Supported by NIH Grants NS14837, R01 NS38537, R24 HD050838 to Dr. Michael E. Selzer; Shriners Research Grant SHC-85220 to Dr. Michael E Selzer; and Shriners Research Grant SHC-85310 to Dr. Michael I. Shifman. Dr. Ant&#243;n Barreiro-Iglesias was supported by the Fundaci&#243;n Barri&#233; (Spain) and the Xunta de Galicia (Galicia, Spain).

이 프로토콜에 사용되는 모든 재료는 표 1을 참조하십시오. 실험은 템플 대학에서 기관 동물 관리 및 사용위원회에 의해 승인되었습니다. 1 동물 <ol><li> 야생형 애벌레 바다 칠성 장어 (Petromyzon 스 marinus L.)를 얻어, 10 - 메인에서 델라웨어 강 (펜실베니아) 또는 스트림 지류에서 미시간 호수를 공급 스트림에서 - (7 세 4)의 길이는 14 센티미터. </li><li> 16 ° C에서 50갤런 담수 탱크 (100) 동물 - 실험실에서, 50 그룹의 유충을 유지한다. 선 자갈 인치와 탱크 애벌레에서 굴을하기 위해, 물은 숯 여과하고 그들 칠성 장어하기 전에 염소 및 기타 불순물을 제거하기 위해 최소 48 시간 동안 공기 돌을 조절한다. 사용 일까지, 자신의 암성 재활용 동물, 먹이를 할 필요는 없다. </li></ol> 2 완전 척수코드 절개 <ol><li> 척수 수술하기 전에 다음과 같은 조성으로 심고 링거 용액 제조 : 110 mM의 염화나트륨, 2.1 mM의 KCl을 2.6 mM의 염화칼슘, 1.8 mM의 MgCl2를, 10 mM 트리스 완충액을; pH가 7.4. </li><li> 척수 절개를 수행하려면 작은 용기에 유충을 배치 얼음 차가운 링거 용액 1 % tricaine 메탄으로 마취합니다. </li><li> 마취 대신 일단 반 실 가드 (실리콘) 가득 배양 접시에있는 동물과 하나가 꼬리에, 동물의 주둥이에 하나씩 배치 (수술 위치에있는 동물을 잡아 4 곤충 핀 (0.15 mm 직경)를 사용 그리고이 다섯 번째 아가미의 수준에서). 거의 링거 용액으로 가기까지 배양 접시를 채 웁니다. 실체 현미경을 이용하여 수술을 수행하는 동안 얼음에 유충으로 요리를 넣습니다. </li><li> 척수 절개에게 등쪽을 수행하려면, 정중선, 5 번째의 레벨 메스 (# 11)와 세로 절개아가미는 척수를 시각화합니다. 초기에 피부가 위로 향하게 척수 이상에서 알 수있는 때까지 근육 조직이 매우 부드럽게 절단 메스 블레이드의 선단을 삽입함으로써 개방된다. 패션이 곤충 핀에서 후크와 척수 절개를 수행하는 동안 몸의 벽이 열려 유지하는 데 사용할. </li><li> 완전히 가위 (8 Castroviejo #)를 이용하여 제 5 아가미의 수준에서 척수를 쪼개다 척수에 수직으로 위를 삽입한다. 척수가 완전히 한 깨끗한 잘라 가로로해야합니다. 척수가 완전히 가로로되어 있음을 확인하기 위해 실체 현미경 척수 절단 단부를 시각화. 그리고, 한 쌍의 집게 (# 5)를 사용하여 절개 후 척수의 절단 단부를 재조정. </li><li> 척수 절개 미세 집게 한 쌍의 지느러미 몸 벽에 상처를 닫고 상처 t 수 있도록 1 시간 동안 얼음에 유충을 넣어 수행 한 후오 건조. 얼음 및 유충 사이 필터 종이 조각을 넣고 심고 링거 용액에 담가. 낮은 온도는 건조 따라서 감염을 방지 할 수 있도록 공기에 상처를 허용 동시에 살아있는 동물을 유지한다. </li><li> 그런 다음 24 시간 동안 얼음 냉수 작은 탱크 유충 (1 조 당 한 유충)를 전송합니다. </li><li> 행동면 부상의 사이트에 더 이동 꼬리가 없음을 확인하기 위해 척추 절개 후 병변 애벌레 일일를 검사합니다. 유충 (5 번째 아가미의 수준에서) 부상의 사이트에만 머리의 주동이를 이동할 수 있습니다 경우 척수 절개가 완료된 것으로 간주됩니다. 이 경우는 현재 23 유생 (실온에서 담수와 탱크 (1 조 당 한 유충)으로 전송 될 수있다 ° C)는 그들이 선택한 시점에 복구 할 수 있습니다. </li><li> 회복 기간 중 매일 2-3일 물을 변경합니다. </li></ol> 3 뇌 해부 및 준비현장 하이브리드 화에 대한 뇌 <ol><li> 부상 후 원하는 시점에서 애벌레 바다 칠성 장어와 장소를 다시 마취 상기와 같이 뇌 해부를 수행하기 위해 링거 용액과 페트리 접시에 그들을 핀. </li><li> 뇌를 해부하려면 먼저 콧 구멍과 뇌의 장기 사이의 메스 횡 절개를합니다. 여기에서 한 쌍의 집게로 잡고 Castroviejo 위의 쌍을 이용하여 뇌를 둘러싸고있는 조직을 절단하기 시작한다. </li><li> 뇌가 노출되면, 한 쌍의 집게를 이용하여 맥락총를 제거하고 뇌 신경을 동물의 머리를 확산하고 노출시키는 후크를 사용한다. 이어서, Castroviejo 가위와 척수 횡 절단. 뇌를 해부 두개골 신경을 잘라 집게의 또 다른 쌍을 사용하는 동안 집게 한 쌍의 척수를 잡습니다. </li><li> 절개 후, 실리콘의 작은 조각에 뇌를 넣고이 기능을 삽입하여 위치를 잡고척수 후각 전구 및 하나의 요법 핀입니다. 그런 다음, 후방 및 가위로 지느러미 중간 선을 따라 뇌의 cerebrotectal commissures을 절감하고 측면 날개의 판을 편향과 4 곤충 핀 (그림 2)와 실리콘에 평평하게 고정. </li><li> 회에 3 시간 동안 방 temparature로 고정 인산염 완충 생리 식염수에 4 % 파라 포름 알데히드 (산도 7.4)를 포함하는 튜브에 뇌와 실리콘의 조각을 전송합니다. </li><li> 전술 한 바와 같이 고정 후 현장 하이브리드 화에 wholemount을 위해 뇌를 사용 (스웨인 외., 1994). 제어 또는 허위 조작 동물의 뇌 부상 동물의 뇌와 병렬로 항상 실행해야합니다. wholemounted 애벌레 뇌 때문에 평평하고 얇은 모양과 인해 뇌가 상당히 투명하게 수초 (10)의 부재에 현장 하이브리드 화에 의해 연구 될 수있다. </li></ol>

<ol>
	<li>Rodicio, M. C., Barreiro-Iglesias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lampreys+as+an+animal+model+in+regeneration+studies+after+spinal+cord+injury.">Lampreys as an animal model in regeneration studies after spinal cord injury.</a> Rev Neurol. 55, 157-166 (2012).</li><li>Davis, G. R., McClellan, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extent+and+time+course+of+restoration+of+descending+brainstem+projections+in+spinalcord-transected+lamprey.">Extent and time course of restoration of descending brainstem projections in spinalcord-transected lamprey.</a> J Comp Neurol. 344, 65-82 (1994).</li><li>Jacobs, A. J., Swain, G. P., Snedeker, J. A., Pijak, D. S., Gladstone, L. J., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+neurofilament+expression+selectively+in+regenerating+reticulospinal+neurons.">Recovery of neurofilament expression selectively in regenerating reticulospinal neurons.</a> J Neurosci. 17, 5206-5220 (1997).</li><li>Shifman, M. I., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+netrin+receptor+UNC-5+in+lamprey+brain+modulation+by+spinal+cord+transection.">Expression of the netrin receptor UNC-5 in lamprey brain modulation by spinal cord transection.</a> Neurorehabil Neural Repair. 14, 49-58 (2000).</li><li>Barreiro-Iglesias, A., Laramore, C., Shifman, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sea+lamprey+UNC5+receptors+cDNA+cloning,+phylogenetic+analysis+and+expression+in+reticulospinal+neurons+at+larval+and+adult+stages+of+development.">The sea lamprey UNC5 receptors cDNA cloning, phylogenetic analysis and expression in reticulospinal neurons at larval and adult stages of development.</a> J Comp Neurol. 520, 4141-4156 (2012).</li><li>Shifman, M. I., Yumu, l. R. E., Laramore, C., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+repulsive+guidance+molecule+RGM+and+its+receptor+neogenin+after+spinal+cord+injury+in+sea+lamprey.">Expression of the repulsive guidance molecule RGM and its receptor neogenin after spinal cord injury in sea lamprey.</a> Exp Neurol. 217, 242-251 (2009).</li><li>Busch, D. J., Morgan, 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=Synuclein+accumulation+is+associated+with+cell-specific+neuronal+death+after+spinal+cord+injury.">Synuclein accumulation is associated with cell-specific neuronal death after spinal cord injury.</a> J Comp Neurol. 520, 1751-1771 (2012).</li><li>Shifman, M. I., Zhang, G., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+death+of+identified+reticulospinal+neurons+after+spinal+cord+injury+in+lampreys.">Delayed death of identified reticulospinal neurons after spinal cord injury in lampreys.</a> J Comp Neurol. 510, 269-282 (2008).</li><li>Swain, G. P., Jacobs, A. J., Frei, E., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+in+situ+hybridization+in+wholemounted+lamprey+brain+neurofilament+expression+in+larvae+and+adults.">A method for in situ hybridization in wholemounted lamprey brain neurofilament expression in larvae and adults.</a> Exp. Neurol. 126, 256-269 (1994).</li><li>Bullock, T. H., Moore, J. K., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+myelin+sheaths:+both+lamprey+and+hagfish+lack+myelin.">Evolution of myelin sheaths: both lamprey and hagfish lack myelin.</a> Neurosci Lett. 48, 145-148 (1984).</li><li>Cohen, A. H., Kiemel, T., Pate, V., Blinder, J., Guan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+can+alter+the+function+outcome+of+spinal+cord+regeneration+in+larval+lampreys.">Temperature can alter the function outcome of spinal cord regeneration in larval lampreys.</a> Neuroscience. 90, 957-965 (1999).</li></ol>

The authors declare that they have no competing financial interests.

여기에서 우리는 애벌레 바다 칠성 장어의 완전 척수 절개 후방 뇌 해부를 수행하기 위해 상세한 프로토콜을 제시한다. 이 절차는 원위치 혼성화에 전체 실장 뇌에 의한 척수 손상 후 척수 식별 돌출 뉴런 간의 유전자 발현의 차이를 분석한다. 절차에서 중요한 단계는 부드럽게 한 쌍의 유충의 주둥이를 터치하여 24 시간 후 stereomicrocope 하에서 척수의 절단 단부를 관찰함으로써 제어되고 확인 될 ?...

포유 동물에서 척수 손상 (SCI)는 부상 축삭은 외상 영역을 통해 재생과 적절한 대상에 다시 연결하지 않기 때문에 부상의 사이트 아래 기능의 영구적 인 손실로 이어지는 치명적인 조건이다. 포유류는 달리, 칠성 장어는 완전한 척수 손상 후 운동력을 복구. 1 흥미롭게 칠성 장어 때문에 그들의 큰 크기 2,3 (도 1)의 뇌 제제 마운트 전체 36 척수에서 개별적으로 식별되어 뉴런 돌출 세트를 가지고 . 이러한 척추-돌출 뉴런은 모두 높은 수준의 완전한 척수 절개하여 axotomized된다. 우리 그룹 등의 이전의 연구는 다른 일반적으로 손상 부위를 통해 자신의 축삭을 재생하는 동안, 심지어 이러한 뉴런의 일부는 매우 낮은 재생 능력을 보여 SCI 후 기능 회복의 존재 (그들이 &quot;나쁜 재생기를&quot;으로 간주된다) 것으로 나타났다 (그들이 간주된다 &quot;g짐 우드 재생기 &quot;). 2,3이 특성은 칠성 장어에게 차례를 시도 뉴런의 고유 재생 능력의 차이로 이어질 것입니다 좋고 나쁜 재생기 척추 - 투사 신경 세포 간의 유전자 발현의 차이를 연구하는 재미있는 척추 동물 모델을 만드는 같은 외부 환경에서 축삭을 재생. 1 우리가 이전에 보여 주었다이 모델을 사용하여 척추 - 투사 각각 netrin와 RGM의 억제 작용을 매개 UNC5 4,5 및 neogenin, 6 등의 축삭지도 분자 수용체의 낮은 재생 능력 쇼 식 뉴런. 또한,이 방법을 사용하여 우리의 그룹은 단지 좋은 재생기가 손상 후 및 재생 공정 동안에 neurofilaments의 표현의 회복을 보여주는 것으로 나타났다. 최근 부시 모건 7은 나쁜 재생기가 increas를 표시하는 면역에 의해 보여 주었다&quot;나쁜 재생기&quot;척추 - 투사 신경 세포가 서서히 완전한 척수 절개의 5,7,8 후 사망 사실을 저자가 관련되어 부상 후 시누 클레인의 에드 표현. 그래서 완전한 척수 손상의 칠성 장어 모델은 척수 투사 신경 세포 axotomy 후 &quot;나쁜 재생기를&quot;무엇이 이해하는 매우 유용한 모델로 등장했다. 우리는 현장 하이브리드 화에 wholemount 수행 손상 후 원하는 시점에서 완전한 척수 절개 수술 프로토콜과 후방 뇌 해부를 수행하는 우리의 연구를 수행합니다. 본 방법론 기사에서 우리는 애벌레 칠성 장어의 완전 척수 손상 수술의 적절한 성능에 대한 자세한 프로토콜을 제시, 동물의 후속 유지 보수 및 현장 하이브리드 화에 wholemount에 대한 최종 뇌의 해부 및 뇌의 준비. 자세한 프로토콜 퍼가기합니다유생 칠성 장어의 뇌에서 원위치 혼성화 wholemount을 rform는 이전에보고 된 바있다. 9는 또한, 척수 손상 및 뇌 해부 본 프로토콜은 또한 다음, 면역 조직 화학 또는 다른 조직 학적 방법에 대한 뇌를 처리하는 데 사용될 수있다.

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			<td height="85" style="height:85px;width:64px;">Name of the reagent</td>
			<td style="width:83px;">Company</td>
			<td style="width:64px;">Catalogue number</td>
			<td style="width:71px;">Comments (optional)</td>
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			<td height="148" style="height:148px;width:64px;">Tricaine methane sulfonate</td>
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			<td style="width:64px;">TR108</td>
			<td style="width:71px;">Benzocaine saturated solution in PBS for sacrifice</td>
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			<td height="85" style="height:85px;width:64px;">Scalpel #3</td>
			<td style="width:83px;">Fine Science Tools (FST)</td>
			<td style="width:64px;">10003-12</td>
			<td style="width:71px;"></td>
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			<td height="85" style="height:85px;width:64px;">Blades for scalpel: #11</td>
			<td style="width:83px;">Fine Science Tools&#160;</td>
			<td style="width:64px;">10011-00</td>
			<td style="width:71px;"></td>
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			<td height="85" style="height:85px;width:64px;">Castroviejo scissors #8</td>
			<td style="width:83px;">Fine Science Tools&#160;</td>
			<td style="width:64px;">15002-08</td>
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			<td height="190" style="height:190px;width:64px;">Forceps #4 &#38; #5</td>
			<td style="width:83px;">Dumont, Switzerland</td>
			<td style="width:64px;">Roboz RS4955</td>
			<td style="width:71px;">#4 for dissection of Spinal cord; #5 for stripping menninges</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Dissecting Microscope</td>
			<td style="width:83px;">Olympus</td>
			<td style="width:64px;">SZ51</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="42">
			<td height="63" rowspan="2" style="height:63px;width:64px;">Sylgard</td>
			<td rowspan="2" style="width:83px;">Dow Corning Co.</td>
			<td rowspan="2" style="width:64px;">184</td>
			<td rowspan="2" style="width:71px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="168">
			<td height="189" rowspan="2" style="height:189px;width:64px;">Insect pins 0.15, 0.20 mm</td>
			<td rowspan="2" style="width:83px;">Austerlitz</td>
			<td rowspan="2" style="width:64px;">No catalogue #</td>
			<td rowspan="2" style="width:71px;">0.15 mm for pinning brain and spinal cord; 0.20 mm for the body</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:64px;">7 ml HDPE Scintillation Tubes with Caps</td>
			<td style="width:83px;">Fisher Scientific</td>
			<td style="width:64px;">03-337-1</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:64px;">Paraformaldehyde 16%</td>
			<td style="width:83px;">Electron Microscopy Science (EMS)</td>
			<td style="width:64px;">19210</td>
			<td style="width:71px;">Dilute to 4% in PBS</td>
		</tr>
	</tbody>
</table>

complete spinal cord injury and brain dissection protocol for subsequent wholemount in situ hybridization in larval sea lamprey

완전 척수 손상 후 처음 바다 칠성 장어는 절개의 수준 이하로 마비된다. 그러나, 몇 주 후에 운동력을 복구하고,이 propriospinal 축삭 및 뇌간으로부터 돌출-척수 축삭의 재생 단거리 (몇 mm)을 동반한다. 36 대 식별 척추 - 투사 신경 세포 중 일부는 좋은 재생기가 있고 다른 사람은 나쁜 재생기입니다. 이 신경 세포는 쉽게 wholemount CNS 준비에 식별 할 수 있습니다. 선호하는 신경 세포 고유의 메커니즘을 이해하거나 척추 동물의 중추 신경계에 손상 후 축삭 재생을 억제하기 위해, 우리는 좋고 나쁜 재생기 간의 유전자 발현의 차이를 결정하는 방법과 표현은 척수 절개에 의해 영향을 받는다. 이 논문은 미세한 비전 아래 완전 척수 transections을 생산하고, BR을 준비하고, 신선한 물 탱크에서 주택 애벌레에 대한 기술과 최근 변화 성인 바다 칠성 장어를 보여줍니다아인 및 현장 하이브리드에 대한 척수 wholemounts. 간단히, 동물은 16로 유지됩니다 &#160; C를 °와 칠성 장어의 벨소리에 1 %의 벤조 카인에 마취. 척수 등쪽 접근 방식을 통해 홍채 절제술 가위로 가로로하고, 동물은 23 ° C에서 신선한 물 탱크에 복구 할 수 있습니다. 현장 하이브리드의 경우, 동물 reanesthetized하고 뇌와 코드가 지느러미 접근 방식을 통해 제거했다.

이 방법을 사용할 때 얻을 수있는 결과의 예를 들어, 식별 척수-돌출 제어의 신경 세포와 이주의 neogenin 성적 증명서의 발현을 보여주는 wholemounted 뇌의 대표 이미지는 병변 애벌레 바다 칠성 장어는 그림 2에 나타내었다 게시 할 수 있습니다. 독자는 완전한 척수 절개 및 자세한 사항은 바다 칠성 장어의 신경 세포를 돌출 식별 척수의 재생 능력 후 neogenin의 표현 사이의 관계를보고 이?...

Watch this Scientific Journal Video about Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey at JoVE.com

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

칠성 장어는 완전 척수 손상 후 운동을 복구 할 수 있습니다. 그러나 일부 척추 - 투사 뉴런 좋은 재생기하고 다른 사람은 없습니다. 이 문서는 완전한 척수 transections 생산 및 현장 하이브리드에 대한 wholemount 뇌와 척수를 준비, 주택 바다 칠성 장어 유생 (최근 변화 성인)에 대한 기술을 보여줍니다.

이후 Wholemount을위한 완벽한 척수 손상 및 뇌 해부 프로토콜<em&gt; 제자리에서</em&gt; 애벌레 바다 장어의 하이브리드

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

complete-spinal-cord-injury-brain-dissection-protocol-for-subsequent

Research

JoVE Journal

Neuroscience

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

9.8K 조회수.  University of Edinburgh.  칠성 장어는 완전 척수 손상 후 운동을 복구 할 수 있습니다. 그러나 일부 척추 - 투사 뉴런 좋은 재생기하고 다른 사람은 없습니다. 이 문서는 완전한 척수 transections 생산 및 현장 하이브리드에 대한 wholemount 뇌와 척수를 준비, 주택 바다 칠성 장어 유생 (최근 변화 성인)에 대한 기술을 보여줍니다. 

비디오: Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

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

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

더블 형광 현장에서</em신선한 두뇌 섹션> 하이브 리다이 제이션

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

측면 유체 타악기 : 마우스의 외상성 뇌 손상 모델

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

닭의 배아 척수 코드 슬라이스 문화 프로토콜

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

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 ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

<em&gt; 전의 VIVO</em&gt; 레이저에 의한 척수 손상 모델은 실시간으로 축삭 변성의 메커니즘을 평가하기 위해

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

Zebrafish의<em&gt; 시츄</em&gt; 척수 척추 감각과 운동 뉴런 전기 생리 녹음 준비

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

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 ...

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 ...

이후 Wholemount을위한 완벽한 척수 손상 및 뇌 해부 프로토콜

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