 3D printing filament</td>
			<td>Inland</td>
			<td>PLA+-175-B</td>
			<td>Black plastic 3D printing material</td>
		</tr>
		<tr>
			<td>3D CAD software</td>
			<td>Dassault Systemes</td>
			<td>Solidworks software</td>
			<td>used to design 3D shapes</td>
		</tr>
		<tr>
			<td>3D printer software</td>
			<td>Raise3D</td>
			<td>Ideamaker software</td>
			<td>software used to interface with the 3D printer</td>
		</tr>
		<tr>
			<td>3D printed oval backplate</td>
			<td>custom</td>
			<td></td>
			<td>Stabilizing imaging field</td>
		</tr>
		<tr>
			<td>Surgical dissecting microscope</td>
			<td>Leica</td>
			<td>M205 C</td>
			<td>Equipped with Leica FusionOptics, Planapo 0.63x M-series objective, and gliding stage</td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Leica</td>
			<td>MC170</td>
			<td>HD color camera for visualizing surgical field</td>
		</tr>
		<tr>
			<td>Gliding stage</td>
			<td>Leica</td>
			<td>10446301</td>
			<td>The gliding stage is constructed of two metal plates. The base plate is fixed. The upper plate slides on greased interface to allow rotational and linear movement.</td>
		</tr>
		<tr>
			<td>Surgical station and stabilization fork</td>
			<td>Whale Manufactoring</td>
			<td>custom</td>
			<td>Laminectomy</td>
		</tr>
		<tr>
			<td>SomnoSuite low-flow isoflurane delivery unit</td>
			<td>Kent Scientific</td>
			<td>SS-01</td>
			<td>Surgical anesthesia administration with integrated digitial vaporizer</td>
		</tr>
		<tr>
			<td>Stainless steel 1.5 inch mounting post</td>
			<td>ThorLabs</td>
			<td>P50/M</td>
			<td>For mounting surgical station onto optical table for two-photon imaging</td>
		</tr>
		<tr>
			<td>Counterbored Clamping Fork for 1.5&#34; mounting Post</td>
			<td>ThorLabs</td>
			<td>PF175</td>
			<td>For stabilizing surgical station mount onto optical table for two-photon imaging</td>
		</tr>
		<tr>
			<td>Ideal bone microdrill</td>
			<td>Harvard apparatus</td>
			<td>72-6065</td>
			<td>Thinning bone for laminectomy</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Fisher Scientific</td>
			<td>15-462-10</td>
			<td>Warming saline</td>
		</tr>
		<tr>
			<td>Cautery gun</td>
			<td>FST</td>
			<td>18010-00</td>
			<td>Cauterizing minor bleeds</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Benchmark</td>
			<td>BF11222</td>
			<td>1.9&#8221; x 4.5&#8221; silicone heater with 20&#8221; Teflon leads, 10W, 5V</td>
		</tr>
		<tr>
			<td>K type thermocoupled rectal probe</td>
			<td>Physitemp</td>
			<td>RET3</td>
			<td>Measuring mouse body temperature</td>
		</tr>
		<tr>
			<td>petroleum jelly</td>
			<td>Sigma</td>
			<td>8009-03-8</td>
			<td>Lubricating rectal probe</td>
		</tr>
		<tr>
			<td>Feedback-regulated thermal controller</td>
			<td>custom</td>
			<td>NA</td>
			<td>Commercially available alternatives include the Physitemp TCAT series</td>
		</tr>
		<tr>
			<td>PVA Surgical eye spears</td>
			<td>Beaver-visitec international</td>
			<td>40400-8</td>
			<td>Absorbing blood</td>
		</tr>
		<tr>
			<td>Electric trimmer</td>
			<td>Wahl</td>
			<td>41590-0438</td>
			<td>Trimming mouse fur</td>
		</tr>
		<tr>
			<td>Blade, #11</td>
			<td>FST</td>
			<td>14002-14</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Forceps, #5</td>
			<td>FST</td>
			<td>11254-20</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Forceps, #4</td>
			<td>FST</td>
			<td>14002-14</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Titatnium toothed forceps</td>
			<td>WPI</td>
			<td>555047FT</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Titanium Iris scissors</td>
			<td>WPI</td>
			<td>555562S</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Vetbond tissue adhesive</td>
			<td>3M</td>
			<td>084-1469SB</td>
			<td>Preparing tissue surface for dental acrylic</td>
		</tr>
		<tr>
			<td>Ceramic mixing tray</td>
			<td>Jack Richeson</td>
			<td>420716</td>
			<td>Mixing dental acrylic agent with accelerant</td>
		</tr>
		<tr>
			<td>Orthojet dental acrylic</td>
			<td>Lang Dental</td>
			<td>1520BLK, 1503BLK</td>
			<td>Permanently bonding backplate to tissue</td>
		</tr>
		<tr>
			<td>Small round cover glass, #1 thickness, 3 mm</td>
			<td>Harvard apparatus</td>
			<td>64-0720</td>
			<td>optical window</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>7447-40-7</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Scientific</td>
			<td>50-99-7</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>7365-45-9</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>Fisher Scientific</td>
			<td>7791-18-6</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>Fisher Scientific</td>
			<td>10035-04-8</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Rimadyl</td>
			<td>QM01AE91</td>
			<td>Analgesia</td>
		</tr>
		<tr>
			<td>Bacteriostatic water</td>
			<td>Henry Schein</td>
			<td>2587428</td>
			<td>Diluent for carprofen</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein</td>
			<td>11695-6776-2</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Lactated ringer solution</td>
			<td>Baxter</td>
			<td>0338-0117-04</td>
			<td>Hydration for mouse</td>
		</tr>
		<tr>
			<td>Agarose High EEO</td>
			<td>Sigma</td>
			<td>A9793</td>
			<td>gel point 34-37 degrees C</td>
		</tr>
		<tr>
			<td>Opthalmic lubricating ointment</td>
			<td>Akwa Tears</td>
			<td>68788-0697</td>
			<td>Prevent corneal drying</td>
		</tr>
		<tr>
			<td>MOM Two-Photon Microscope</td>
			<td>Sutter</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

laminectomy and spinal cord window implantation in the mouse

이 프로토콜은 마우스 척수의 생체 내 이미징을 위한 척수 하구 절제술 및 유리 창 이식방법을 기술한다. 통합 된 디지털 기화기는 이소플루란의 낮은 유량에서 마취의 안정적인 평면을 달성하기 위해 활용된다. 단일 척추척추가 제거되고 시판되는 커버 글래스가 얇은 아가로즈 침대에 겹쳐져 있습니다. 3D 인쇄 플라스틱 백플레이트는 조직 접착제 및 치과 시멘트를 사용하여 인접한 척추 척추에 부착됩니다. 안정화 플랫폼은 호흡과 심장 박동에서 모션 아티팩트를 줄이기 위해 사용됩니다. 이 신속하고 클램프가없는 방법은 급성 다중 광자 형광 현미경 검사법에 적합합니다. 대표적인 데이터는 eGFP:Claudin-5 -단단한 접합 단백질을 발현하는 형질전환 마우스에서 척수 혈관의 2광자 현미경 검사법에 이 기술의 적용을 위해 포함된다.

이식된 유리 창과 활력 내 2광자 현미경 검사는 CNS 단백질의 동적 변화를 평가하는 데 유용한 도구를 제공합니다. BBB의 기능적 무결성은 타이트한 접합 단백질의 발현, 세포외 국소화 및이직률7에의해 영향을 받습니다. 이전 연구는 단단한 접합 단백질이 정상 상태8에서신속하고 역동적 인 리모델링을 겪는다는 것을 입증했습니다. 현재...

Watch this Scientific Journal Video about Laminectomy and Spinal Cord Window Implantation in the Mouse at JoVE.com

Laminectomy and Spinal Cord Window Implantation in the Mouse

이 프로토콜은 쥐의 척수에 유리 창을 이식하여 분사 내 현미경 검사법에 의한 시각화를 용이하게 합니다.

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 ...

이 방법은 신경 과학 및 면역학에 있는 질문을 해결하기 위하여 그대로 중앙 신경계 내의 생체 내의 단백질 역학의 검사를 가능하게 합니다. 이 절차의 주요 장점은 이미지 수집에 대한 우수한 모션 안정성, 짧은 수술 시간, 기체 마취에 대한 작업자 노출 감소, 저렴한 사용자 정의 백플레이트입니다. 이 방법의 시각적 데모는 절차의 라미네절제술 단계 동안 밀접하게 근본적인 신경계 조직을 손상시키지 않고 뼈를 제거하기 어렵기 때문에 중요합니다.절차를 시작하기 전에 3D 컴퓨터 지원 설계 소프트웨어를 사용하여 표시된 차원에 대한 모델을 만듭니다. 그래서 우리는 3 차원 개체인 객체를 가지고 있으며, 이것이 우리가 시작할 수 있는 것입니다. 내가 할 수있는 것은 우리가 인쇄 버전에 대한 설정에 넣을 수 있다는 것입니다.그래서 이 편집을 열면 Advanced로 이동하여 이러한 설정을 모두 볼 수 있는 창을 엽니다. 노즐 온도를 섭씨 205도, 침대 온도를 섭씨 45도로 설정하고 인쇄 속도를 3D 프린터에서 초당 45mm로 설정합니다. 0.4 밀리미터 의 뜨거운 엔트 노즐과 0.2 밀리미터 레이어 높이를 사용하여 뒷면 플레이트를 인쇄합니다.그런 다음 인쇄된 백 플레이트를 시각적으로 평가하여 구조적 무결성을 위해 평가합니다. 총 구조적 고장은 인쇄 결함을 나타냅니다. 백 플레이트가 준비되면 수술 용 제지자의 이어 바 사이에 열 패드에 8 -12 주 된 마취 마우스의 머리를 놓고 동물의 눈에 연고를 적용하십시오.발가락 핀치에 대한 반응의 부족을 확인 한 후, 숫자 11 블레이드를 사용하여 하부 흉부 상부 요추 부위에 걸쳐 코달 미드 라인 절개에 1.5 센티미터 로스트랄을 만들고 피부와 배후로부터 피부를 분리하십시오. 집게를 사용하여 피부 아래에 남아있는 투명한 결합 조직을 벗겨 피상적 인 근육을 노출하십시오. 대상 척추의 나머지 깊은 근육과 함께 거품 수술 창으로 근육을 대체합니다.백 플레이트에 대한 좌석을 만들려면 흉부 11의 후방 측면과 흉부 13의 전방 측면에서 근육을 제거하십시오. 필요에 따라 힘줄에서 나머지 근육을 제거하기 위해 집게를 사용하여. 모든 근육이 제거되면 척수를 시각화하고 조작하기에 충분한 공간이 될 때까지 힘줄을 집게로 조심스럽게 잘라냅니다.추간판의 두라 메이트, 반투명 라미나르 뼈, 뼈 아래중앙 피상성 혈관, 전방 방사 동맥이 명확하게 볼 수 있는지 확인한다. 따뜻한 인공 뇌 척수액으로 부위를 적시고 미세 드릴을 사용하여 척수의 긴 축과 평행한 직선 스트로크를 사용하여 라미나르 뼈를 얇게 만듭니다. 집게로 피상적 인 가시 공정을 부드럽게 잡고 척추를 들어 올립니다.뼈는 쉽게 들어 올려야합니다. 네 번의 집게를 사용하여 뼈 조각을 제거하고 수술 창으로 출혈을 제어하고 필요에 따라 부드러운 꾸준한 압력을 제어하십시오. 그런 다음 따뜻한 인공 뇌 척수액으로 조직을 헹구습니다.커버 글래스 이식의 경우, 노출된 코드에 3mm 보로실리케이트 커버 글래스를 부드럽게 발라주세요. 작은 주걱을 사용하여 39도 따뜻하게 데운 아가로즈를 유리 가장자리에 적용하고 모세관 작용이 커버 유리 표면 아래에 아가로즈를 그릴 수 있도록 하십시오. 흉부 레벨 11 및 13 척추 척추에서 그대로 인접한 척추의 노출된 뼈 관절 과정에 조직 접착제를 적용합니다.인접한 힘줄과 횡방향 과정을 통해 라미네절 절제술 부위 주변의 링에 추가 조직 접착제를 적용하십시오. 다음으로, 새로운 주걱을 사용하여 조직 접착제 층에 가속기와 혼합된 치과 시멘트를 적용하고 표지 유리 창을 중심으로 수술장에 백 플레이트를 놓습니다. 여러 개의 얇은 층의 치과 시멘트를 적용하면 백 플레이트 접착력이 강해지지만 치과 시멘트가 건조되기 전에 백 플레이트를 신속하게 배치해야합니다.치과 시멘트가 10 분 동안 치료 할 수 있도록 한 후, 추가 접착제와 뒤의 장소의 내부 베이스와 밑면을 채웁니다. 얇은 층에 치과 시멘트를 적용하면 시멘트가 커버 유리와 아가로즈로 퍼지는 위험을 줄이는 데 도움이 될 수 있으며, 이는 전체 프로토콜을 손상시킬 수 있습니다. 치과 시멘트가 건조되면, 포크 백 플레이트 홀더를 창 위에 적절한 위치에 적용하고 백 플레이트를 백 플레이트 홀더에 나사로 넣습니다.그런 다음 누설 을 테스트하기 위해 뒷면 플레이트에 식염수를 적용합니다. 동물과 수술 플랫폼은 표지 유리 창을 통해 이미징을 위한 2개의 광자 현미경의 광학 테이블로 옮겨질 수 있습니다. 본 대표적인 실험에서 EGFP 양성 클라이넥-5는 혈관 신경총 전체에 걸쳐 형광으로 부착된 단단한 접합 내에서 시각화되었다.Z 프로젝션의 단단한 접합 구조물의 명확한 묘사는 성공적인 라미독, 창 배치 및 백 플레이트 이식 후에 최소한의 XY 이미지 변위가 생성된다는 것을 나타냅니다. 이 절차를 시도하는 동안이 미세 수술이 어렵고 마스터하는 데 시간이 걸릴 수 있음을 기억하는 것이 중요합니다. 그러나 일단 마스터되면,이 수술은 약 30 분 안에 수행 될 수 있습니다.이 절차에 따라, 다른 방법, 두 광자 현미경 검사법과 생체 내 이미징 처럼, 신경 혈관 리모델링 및 척수와 관련 된 다른 질병 프로세스에 대 한 추가 질문에 대답 하기 위해 수행 될 수 있다.

laminectomy-and-spinal-cord-window-implantation-in-the-mouse

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

배아 척수에서 뉴런의 Commissural 해부와 문화

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

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 ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

마우스 척추 조건부 유전자 조작을위한 바이러스 성 벡터의 Stereotaxic 주입

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

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 ...

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 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 ...

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 ...

Extraction and Enzymatic Purification of Microglia from the Mouse Spinal Cord

Rapid and Efficient Enrichment of Mouse Spinal Cord Microglia

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and function of the mammalian central nervous system rely significantly on the activity of resident macrophages known as microglia. Microglia display heterogeneity and multifunctionality, enabling distinct gene expression and behavior within the spinal cord and brain. Numerous studies have explored cerebral microglia function, detailing purification methods extensively. However, the purification of microglia from the spinal cord in mice lacks a comprehensive description. In contrast, the utilization of a highly purified collagenase, as opposed to an unrefined extract, lacks reporting within central nervous system tissues. In this study, the vertebral column and spinal cord were excised from 8-10 week-old C57BL/6 mice. Subsequent digestion employed a highly purified collagenase, and microglia purification utilized a density gradient. Cells underwent staining for flow cytometry, assessing viability and purity through CD11b and CD45 staining. Results yielded an average viability of 80% and a mean purity of 95%. In conclusion, manipulation of mouse microglia involved digestion with a highly purified collagenase, followed by a density gradient. This approach effectively produced substantial spinal cord microglia populations.

저자 스포트라이트: 척수 이질성 및 정제에 대한 미세아교세포 연구 

Microglia are regarded as some of the most versatile cells in the body, capable of morphological and functional adaptation. Their heterogeneity and multifunctionality enable the maintenance of brain homeostasis, while also being linked to various neurological pathologies. Here, a technique for purifying spinal cord microglia is described.

마우스 척수 미세아교세포(Mouse Spinal Cord Microglia)의 빠르고 효율적인 농축

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and ...