Name: an approach to enhance alignment and myelination of dorsal root ganglion neurons
Uploaded: 2016-08-24T14:00:00
Description: Watch this Scientific Journal Video about An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons at JoVE.com

저자는 PDMS 기판의 제조에 그의 도움을 에릭 바스 감사드립니다, 유용한 제안 및 DRG 분리의 훈련을위한 미시간 대학에서 박사 마리나 마타의 실험실에서 박사 Shiyong 왕, 박사 마크 Tuszynski 박사 . UC 샌디에고 W. 마리 캄파는 SC 격리를위한 유용한 제안 및 프로토콜. 이 연구는 국립 과학 재단 (CBET 0,941,055 및 CBET 1,510,895), 국립 보건 연구소 (R21CA176854, R01GM089866 및 R01EB014986)에 의해 부분적으로 지원되었다.

세포의 분리에 관한 모든 절차는 미시간 주립 대학에서 기관 동물 관리 및 사용위원회에 의해 승인되었습니다. 사전 뻗어 이방성 표면의 1. 준비 <ol><li> 베이스와 경화제의 1 용액과 조직 배양 접시 (12cm 직경)로 혼합물을 부어 : 10을 섞는다. 4,900 mg의베이스와 총 가교 혼합물에 대한 경화제 490 mg을 사용합니다. </li><li> 기포를 제거하기 위해 20 분 동안 진공 하에서 겔 혼합물을 유지한다. </li>...

<ol>
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P., Papautsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+stability+of+plasma+oxidized+PDMS+surfaces.">Long-term stability of plasma oxidized PDMS surfaces.</a> Conf Proc IEEE Eng Med Biol Soc. 7, 5013-5016 (2004).</li><li>Lopera, S., Mansano, 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=Plasma-Based+Surface+Modification+of+Polydimethylsiloxane+for+PDMS-PDMS+Molding.">Plasma-Based Surface Modification of Polydimethylsiloxane for PDMS-PDMS Molding.</a> ISRN Polymer Science. 2012, (2012).</li><li>Chandra, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organosilicon Materials. , (2013).</li><li>Wu, M. 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사전 뻗어 표면에 축삭 배향을 유도하기 위해 두 가지 중요한 단계가 있습니다 : 1) PDMS 막을와 균일 한 두께의 평면이어야합니다 2) 신경 교세포가 DRG로부터 제거되어야한다. PDMS의 가교제와 혼합하고 오븐에서 경화 후, PDMS 가교 겔 평평한 작업대 위에 보관 및 틸팅을 피하기 위해주의 깊게 취급되어야한다. 플라즈마 처리 후 (세포 부착을 위해 요구되는) 표면의 친수성이 시간에 감쇠 이후 PDMS 막?...

신경 손상에서, 근위 및 원위 신경 그루터기는 종종 인해 병변 사이트에 1-2로 신경 다발의 직접 재배치 방지 할 수있다. 일반적으로, 축삭 책자는 연결의 복잡한 네트워크를 형성 축삭의 높은 순서 정렬 번들로 구성된다. 그러나, 신경 재생이 제대로 구성 축삭 정렬 3-4으로 인해 혼란스러운 과정이다. 따라서, 병변 부위를 해소 축삭 재생의 충분한 수를 생성하는 것이 아니라 축삭 조직 배향을 유도 할 필요가있다. 또한, 탈수 초화는 부상 사이트에서 myelinating 세포의 죽음으로 인한 신경 손상을 수반. 탈수 초화가 강하게 축삭 생존의 도전 능력을 손상 때문에, 탈수 초화를 대상으로 또는 재유 수화를 촉진 치료는 신경 손상 (5) 후 기능 회복을 위해 중요하다. 따라서이 프로토콜의 목적은 두 가지 문제를 해결하는 설계 방법을 예시하는 것이다신경 재생의. 재료의 물리적, 기계적 성질, 상이한 축을 따라 측정 할 때의 차이로 정의되는 표면 이방성은 셀의 배향의 성장 및 이동 6-7 영향을인가하고있다. 지형뿐만 아니라, 이방성을 유도하는 다른 방법이있다. 이전에, 우리는 폴리 디메틸 실록산 (PDMS) 멤브레인의 기계적 정적 사전 스트레치에 의해 유도 된 표면 이방성을 조사 하였다. &quot;큰 부과 작은 변형 SUPER&quot;이론은 세포가 연신 방향 감지 유효 강성은 수직 방향과 다른 것을 예측하고 효과적인 강성 차이는 이방성 8면에 기인한다. 미리 연신 PDMS 막에서 배양 된 중간 엽 줄기 세포 (MSC들)을 적극적 표면 당겨 그 결과 이방성을 감지 전 연신 방향 (9)으로 정렬 할 수있다. 마찬가지로, 이방성 표면 AR기계 사전 뻗어면에서 유망한 것은 후근 신경절의 정렬뿐만 아니라 성장과 수초 (DRG) 10 축삭에 영향을 미칩니다. 여기에서 우리는 축삭 재생 (10)을 향상시키기 위해 정적 사전 뻗어 PDMS 기판에 표면 이방성을 유도하기위한 프로토콜을 제공합니다. 축삭 정렬, 원하는 패턴 위상 기능을 유도하기 위해 정렬 섬유 및 채널 6,11-12를 통해 연락처 지침을 제공하는 것으로보고, 축삭 정렬 11,13을 용이하게 입증되었다. 그러나, 섬유, 채널 및 패턴 등의 위상 기능을 통해 축삭 정렬을 유도하는 기술을보고, 길게과 축삭의 두께를 증가 할 수 없습니다. 반면에, 기계는 점진적 스트레치 (14)의 크기를 증가 길고 두꺼운 축색과 스트레칭 방향으로 정렬 축삭 연신되었다. 그러나, 생체 내에서 모터 구동 장치를 포함하는 것은 아니다실행할 수 있는. 반대로, 정적 프리 연신 유도 이방성은 덜 복잡하고보다 용이하게 생체 내 응용 프로그램의 장래 지지체 디자인에 혼입 될 수있다. 이 프로토콜에서, 고정 전 신장 세포 배양 시스템은 위상 기능이없는 표면 이방성을 유도하기 위해 사용된다. 막 프레임 상에 고정되고 소정의 스트레칭 정도는 연신 단계에 적용 그러자 예비 연신 배양 시스템하는 PDMS 막, 신축성 프레임 및 연신 단계로 구성된다. 최대 21 일간 전 배양 연신 표면에 갓 절연 DRG 신경 세포 축삭 정렬 및 두께에 대해 모니터링된다. 그 후, 슈반 세포 (희주) 수초에 대한 모니터링 정렬 된 축삭와 공동 배양. 사전 스트레칭에 의한 표면 이방성을 통합함으로써 우리는 중간 엽 줄기 세포 및 DRG 뉴런 각각 9 ~ 10의 세포 정렬 분화 및 축삭 정렬 성장을 향상 할 수 있었다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Polydimethylsiloxane &#65288;PDMS)</td>
			<td>Dow Corning</td>
			<td>SYLGARD 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium 1X</td>
			<td>GibcoBRL</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement 50X</td>
			<td>GibcoBRL</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax-I 100X</td>
			<td>GibcoBRL</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumax-I</td>
			<td>GibcoBRL</td>
			<td>11020-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Nerve Growth Factor-7S</td>
			<td>Invitrogen</td>
			<td>13290-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>GibcoBRL</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA/1mM EDTA</td>
			<td>GibcoBRL</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Trevigen</td>
			<td><a href="http://www.trevigen.com/item/2/7/64/220/Cultrex_PolyLLysine/">3438-100-01</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma</td>
			<td>p-6407</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoro-2 deoxy-uridine</td>
			<td>Sigma</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma</td>
			<td>U3003</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Invitrogen</td>
			<td>14170-112</td>
			<td>Isolation Buffer</td>
		</tr>
		<tr>
			<td>Type I Collagenase</td>
			<td>Worthington</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11885</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat inactivated Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH30080.03</td>
			<td></td>
		</tr>
		<tr>
			<td>BPE</td>
			<td>Clonetics</td>
			<td>CC-4009</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Calbiochem</td>
			<td>344270</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone chamber</td>
			<td>Greiner bio-one</td>
			<td>FlexiPERM ConA</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaning/etching system</td>
			<td>March Instruments</td>
			<td>PX-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Thy 1.1 antibody</td>
			<td>Sigma- Aldrich</td>
			<td>M7898</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Complement</td>
			<td>Sigma- Aldrich</td>
			<td>S-7764</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard growth medium</td>
			<td></td>
			<td></td>
			<td>For 500 ml Neurobasal Medium 1X, add 10 ml of B-27 50X, 5 ml of Glutamax-I 100X,</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>2.5 ml of Penicillin/Streptomycin (Penn/Strep), 1 ml of Albumax-I, and 1 &#956;l of NGF-- 7S (50 ug/ml).</td>
		</tr>
		<tr>
			<td>FDU and Uridine stock solution</td>
			<td></td>
			<td></td>
			<td>FDU 100mg in 10ml of ddw (10mg/ml), filter in the hood and divided in 500ul aliquots and store at -20 &#186;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Uridine 5g in 166.7ml of ddw (33mg/ml), filter in hood, divide in 200ul aliquots and store at -20 &#186;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Take 61.5ul of FDU (10mg/ml) and 20.5ul of Uridine(33mg/ml), and add 4918ul of ddw to a final stock concentration,</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>then divide in 1 ml aliquots and store at -20 &#186;C.</td>
		</tr>
	</tbody>
</table>

an approach to enhance alignment and myelination of dorsal root ganglion neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

사전 신장 세포 배양 시스템 DRG 축삭 배향 (10)을 추진. DRG 뉴런 12 일 동안 사전 뻗어 미연 신 표면에 배양 하였다. 축삭은 정렬을 입증하는 β-III-tubulin에 대한 염색 하였다. (2) 문화의 12 일 후 사전 뻗어 미연 신 PDMS 기판에 축삭 방향을 비교 한 그림. 그들은 임의의 배향을 보여 미연 신 PDMS 기판상의 상호 연결된 네트워크를 형성하는 반면, ...

Watch this Scientific Journal Video about An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons at JoVE.com

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

접근 방법은 등쪽 뿌리 신경절 뉴런의 정렬 및 수초를 강화하는

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

The overall goal of this protocol is to enhance axon alignment and myelination of dorsal root ganglion neurons using a static, pre-stretched cell culture system. This method can help answer key question in the nerve tissue engineering field, such as axon alignment and thickness growth. The main advantage of this technique is that this pre-stretched method not only enhances axon alignment but also promotes myelination.To begin this procedure, pour the mixture of base and curing agent into a tissue culture dish. Keep the gel mixture under vacuum for 20 minutes to remove air bubbles. Then place the gel mixture in the oven at 60 degrees Celsius for overnight curing.After the PDMS membrane has cured, treat the surface with oxygen plasma in the plasma cleaning and etching system for three minutes. Subsequently, cut a piece of rectangular membrane from the dish. Fix it onto a stretching frame with the oxygen-treated side facing up.Then place the frame on a stretching stage and evenly stretch it by turning the knob on the stage until it reaches 10%elongation in the longer axis. Next, fix the stretch by tightening the screws on the frame. After that, remove the frame from the stage.Ensure the membrane surface is dry and free of dust before placing a silicone chamber onto the membrane. Allow the sticky side of the chamber to attach tightly to the membrane. Then sterilize the surface with UV for 10 minutes.Afterward, add one milliliter of PLL to the PDMS surface in the chamber within six hours of plasma treatment. Next, incubate it for two hours at 37 degrees Celsius prior to seeding the DRG neurons to enhance cell attachment. In this step, prepare the standard grow medium for the DRG neurons.Prior to isolation, autoclave the isolation equipment and filter reagents. Add five milliliters of isolation buffer into one well of a six-well plate and place the plate on ice. Then prepare 10 milliliters of dissociation medium by adding one milliliter of collagenase A to nine milliliters of 0.05%Trypsin-EDTA.Filter the solution with a 0.22 micrometer filter and place it on ice. Afterward, sacrifice 10 to 12 five to seven days old SD rats and cut away the skin overlying the spinal cord from the back of each one. Remove any excess tissue around the spine.Under a surgical magnifier, make the first incision from the neck then make one cut along the spine on both sides with scissors. Detach the spine from the body of the pup and remove excess muscles. Then cut along the long axis of the spine and use tweezers to open the spine completely to extract the spinal cord.Next, remove the ganglion from the bone pocket. Trim the nerve roots and transfer the ganglia to the ice-cold isolation buffer. Collect approximately 10 to 16 DRGs from both sides.Repeat the dissection procedure for the rest of the pups. Now, transfer the DRGs with isolation buffer into a sterile 15-milliliter tube. Let the tissue settle to the bottom of the tube and gently remove the isolation buffer on top.Then add 10 milliliters of dissociation medium into the tube. Incubate the dissected tissues in the dissociation medium in a 37-degree Celsius water bath for one hour while shaking the tube every five to 10 minutes. After the chemical dissociation, centrifuge the ganglia at four degrees Celsius for five minutes.After five minutes, remove the supernatant, resuspend the pellet in 10 milliliters of standard growth medium and vortex it. Subsequently, centrifuge the dissociated cells once again. Then remove the supernatant, resuspend the pellet in 12 milliliters of standard growth medium and vortex it.Prior to seeding the cells, remove the PLL solution from the chamber. Rinse the PDMS surface with sterile water and air-dry it. After resuspending the cells, let the suspension sit for one to two minutes to allow the debris to settle to the bottom of the tube.Next, add 1.5 milliliters of cell suspension into each stretched chamber and incubate it at 37 degrees Celsius with 5%CO2. To eliminate glial cells at one day in vitro, add 10 microliters or 15 microliters of the FDU-U mixture stock solution to each well. After seven hours, replace this medium with fresh standard growth medium and place the pre-stretched culture device in a 37-degree Celsius incubator in 5%CO2.During the cell culture period, change the medium every two days by replacing half the spent medium with fresh medium. In this procedure, remove the culture medium from the Schwann cells and add five milliliters of 0.05%Trypsin-EDTA into the flask. Incubate the cells at 37 degrees Celsius in 5%CO2 for two to three minutes.Check the cells under the optical microscope using a 10X objective to see if they lift up from the flask. Then add five milliliters of culture medium to the cells in the flask and mix. Subsequently, add the cell suspension to a 15-milliliter centrifuge tube and centrifuge at 20 degrees Celsius for five minutes.Then remove the supernatant and resuspend the pellet in seven milliliters of standard DRG growth medium. Following this, transfer 10 microliters of the cell suspension into a 0.5-milliliter microcentrifuge tube. Mix it with 10 microliters of Trypan blue and then count the cell number with a hemocytometer using a 10X objective.Add the DRG growth medium to dilute the cell suspension to 5, 000 cells per milliliter. Next, remove 0.5 milliliters of the medium from the DRG culture in the stretched chamber and add 0.5 milliliters of Schwann cell suspension to the DRG culture. Culture the cells for one week at 37 degrees Celsius in 5%CO2.Change the medium every two days by replacing half of the spent medium with fresh standard growth medium. After culturing on the stretched and unstretched surfaces for two weeks, purified Schwann cells were added to the chamber and cultured for another week. Shown here is the beta-III tubulin staining for axons on the stretched surface.And this is the overlay of beta-III tubulin and P0 staining on the stretched surface, suggesting the Schwann cells are attached to and likely myelinating the axons. This image shows the beta-III tubulin staining for axons on the unstretched surface. And the overlay of beta-III tubulin and P0 staining on the unstretched surface suggests that the Schwann cells are not attached to the axons.Once mastered, this technique can be done in three to four hours if it is performed properly. While attempting this procedure, it is important to remember to keep the PDMS membrane flat and of homogeneous thickness. Following this procedure, atomizers like pre-stretched microchannels can be performed in order to answer additional questions like axon lens and Schwann cells migration.After its development, this technique will pave the way for researchers in the field of tissue engineering to explore the role of surface anisotropy on nerve regeneration in spinal cord and sciatic nerve. After watching this video, you should have a good understanding of how to induce axon alignment and myelination using pre-stretched device.

Research

JoVE Journal

Neuroscience

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

교양 성인 지느러미 루트 신경절의 신경 세포와 축삭 재생의 유전 연구

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

연구

신경과학

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

루트 신경절 신경 세포와 차별화 된 지방 유래 줄기 세포를 이룬다 :<em&gt; 체외</em&gt; 공동 문화 모델은 주변 신경 재생을 연구하는

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

등쪽 뿌리 신경절 모델 : 암성 신경 침략의 생체 외 모델링에서

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

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

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

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

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Laminotomy 허리 지 루트 신경 절의 액세스 및 돼지에 주입

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

단일 좌 골 신경 및 Cytometry 사용 하 여 단일 마우스에서 지 루트 신경 절에 면역 세포의 분석

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

신경 전달 물질 방출 공부 하 느 루트 중추 절연 및 주 문화

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

성인 마우스 지 루트 신경 절의 Electroporation Vivo에서 조사 포유류 축 삭 재생:

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

전도 실패의 메커니즘을 검사하기 위해 연결된 Sciatic 신경을 가진 생체 내 단일 섬유 기록 및 손상되지 않은 등철 루트 신경절의 사용

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

등갈 뿌리 신경절 대식세포의 신속한 절연

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...