우리는 기술적 인 도움과 조언을 밥 Nowak (ANDOR 기술)을 감사드립니다. SA-S. 독일 Forschungsgemeinschaft (DFG)의 하이젠 베르크 친목에 의해 지원됩니다. 이 작품은 기금 SE2016/7-1을 DFG의 지원을했습니다.

1. 단일 셀 라벨 β-고를 프로모터의 조절하에 플라스미드 인코딩 GFP 또는 GFP-융합 단백질과 함께 한 세포 단계 배아를 삽입. 개발 과정, GFP는 다음 모자이크 방식으로 표현된다. 여기, 우리는 사용 된 유전자 변형 구조 TG [β-고를 : α-actinin-GFP], 그 β-고를 프로모터의 제어에 따라 α-Actinin-GFP 융합 단백질을 남깁니다.에게 2. 배아 퍼가기 <ol class="lalpha"><li> zebrafish의 배아를 수집 및 28 hpf (준비는 12에 따라)까지 정상적인 조건에서 유지합니다. </li><li> 포셉 (뒤몽 # 5)으로 stereomicroscope에 따라 Dechorionate 배아. </li><li> 석유 젤리 링과 현미경 슬라이드를 준비합니다. </li><li> 1 %의 낮은 녹는 점 아가로 오스 / E3 매체가 10% tricaine 솔루션 준비 : 액체와 균일 한 아가로 오스 솔루션을 얻기 위해 10 ML E3 솔루션에 종기가 100 MG 아가로 오스를, 나누어지는 및 매장자세한 사용을위한 -20 ° C에서 사용하지 않는 아가로 오스 솔루션입니다. 즉시 사용을 위해, 삶은 솔루션은 냉각과 맥스를 유지 보자. 39 ° C. 10% tricaine의 최종 농도를 얻기 위해 tricaine 주식 솔루션을 추가합니다. tricaine와 아가로 오스는 모두 배아 움직임을 방지하기 위해 필요합니다. </li><li> 배아은 옆에 자연스럽게 놓고와 함께, 1 % 석유 젤리 링에서 낮은 용융 아가로 오스 / 10 % tricaine / E3 매체에 하나의 배아를 삽입, 부드럽게 제자리에 배아를 유지하고 볼록을 피하기 위해 coverslip으로 커버 빛을 깰 것이다 표면. 이상적으로, 타겟팅됩니다 배아 근육 조직은 coverslip을 터치해야합니다. </li></ol> 3. 태아 레이저 남긴 상처 <ol class="lalpha"><li> micropoint 레이저와 현미경을 준비 : <ol class="lroman"><li> 현미경과 함께 설정하고 레이저를 사용하는 방법에 대한 자세한 내용은 micropoint 레이저 설명서를 참조하십시오. </li><li> 올바른을 선택435 nm의 파장 레이저 빔의 생성을위한 염료. </li><li> 감쇠 슬라이더를 사용하여 레이저 파워를 조정합니다. 레이저 전원은 단일 펄스 (이 테스트 샘플의 coverslip에 예를 들면 초점)로 유리 표면을 아프게 할 수있을 정도로 회복이 있어야합니다. 이 세포 절제에 필요한 레이저 파워입니다. </li></ol></li><li> 현미경의 접안 렌즈에 설치된 reticle을 사용하여 관심있는 지역이나 셀에 초점을 맞 춥니 다. 20x 목표를 (40x 더 정확한 손상을 수 있지만, 종종 작동 거리가 렌즈에 충분하지 않습니다)를 사용하십시오. </li></ol> 주 1 : 레이저는 초점 비행기 내에서 세포를 손상뿐만 아니라, 위의 셀과 레이저 빔 내에있는 아래에되지 않습니다. 레이저가 최적으로 설정하고 Z 축에서 최대 초점을 조정하는 실험을하기 전에 있는지 확인합니다. 그것은 알기 위해 실험을 수행하기 전에 레이저 부상의 깊이를 결정하는 가장 적합한 셀s이 (가) 정확히 부상 될 것입니다. <ol class="lalpha" start="3"><li> 휘황 - 라벨 세포를 타겟으로하는 경우 정상적인 사용, 또는 형광에 대한 brightfield : 실험을 시각화에 필요한 빛을 설정합니다. </li><li> 부상 원하는 정도에 따라 대상 지역 또는 여러 펄스에 레이저 빛의 펄스를 보냅니다. </li></ol> 4. 남긴 상처 및 복구 분석 <ol class="lalpha"><li> 원하는 경우, 레이저 부상의 절차는 라이브 (예 : Metamorph 소프트웨어의 스트림 인수 옵션을 사용) 6.를 기록 할 수 있습니다 </li><li> 즉시 남긴 상처 후, 배아는 포셉으로 아가로 오스에서 부드럽게 제거 및 복구에 달걀 물에 다시 배치해야합니다. </li></ol> 2 참고 : 근육질 활동 (수영 꿈틀) 골격근 복구를 위해 특히 중요합니다. 배아는 tricaine 노출과 치열한 아가로 오스 퍼가기 때문에 이동할 수 없습니다 때문에, recomme하지 않습니다너무 오래 슬라이드와 coverslip 사이의 배아을 유지하기 위해 nded. <ol class="lalpha" start="3"><li> 표시로 복구 후, 배아는 빛, 형광 또는 공 촛점 현미경에 의해 수정하고 원하는대로 분석 할 수 있습니다. </li></ol>

<ol>
	<li>Charge, S. B., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+regulation+of+muscle+regeneration.">Cellular and molecular regulation of muscle regeneration.</a> Physiol. Rev. 84, 209-238 (2004).</li><li>Steffen, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+runzel+muscular+dystrophy+is+linked+to+the+titin+gene.">The zebrafish runzel muscular dystrophy is linked to the titin gene.</a> Dev. Biol. 309, 180-192 (2007).</li><li>Bassett, D., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+zebrafish+model+of+muscular+dystrophy.">Identification of a zebrafish model of muscular dystrophy.</a> Clin. Exp. Pharmacol. Physiol. 31, 537-540 (2004).</li><li>Kawahara, G., Serafini, P. R., Myers, J. A., Alexander, M. S., Kunkel, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+zebrafish+dysferlin+by+morpholino+knockdown.">Characterization of zebrafish dysferlin by morpholino knockdown.</a> Biochem. Biophys. Res. Commun. 413, 358-363 (2011).</li><li>Behra, M., Etard, C., Cousin, X., Str&#228;hle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+zebrafish+mutants+to+identify+secondary+target+effects+of+acetylcholine+esterase+inhibitors.">The use of zebrafish mutants to identify secondary target effects of acetylcholine esterase inhibitors.</a> Toxicol. Sci. 77, 325-333 (2004).</li><li>Otten, C., 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=Xirp+proteins+mark+injured+skeletal+muscle+in+zebrafish.">Xirp proteins mark injured skeletal muscle in zebrafish.</a> PLoS One. 7, e31041 (2012).</li><li>Roostalu, U., Str&#228;hle, U. <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+molecular+interactions+at+damaged+sarcolemma.">In vivo imaging of molecular interactions at damaged sarcolemma.</a> Dev. Cell. 22, 515-529 (2012).</li><li>Stickney, H. L., Barresi, M. J., Devoto, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somite+development+in+zebrafish.">Somite development in zebrafish.</a> Dev. Dyn. 219, 287-303 (2000).</li><li>Stellabotte, F., Dobbs-McAuliffe, B., Fernandez, D. A., Feng, X., Devoto, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+somite+cell+rearrangements+lead+to+distinct+waves+of+myotome+growth.">Dynamic somite cell rearrangements lead to distinct waves of myotome growth.</a> Development. 134, 1253-1257 (2007).</li><li>Choi, W. Y., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+regeneration.">Cardiac regeneration.</a> Curr. Top. Dev. Biol. 100, 319-344 (2012).</li><li>Stewart, S., Stankunas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limited+dedifferentiation+provides+replacement+tissue+during+zebrafish+fin+regeneration.">Limited dedifferentiation provides replacement tissue during zebrafish fin regeneration.</a> Dev. Biol. 365, 339-349 (2012).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. , 203-253 (1995).</li></ol>

관심 없음 충돌이 선언 없습니다.

레이저로 인한 손상은 zebrafish 배아의 제어 조건 하에서 재생을 연구하기 위해 셀을 흡열하여 원하는 크기의 상처를 가하는위한 강력한 방법입니다. 특히, 세포는 정확하게 타겟팅 할 수 있습니다 (그림 2)와 상해 지역뿐만 아니라 타이밍이 모두 제어 할 수 있습니다. 그 후, 부상 사이트 및 재생 프로세스는 쉽게, 모니터링 기록 (그림 3) 및 (그림 1) 분석됩니다...

<table border="1"><tbody><tr><td> 가열 블록 </td><td></td><td></td><td></td></tr><tr><td> 짝 # 5 포셉 </td><td> 뒤몽 </td><td></td><td></td></tr><tr><td> 유리 슬라이드 </td><td> Menzel </td><td></td><td> 76 X 26mm </td></tr><tr><td> Coverslips </td><td> 로스 </td><td></td><td> 50 X 24mm # 1 </td></tr><tr><td> 석유 젤리 </td><td></td><td></td><td></td></tr><tr><td> Stereomicroscope </td><td> Leica </td><td></td><td> MZFLIII </td></tr><tr><td> Micropoint 레이저 </td><td> ANDOR 기술 </td><td></td><td></td></tr><tr><td> 형광 현미경 </td><td> Zeiss </td><td></td><td> Axioplan II </td></tr><tr><td> Metamorph 소프트웨어 </td><td> 분자 장치 </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> 시약 <ul><li> 낮은 녹는 점 아가로 오스 (# 50081,Lonza) </li><li> Tricaine 재고 솔루션 : 400 밀리그램 Tricaine (# A-5040, 시그마 - 알드리치) / 100 ML DH 2 O pH를 9.0 </li><li> E3 중간 (5 MM NaCl, 0.17 MM KCl, 0.33 MM CaCl 2, 0.33 MM MgSO 4) </li></ul></td></tr></tbody></table>

laser-inflicted injury of zebrafish embryonic skeletal muscle

다양한 실험 방법은 손쥐 근육 영양 장애 모델도 myotoxin 주사 (bupivacaine, cardiotoxin 또는 notexin), 근육 transplantations (denervation-devascularization 유도 재생), 집중적 인 운동을 포함하여 근육 재생을 연구하는 목적으로 근육 부상을 유발하기 위해 마우스를 사용하지만, 한 MDX 마우스 (이 방법의 검토 1 참조) 등. zebrafish에서 유전자 접근법은 근육의 영양 장애의 phenotypes (예 : runzel 2 sapje 3 등) 전시 및 영양 장애 - 관련 유전자의 표현 4 차단 oligonucleotide morpholinos를 안티센스 돌연변이가 포함되어 있습니다. 또, 화학 접근함으로써 결국 근육 영양 장애 5 연결 hypercontraction, 그 결과도 Galanthamine, 화학 화합물 억제 acetylcholinesterase와 예 가능합니다. 그러나, 유전 및 약리 접근 g물리적으로 입은 부상의 정도가보다 쉽게 spatially과 시간적으로 한 통제하는 반면 enerally, 개인 내의 모든 근육에 영향을 미칩니다. 현지화 신체적 부상은 내부 통제 등의 contralateral 근육 평가를 할 수 있습니다. 다른 그룹은 최근 매우 로컬 개별 배아 zebrafish 근육의 플라즈마 막에 손상을하기 위해 두 광자 레이저 (822 nm 정도)의 사용을보고하면서 실제로, 우리는 최근 zebrafish 배아 6 골격근 재생을 공부하는 레이저로 인한 세포 절개를 사용 세포 7. 여기, 우리는 zebrafish 배아의 골격근 세포 손상에 대한 micropoint 레이저 (ANDOR 기술)를 사용하는 방법을보고합니다. micropoint 레이저는 435 nm의 파장을 대상으로 휴대 절제에 적합한 고 에너지 레이저입니다. 레이저는 현미경이 동일한 시간 F에서 사용할 수있는 방식으로 (우리의 설치, Zeiss의 광학 현미경)을 현미경에 연결되어또는 샘플에와 남긴 상처 (brightfield 또는 형광)의 효과를 시각화를위한 레이저 빛을 초점을 맞추고. 레이저 펄스를 제어하기위한 매개 변수 파장, 강도, 그리고 펄스의 수를 포함합니다. 의 투명성과 외부 배아 발달로 인해, zebrafish 배아는 레이저 유도 부상 및 모두 후속 복구를 공부 높은 의무가 있습니다. 1 ~ 2 일 후 수정을, somitic 골격근 세포는 점차적으로 꼬리 8, 9 트렁크에서 somitogenesis의 진행으로 인해 뒤쪽하기 위해 앞의 성숙을 받고있다. 이 단계에서 배아는 자발적으로 경련과 수영을 시작합니다. zebrafish는 최근 성인 zebrafish 10, 11 부상 후 다시 생성 할 수 있습니다 조직의 여러 유형 (등 혈관, neuronal, 심장)으로, 조직 재생 연구의 중요한 척추 모델 생물체로 인식되었습니다.

레이저로 인한 부상은 고정 일일 된 배아에서 수행되었다. 그림 1에 도시 된 바와 같이, 몇 레이저 펄스는 일반적으로 체절 경계 사이에 뻗어있는 손상된, 코일, 고를 풍부한 myofibrils로 쉽게 알아볼 수, 작은 상처를 생성 할 수 있습니다. 레이저 펄스의 더 많은 그러나 대부분의 myofibrils이 파괴되어 크게 손상 체절 블록에서 발생합니다. 그럼에도 불구하고, 우리는 그대로 큰 사람보다 ?...

Watch this Scientific Journal Video about Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle at JoVE.com

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

여기에 제시된 방법은 고 에너지 레이저 펄스와 시간을 통해 이러한 부상과 회복의 후속 분석 라이브 zebrafish 배아의 정확한 부상을 포함한다. 우리는 또한 유전자 하나의 표시 또는 골격근 세포의 그룹이 동안 레이저 광 유도 손상 후 추적 할 수있는 방법을 보여줍니다.

Zebrafish 배아 골격근의 레이저 쏜 부상

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

laser-inflicted-injury-of-zebrafish-embryonic-skeletal-muscle

Research

JoVE Journal

Biology

10.4K 조회수.  Max Delbrück Center for Molecular Medicine. 여기에 제시된 방법은 고 에너지 레이저 펄스와 시간을 통해 이러한 부상과 회복의 후속 분석 라이브 zebrafish 배아의 정확한 부상을 포함한다. 우리는 또한 유전자 하나의 표시 또는 골격근 세포의 그룹이 동안 레이저 광 유도 손상 후 추적 할 수있는 방법을 보여줍니다.

비디오: Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

성인 및 배아 골격근의 Microexplant 문화 및 골격 근육 줄기 세포의 분리

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

연구

생물학

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

골격근에서 Mitochondrial 절연

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

골격근에서 Progenitors의 정화

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

해리 된 준비에서 배아와 애벌레 Zebrafish의 골격 근섬유의 분석

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Zebrafish의에서 콩팥 세포 독소 미세 주입은 급성 신장 손상을 모델링하기

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.

플라스미드 DNA를 마우스 골격근으로 전기천공

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological ...

High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring mitochondrial function is fundamental in biomedical research. Skeletal muscle is a robust source of mitochondria, particularly in animals with a very high aerobic capacity, such as horses, making them ideal subjects for studying mitochondrial physiology. This article demonstrates the use of high-resolution respirometry with concurrent fluorometry, with freshly harvested skeletal muscle mitochondria, to quantify the capacity to oxidize substrates under different mitochondrial states and determine the relative capacities of distinct elements of mitochondrial respiration. Tetramethylrhodamine methylester is used to demonstrate the production of mitochondrial membrane potential resulting from substrate oxidation, including calculation of the relative efficiency of the mitochondria by calculating the relative membrane potential generated per unit of concurrent oxygen flux. The conversion of ADP to ATP results in a change in the concentration of magnesium in the reaction chamber, due to differing affinities of the adenylates for magnesium. Therefore, magnesium green can be used to measure the rate of ATP synthesis, allowing the further calculation of the oxidative phosphorylation efficiency (ratio of phosphorylation to oxidation [P/O]). Finally, the use of Amplex UltraRed, which produces a fluorescent product (resorufin) when combined with hydrogen peroxide, allows the quantification of reactive oxygen species production during mitochondrial respiration, as well as the relationship between ROS production and concurrent respiration. These techniques allow the robust quantification of mitochondrial physiology under a variety of different simulated conditions, thus shedding light on the contribution of this critical cellular component to both health and disease.

Horses have an exceptional aerobic exercise capacity, making equine skeletal muscle an important tissue for both the study of equine exercise physiology as well as mammalian mitochondrial physiology. This article describes techniques for the comprehensive assessment of mitochondrial function in equine skeletal muscle.

말 골격근의 고분해능 불소 호흡 측정

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring ...

탈세포화(decellularization)를 통한 골격근 지방 침투의 정량화

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

저자 스포트라이트: 골격근 지방 침투의 탈세포화 기반 정량화  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.