Name: induction of acute skeletal muscle regeneration by cardiotoxin injection
Uploaded: 2017-01-01T13:00:00.000+00:00
Duration: 7 min 39 s
Description: Watch this Scientific Journal Video about Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection at JoVE.com

We thank the Animal House and the Integrated Microscopy Facilities of IGB-CNR. This work has benefited from research funding from the European Community's Seventh Framework Programme in the project ENDOSTEM (Activation of vasculature associated stem cells and muscle stem cells for the repair and maintenance of muscle tissue, grant agreement number 241440), the Italian Ministry of Education-University-Research (MIUR-PRIN2 010-2011) to G.M. and S.B. and PON Cluster IRMI to G.M., and the CARIPLO foundation to G.M. and S.B.

모든 실험 동물 연구를위한 제도적 지침에 엄격한에 따라 실시 및 동물 실험에 관한 법률에 따라 공중 보건, 동물 건강, 영양과 건강의 이탈리아 정부의 식품 안전의 부서에 의해 승인되었다. 경추 탈구 절차 IACUC 또는 이에 상응하는 요구 사항에 따라 기관의 기관에 따라 다를 수 있습니다. 경골 전방 근육 1. Cardiotoxin 주입 <ol><li> 절차를 시작하기 전에 멸균 인산 완충 용액 (PBS) 또는 물에 cardiotoxin 원액을 희석함으로써 cardiotoxin (CTX)의 용액을 10 μM 작업을 준비한다. 주 : CTX는 1 ㎎ / ㎖의 물에 용해된다. 70 μm의 주식 솔루션을 준비합니다. -20 ° C에서 나누어지는 및 저장. 이 프로토콜에 사용 CTX 약 7,000 다 분자량 cardiotoxins의 혼합물이다. 착용하는 것이 좋습니다 :주의이중 장갑, 위생 마스크, 보호 안경 희석 경우에도, 상기 용액과 접촉을 피하기 위해 매우 조심해야 (또는 안전 안경에, 후드에서 작동하는 것이 좋습니다). </li><li> 케타민과 자일 라진 (80 ㎎ / ㎏ 및 / kg 몸 질량의 10 mg)을 또는 승인을 사용할 수 마취제의 복강 내 주사하여 마우스를 마취. </li><li> 마우스 얼굴이 위로, 70 % 에탄올로 뒷다리를 스프레이. 메스의 도움으로 전 경골근 (TA) 근육의 정확한 위치를 시각화하기 위해 무릎 아래 낮은 다리의 앞쪽 부분에서 떨어져 머리를 잘라. 참고 : TA 근육이 뼈에 측면을가는 발목에서 끝나는, 경골의 근위 몸에서 발생한다. 긴 힘줄 (그림 1A 참조) 발목에서 다리로 확장합니다. </li><li> 다시 천천히 플런저를 잡아 당겨 주사기로 솔루션의 ~ 100 μL를 그립니다. </li><li> (기포 제거 수직 주사기 유지바늘과 위쪽). 조심스럽게 아래로 플런저를 끌어와 튜브의 내부에 부착 된 기포가 위쪽으로 올 수 있도록 주사기의 측면을 누릅니다. 천천히 큰 공기 방울을 해제 플런저를 누릅니다. 액체의 몇 방울 바늘을 올 것이다. 이 시점에서, cardiotoxin의 독성 주어, 그것을 분산 튜브로 다시 액체를 픽업하지해야합니다. </li><li> 원하는 투여 량에 도달 할 때까지 주사기로 상기 용액을 철회. 각 TA 근육에 대한 CTX 용액 20 μL를 주입한다. </li><li> 무릎을 향해 건에서 지침과 경골 뼈의 위치에 따라 TA의 중심에 바늘을 삽입합니다. 조교가 얇은 조직이기 때문에, (2/3 mm 깊이 바늘, 약 10 ° ~ 20 °의 각도를 삽입) 너무 깊이하지 않고 주사를 수행하고, 근육 자체 (그림 1A)을 넘어 마십시오. </li><li> 즉시 주사 후 바늘을 꺼내하지만 2 기다리지 말고 - 누출을 방지하기 위해 3들CTX의 나이는 근육에서 삭제합니다. </li><li> 쥐가 마취 때문에, 깨어 사용 된 에탄올 체온을 줄일 때까지 절차 후 (37 ° C에서) 가열 접시에 새장을 배치합니다. </li></ol> 2. 앞정 전방 격리 주 : 근육 실험 조건에 따라 cardiotoxin 주사 후에 여러 시점에서 분리 될 수있다. <ol><li> 자궁 경부 전위로 마우스를 희생. </li><li> 70 % 에탄올로 뒷다리 스프레이. 절차 동안, 동물을 누르고 지지체에 고정 될 수있다. </li><li> (다리 위) 근위 힘줄 레벨에서 피부를 잘라 피부 아래 위의 블레이드를 삽입하고, 무릎의 방향을 위쪽으로 피부를 잘라. 집게의 도움으로, 피부를 위로 당겨 완전히 근육을 노출. 조심스럽게 피부와 관심의 영역에서 머리를 모두 제거합니다. </li><li> 일에 천천히 끼거나 절단하여 근막을 (제거핀셋 팁 또는 경골 (도 1b)의 대향 측) 근육의 극단적 외주 위 해부와 미세한 마이크로. 한 번 깨진 부드럽게 얇은 팁 핀셋의 도움으로 근막을 제거; 기본 근육의 손상을 방지. 주의 : 다른 근육 및 기관과 마찬가지로, TA는 밴드 (다리 깊은 근막) 깊은 피부 아래에 놓여 있고 그 쉽게 기본 근육을 분리 제거 할 필요가 결합 조직의 얇은 판으로 덮여있다. </li><li> 말단 TA 건과 병치되어 다리 위의 신근 심지 건, 시각화합니다. 신근 심지 Longus (EDL) 근육의 힘줄이 약간 TA 건 아래에 있습니다. 성인 쥐의 힘줄은 육안으로 볼 수 있습니다. </li><li> 다음 두 가지 방법 중 하나 다음 TA 근육을 제거합니다 : <ol><li> 머무르고, 미세 마이크로 가위를 해부와 두 힘줄을 잘라 전방 측을 향해을 당겨g 집게와 힘줄. 두 힘줄 별개 근육을 분리 반대 방향으로 부드럽게 당겨. </li><li> 얇은 팁 핀셋의 도움으로, 힘줄 자체 (그림 1C, 위 사진) 아래의 팁을 전달하여 신근 심지 건에서 말단 TA 건을 분리합니다. 부드럽게 기본 근육 (그림 1C, 아래 사진)에서 분리하기 위해 TA 아래 핀셋을 밀어 넣습니다. </li><li> 약간 상승을 유지 조교 건 잘라 근육 아래에있는 핀셋을 잡고 조심스럽게 무릎 (그림 1D, 왼쪽 사진)를 부착 이하 만 지역까지 위쪽으로 당깁니다. 가위 (그림 1D, 오른쪽 사진)와 근육의 가장자리 다음, 무릎 아래 TA를 잘라. 참고 : 근육이 약간 측면에 부착 된 상태를 유지하는 경우를 분리하기 위해 가위를 해부 미세 마이크로를 사용합니다. 참고 : 집게 쉽게 TA 아래에 맞지 않으면,이는 밴드가 완전히 제거되지 않은 것으로 가능하고, 그것은 여전히 근육 위에 표시 될 수있다. 이 경우, 완전히 진행하기 전에 밴드를 제거합니다. </li></ol></li></ol> 3. 신선한 냉동 근육 기술 <ol><li> 트라가 칸트 검 또는 코르크의 조각에 비슷한 접착제 동결 화합물 (예를 들어, 최적의 절삭 온도 OCT () 화합물)의 작은 금액을 놓습니다. 동결하기 전에 뒷면에 코르크의 조각 레이블, 필요한 (그림 2A)의 경우. </li><li> 근육의 횡단면을 얻기 위해, 코르크에 대하여 수직 위치에서 근육을 확실하게 선단 힘줄 넣고 외부 3/4 근육을 떠나는 트라가 칸트 검으로 TA 싱크 (도 2A). </li><li> 알루미늄 캔을 작성 (또는, 금속 컵이나 유리 비이커) 이소 펜탄과. 듀어 포함하여 Liqui에 이소 펜탄의 캔을 일시 중단D 질소는 액체 질소는 캔에 입력 할 수 없습니다. 참고 : 이소 펜탄은 조직의 최적의 동결에 대해 (-150에서 -160 ° C에) 적절한 온도에 도달해야합니다. 일부 흰색 고체 입자가 상기 캔의 바닥에 형성되고 펜탄 약간 점성이되면 정확한 온도에 도달한다. 참고 : 냉동 유물로 이어질 수 있으므로, 고체 입자의 형성 전에 샘플을 동결하지 마십시오. 펜탄 완전히 단단한지면이 해동 될 때까지 한편, 약간 백색 입자가 여전히 존재하는지 확인하고, 실온에서두고. </li><li> Mixter 집게를 사용하여 빠르게 근육 위치 아래를 유지하면서 이소 펜탄에 근육과 코르크를 찍어, 그리고 완전히 액체에 침지 때 코르크를 해제해야합니다. 시료가 떠있는 동안 이러한 방법으로, 코르크의 뒷면은 액체에 표시해야한다. </li><li> 는 sa를 남겨1 분 동안 이소 펜탄에 mple. </li><li> Mixter 집게 (또는 유사한 집게)을 사용하여 근육을 적어도 2 분 동안 액체 질소에 담궈. </li><li> A의 보관 -80 C의 냉동고 ° 직접 표시 알루미늄 호일에 개별적으로 표본을 감싸 즉시 드라이 아이스 (-70 °에 C)에 배치하거나. 주 : 절차를 통하여, 단계 3.4에서 3.7로, 그것은 정확한 온도를 유지하고 (. C 냉동기로 -80 ° 이송 중에 예) 해동로부터 시험편을 방지하는 것이 매우 중요하다. 제어되지 해동 조직 내에 존재하며, 결국 인공물의 형성 또는 근육 섬유의 파괴를 야기하는 물 미결정의 재 냉각의 온도 상승 결과 (섬유의 중심에 큰 화이트 홀로 조직 절편을 가시화). </li></ol> 냉동 근육의 (4)의 저온 단면 <ol><li> 그라 이오 스탯 챔버 온도 t 설정오 -20 -22 ° C까지. </li><li> 이를 최소 30 분간 평형을 허용 시험편 스터브 블레이드 및 저온 유지 챔버 내의 시료를 넣어. </li><li> 시편 스텁에 냉동 화합물 (예를 들어, 10월 화합물)의 작은 금액을 놓고 냉동 화합물 응고 전에 시편으로 코르크를 체험. 한편, 블레이드 홀더 블레이드를 배치합니다. </li><li> 블레이드 홀더 (도 2B)로 시험편 홀더 방향을 제공된 나사를 사용하여 시험편 및 블레이드 정렬. </li><li> 10 μm의 섹션 두께를 설정합니다. 조직을 잘라 진행합니다. 구동 휠의 각각의 차례를 시편 홀더 블레이드를 향해 제어 거리를 진행한다. </li><li> 편광 슬라이드 (슬라이드 당 8 ~ 10 절) (그림 2C)에 섹션을 놓습니다. </li><li> 절편 한 후, -80 ° C에서 슬라이드를 저장합니다. 주 : 시료는 -80 ° C에 냉동 저장 furthe 위해 재사용 될 수있다제대로 처리하고 저장하는 경우 R, 절편. </li></ol> 5. 일상적인 조직 학적 염색 (헤 마톡 실린 및 에오신 얼룩) 주 : 여러 학적 얼룩 분석에 의한 근육의 부분에서 수행 될 수있다. 형태 학적 및 형태 계측 학적 분석을위한 일상적인 조직 학적 염색은 헤 마톡 실린 및 에오신 (H &amp; E) 중크롬산 얼룩입니다. 헤 마톡 실린은 핵에게 깊은 보라색 얼룩. 핵 염색 에오신으로 대조되는 이러한 세포질의 근섬유 같은 산성 구조, 얼룩 (핑크 / 레드). <ol><li> 완전히 자연 건조 10 절 냉동 슬라이드 - 실온에서 15 분, 염색 트레이에 슬라이드를 제출하고, 염색 통에 트레이를 넣어. </li><li> 헤 마톡 실린 1 분 동안 얼룩. </li><li> 10 분은 헤 마톡 실린를 세척하는 상당히 약한 수돗물 제트에서 염색 통을 놓습니다. </li><li> 에오신의 알콜 용액을 사용할 경우 t을 이동 (5 분 95 % 에탄올에 대한 슬라이드를 빠져수성 에오신 솔루션의 경우 자신의 단계). </li><li> 에오신 용액으로 1 분간 Counterstain과. </li><li> 과도한 에오신 용액을 제거하기 위해 물로 씻어. 참고 : 헤 마톡 실린이나 에오신 중 하나와 염색의 최적의 타이밍은 각각의 새 배치에 대해 정의해야합니다. </li><li> 채점 에탄올 시리즈 샘플 탈수 50 % 및 70 % (몇 초간 각각). 신속하게 100 %의 에탄올이 변화를 5 분마다 한 후, 95 % 에탄올에 담가. </li><li> 자일 렌의 2 변경, 각 5 분 이상에 걸린. 이 단계는 화학 후드 하에서 수행되어야한다. </li><li> 슬라이드에 미디어를 장착하고 커버 슬립으로 다루는 몇 방울을 적용, 크실렌 기반 장착 매체 슬라이드를 탑재합니다. 장착 매체는 커버 슬립 슬라이드에 넣어 커버 슬립에 적용될 수있다. <ol><li> 슬라이드와 커버 슬립 사이의 공기 방울을 형성하지 마십시오. 기포를 제거 (설치 후) 수직 슬라이드를 유지하고 과잉 개월을 짜려면부드럽게 무딘 집게 (또는 유사한 무딘 도구)와 커버 슬립 눌러서 unting 매체 기포. </li></ol></li><li> 크실렌이 분석을 진행하기 전에 완전히 증발 할 수 있도록 밤새 몇 시간 동안 후드 슬라이드를 유지하거나. </li><li> 마우스 당 적어도 하나의 전체 근육 부의 겹치지 일련의 이미지를 이용하여 H &amp; E 염색 된 근육의 재생 구간의 형태 학적 및 형태 계측 학적 분석을 수행한다. 20X 배율 밝은 필드 현미경 이미지를 캡처. 주 : 5 마우스의 최소 개수는 측정의 통계적 유의성을 구하는 것을 권장한다. 참고 : 이미지 분석 소프트웨어는 (http://imagej.nih.gov/ij/)를 무료로 다운로드 소프트웨어 ImageJ에,이 목적을 위해 사용할 수 있습니다. </li></ol>

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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 (1), 209-238 (2004).</li><li>Arnold, L., 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=Inflammatory+monocytes+recruited+after+skeletal+muscle+injury+switch+into+antiinflammatory+macrophages+to+support+myogenesis.">Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis.</a> J Exp Med. 204 (5), 1057-1069 (2007).</li><li>Chang, C. C., Chuang, S. T., Lee, C. Y., Wei, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cardiotoxin+and+phospholipase+A+in+the+blockade+of+nerve+conduction+and+depolarization+of+skeletal+muscle+induced+by+cobra+venom.">Role of cardiotoxin and phospholipase A in the blockade of nerve conduction and depolarization of skeletal muscle induced by cobra venom.</a> Br J Pharmacol. 44 (4), 752-764 (1972).</li><li>Meng, H., 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=Tissue+triage+and+freezing+for+models+of+skeletal+muscle+disease.">Tissue triage and freezing for models of skeletal muscle disease.</a> J Vis Exp. (89), (2014).</li><li>Mann, C. 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=Aberrant+repair+and+fibrosis+development+in+skeletal+muscle.">Aberrant repair and fibrosis development in skeletal muscle.</a> Skelet Muscle. 1 (1), (2011).</li><li>Pessina, P., 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=Novel+and+optimized+strategies+for+inducing+fibrosis+in+vivo:+focus+on+Duchenne+Muscular+Dystrophy.">Novel and optimized strategies for inducing fibrosis in vivo: focus on Duchenne Muscular Dystrophy.</a> Skelet Muscle. 4 (1), 7 (2014).</li></ol>

The authors have nothing to disclose.

Here, we describe a protocol to induce acute injury in skeletal muscle (i.e., the intramuscular injection of CTX). It is widely used as a powerful tool to study the dynamics of skeletal muscle regeneration in vivo. CTX injection induces the degeneration of muscle fibers, which is caused by the depolarization of the sarcolemma and the contraction of the fibers12, and triggers the cascade of events that leads to muscle regeneration. Skeletal muscles are dissected at desired time points after the injection and injury, according to experimental requirements, and used for subsequent histological analyses. The dissected muscles can either be frozen, after inclusion in cryo-protectant media, or included in paraffin. However, these procedures require a step of pre-fixation with paraformaldehyde, which can generate artifacts and can eventually affect the subsequent analyses. Freezing the tissue directly can prevent this problem. Of note, many primary antibodies work exclusively or more effectively on fresh frozen muscle sections13, which makes this procedure more reliable. The morphometric analysis is usually performed on time course experiments, which allow for the evaluation of differences in the regeneration processes between groups of mice with the same age and with specific genetic mutations and/or pharmacological treatments. Although the protocol of CTX-induced damage is highly reproducible per se, a possible limitation is due to the operator-dependent variability of CTX injection. To overcome this limitation, it is preferable that the entire time course experiment is performed by the same operator.
The extent of muscle regeneration can be quantified as percentage of healthy fibers, necrotic areas and inflammation, and regenerating and regenerated areas over the total area. However, this approach is mostly used when studying events of chronic damage, such as in dystrophic mdx mice, rather than in models of acute injury. Indeed, while dystrophic muscles are characterized by asynchronous events of degeneration and regeneration, acute injury is followed by well-defined and consequential events. One limitation of these analyses, which require an empirical identification of the morphological features described above, is that they are not completely unbiased. To support experimental conclusions, the morphological analysis should always be complemented with further analysis, such as immunofluorescence analysis of specific molecular markers, to support experimental conclusions. For this reason, it is preferred to use parallel analysis to unequivocally interpret the results. For instance, regenerating myofibers are positively stained for embryonic Myosin Heavy Chain (eMyHC) that is expressed specifically in newly formed myofibers. Thus, quantification of eMyHC-stained myofibers can be performed side-by-side with the morphological analysis.
The CSA analysis is a more reliable quantification and can be performed either on H&#38;E-stained sections or on muscle sections stained with laminin, which marks the edge of the muscle fibers; quantification is performed using appropriate macros of ImageJ, as described above. In both cases, it is always necessary to first assess the quality of the sections and to exclude areas of tissue that appear deformed or curled.
Beside the morphological analyses of the tissues, H&#38;E-stained sections allow for the identification of other specific features, such as the presence of fibrosis and/or fatty tissue. Indeed, fibrosis derives from the excessive accumulation of extracellular matrix that occurs either if the regeneration is impaired or in chronic diseases14. Indeed, extracellular matrix accumulation is visible as pale material deposited between the regenerated myofibers in H&#38;E-histological staining. Multiple rounds of CTX injection can be used to mimic chronic disease in which the myofibers undergo waves of degeneration and regeneration and scar tissue accumulates aberrantly. However, more reliable protocols are available to induce muscle fibrosis15, and specific histological staining can be performed to identify and quantify these structures, such as Masson&#39;s Trichrome or Sirius Red staining. Formation of fatty tissue is clearly visible in H&#38;E-stained sections as rounded white structures between the myofibers, giving an important indication of the presence of fatty tissue, which is quantified by Oil Red staining. Finally, skeletal muscle sections obtained following the protocol described can be used to perform immunofluorescence analysis using specific antibodies and protocols.

포유류 성인 골격 근육 수축 전문화 다핵 근육 세포 (근섬유) fascicules의 그룹에 의해 형성된다. 각각의 근섬유는 정기적으로 반복 조직 수축성 단백질 (액틴과 미오신 필라멘트)로 구성되는 sarcolemma (혈장 막)과 포함 근원 섬유에 의해 둘러싸여 긴 syncytium입니다. 성인의 삶과 휴식 조건에서, 골격 근육은 myonuclei 1의 매우 낮은 회전율을 가지고, 실제로 sarcolemma 아래 근섬유의 주변에 위치하는 myonuclei은, 세포주기의 G0 단계에서 검거 및 1,2- 증식 할 수없는된다. 골격 근육 손상을 따라 서로 밀접하게 관련되어 조직 리모델링 여러 이벤트 후에 항상성 도달 재생하는 독특한 능력을 가지고있다. 급성 손상 또는 외상 후 변성이 유발되어 재생 공정 뒤에근육 세포 집단의 거주자 등 다양한 세포 집단을 포함하는, 상기 위성 세포 SCS (). 실제로, 어떠한 환경 적 자극의 부재 하에서, 위성 셀은 정지 상태에 있고 sarcolemma 상기 기저판 사이에 3,4- 전문 틈새에 위치하고있다. 부상 또는 질병, 활성화, 새로 형성 근섬유 5 상승을주고, 손상된 지역으로 이주, 결국 차별화 확산 될 희주 다음과 같습니다. 활성화 SCS는 외상 6-8의 사이트에서 모집하는 서로 다른 세포 집단, 주로 염증 세포와 크로스 토크 (cross-talk)을 설정합니다. 이 크로스 토크는 세포가 결국 9 항상성 선도 분자 신호 구조 변경을 유도하는 규제 패러다임을 수행 할 수 있습니다. 희주, 염증 및 간질 세포, 혈관 신생 과정 및 재 신경 분포 이벤트 외에도이 높은 조직의 복구를 조율 된 방식으로 행동, 참여pecialized들 구조. 근육의 생리를 이해하고, 또한 전체 공정의 깊은 지식을 필요로 치료 전략을 향상시킬뿐만 아니라, 골격근 재생의 다양한 측면을 연구하는데 관심이있다. 여러 가지 실험 방법은 현재 서로 다른 세포 집단의 신호 경로, 및 관련된 분자 메커니즘의 정체성과 기능을 연구하는 데 사용할 수 있습니다. 급성 손상의 마우스 모델은이 과정의 여러 측면을 조사하는 강력한 도구를 나타냅니다. 다른 일반적으로 급성 근육 손상 연구자 프로세스의 말미에 초기 단계에서, 생체 내에서 재생 공정을 수행 할 수 있도록 유도하는 방법을 사용 하였다. 이 프로토콜은 용해술 유도 및 조직 샘플의 분석까지 재생 공정을 트리거 뱀독 - 유래 cardiotoxin의 근육 내 주사 (CTX)의 단계를 설명한다. CTX 주입 후, 마일CE는 실험 조건에 따라 서로 다른 시점에서 희생 될 수 있고, 골격 근육 해부하고 추가 분석을 위해 처리 될 수있다. 마지막으로, 우리는 기본 형태 학적 관찰과 정량적 분석을 수행하도록 조직 절편의 염색 프로토콜을 설명한다. 이 프로토콜은 높은 재현성으로 10 생체 내에서 급성 골격 근육 재생의 연구 수 있습니다.

<table><tbody><tr><td>Cardiotoxin from&amp;nbsp;&lt;em&gt;Naja mossambica mossambica&lt;/em&gt;</td><td>SIGMA ALDRICH</td><td>C9759</td><td></td></tr><tr><td>Syringe For Insulin BD Micro-Fine+ Needle 30 G x 8 mm - Da 0.3 mL</td><td>BD</td><td>324826</td><td></td></tr><tr><td>Tragacanth Gum</td><td>MP BIOMEDICALS,LLC</td><td>104792</td><td></td></tr><tr><td>2-Methylbutane (Isopentane)</td><td>SIGMA ALDRICH</td><td>78-78-4.</td><td></td></tr><tr><td>OCT Killik Solution For Inclusion Cryostat</td><td>Bio-optica</td><td>&amp;nbsp;05-9801</td><td></td></tr><tr><td>Feather Microtome Blade S35</td><td>Bio-optica</td><td>&amp;nbsp;01-S35</td><td></td></tr><tr><td>Glass Slide Superfrost Plus</td><td>Menzel-Gl&amp;auml;ser</td><td>09-OPLUS</td><td></td></tr><tr><td>Dumon #5 Mirror Finish Forceps&amp;nbsp;</td><td>2BIOLOGICAL INSTRUMENTS</td><td>11251-23</td><td></td></tr><tr><td>Scissors Straight Sharp/Sharp</td><td>2BIOLOGICAL INSTRUMENTS</td><td>15024-10</td><td></td></tr><tr><td>Scissors Noyes Straight</td><td>2BIOLOGICAL INSTRUMENTS</td><td>15012-12</td><td></td></tr><tr><td>Fine Iris Scissors Straight Sharp/Sharp 10.5 cm</td><td>2BIOLOGICAL INSTRUMENTS</td><td>14094-11</td><td></td></tr><tr><td>Eukitt</td><td>Bio-optica</td><td>09-00100</td><td></td></tr><tr><td>Slide Coverslip</td><td>BIOSIGMA</td><td>VBS651</td><td></td></tr><tr><td>Xylene</td><td>SIGMA ALDRICH</td><td>214736</td><td></td></tr><tr><td>Ethanol 100%</td><td>sigma-Aldrich</td><td>02860-2.5L</td><td></td></tr><tr><td>Hematoxyline</td><td>J.T. BAKER</td><td>3873</td><td></td></tr><tr><td>Eosin</td><td>SIGMA ALDRICH</td><td>HT110116</td><td></td></tr><tr><td>Cryostat</td><td>LEICA</td><td>CM3050 S</td><td></td></tr></tbody></table>

induction of acute skeletal muscle regeneration by cardiotoxin injection

골격 근육 재생이 부상 또는 질병에 대한 응답으로 성인 골격 근육에서 발생하는 생리적 과정이다. 급성 손상 - ​​유도 골격근 재생 근육 재생뿐만 아니라 메커니즘과 다른 플레이어와 관련된 사건을 연구하기 위해 널리 사용되는 강력한 모델 시스템이다. 사실상,이 프로세스의 상세한 기술은 골격근 변성을 초래할 병적 조건의 이해에 필수적이며, 새로운 표적 치료 전략을 식별 돕는다. 본 연구는 cardiotoxin의 단일 근육 내 주사 (CTX)를 통해 마우스의 급성 골격 근육의 재생을 유도하는 상세하고 재생 가능한 프로토콜을 설명합니다. CTX는 뱀 독 독소의 가족에 속하고 결국 재생 이벤트를 트리거 근섬유의 근종 용해술을 발생합니다. 골격 근육 재생의 역학은 근육 부분의 조직 학적 분석에 의해 평가된다. 또한 프로토콜해부 동결 및 경골근 근육뿐만 아니라 널리 후속 학적 및 형태 계측 학적 분석에 사용되는 루틴을 헤 마톡 실린 및 에오신 염색 절단 실험 절차를 도시한다.

H&#38;E staining allows for the evaluation of the morphology of the regeneration process at specific time points during skeletal muscle regeneration. Figure 3 shows the time course analysis performed on injured TA muscles of wild type mice. Muscles have been isolated at 3, 7, 15, and 30 days after CTX injection, as schematized in Figure 3A. Representative pictures of H&#38;E-stained transverse sections show the dynamics of skeletal muscle repair over time (Figure 3B-E). At day 3 after injury, the structural architecture of the muscle is completely destroyed, and both degenerated myofibers (necrotic area) and mononucleated cells are clearly visible (Figure 3B). This heterogeneous group of cells mainly consists of transient amplifying satellite cells (myoblasts), inflammatory cells recruited from blood11, and interstitial and endothelial cells that participate in the regeneration process, which is triggered at the site of injury. In this context, new regenerating myofibers are generated by fusion and differentiation of myoblasts. The main feature of the regenerating area is the presence of small basophilic myofibers with centrally located nuclei and a dark-stained cytoplasm (Figure 3C). At this stage, inflammation is still visible, although it progressively decreases. The size of regenerating myofibers increases over time, giving rise to eosinophilic regenerated myofibers characterized by centrally located nuclei (Figure 3D, E). The regenerated myofibers are visible at later stages of regeneration and until the end of the process. The healthy myofibers appear highly organized and close to each other, with a pink cytoplasm and with the nuclei placed at the periphery of the fibers, under the sarcolemma. These myofibers are usually present far from the site of CTX injection and injury, and they more rarely represent areas where the regeneration is definitively complete. Indeed, regenerated fibers are characterized by the presence of centrally located nuclei that move to the periphery only several months after injury.
Digital images of the stained muscle sections are captured by adding the scale bars for spatial calibration, which is necessary to accurately measure the areas of interest by outlining the contours of the interested areas. The measurement of the myofiber cross-sectional area (CSA) is a reliable morphometric analysis, which is widely used to quantify the differences in the regeneration trend between different groups of mice. This analysis requires the measurement of areas of centrally nucleated myofibers, including at least 500 and up to 1,000 fibers per section. Both the average of the regenerating and regenerated myofiber areas and the Gaussian distribution of these areas are indicators of the regeneration process. Increased myofiber CSA is generally associated with an improved and/or accelerated regenerative response. On the contrary, failure of proper regeneration is associated with a decrease in CSA. Here, we reported the myofiber CSA analysis, indicated as both the average (Figure 3F) and distribution (Figure 3G) of myofiber CSA at different time points. The analysis highlights how the distribution of myofiber areas changes over time and how the CSA shifts towards larger-sized fibers as the process of regeneration proceeds.
<img alt="Figure 1" src="/files/ftp_upload/54515/54515fig1.jpg" /> 
Figure 1. Intramuscular Injection and Isolation of the Tibialis Muscle. A. Intramuscular injection of cardiotoxin in the Tibialis Anterior muscle. B. Removal of muscle fascia. The white arrow indicates the thin-tipped tweezers that pinch the muscle to remove the fascia. C. The top picture shows the anatomical position of the distal TA tendon. The bottom picture shows how to lift the TA and remove it while avoiding muscle damage. D. Left picture: the TA is lifted by pulling the distal tendon upward. The white arrow indicates the distal EDL tendon. Right picture: by holding the tendon with forceps, the top edge of the TA is cut off below the knee. The white arrow indicates the cutting site. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54515/54515fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54515/54515fig2.jpg" /> 
Figure 2. Fresh Freezing Muscle and Cryostat Sectioning of Frozen Muscles. A. Technique of fresh muscle inclusion in tragacanth gum. The TA muscle is immersed in the gum from the tendon, with about 3/4 outside and maintained in a perpendicular position with respect to the cork. B. Representative images of the procedure for cryostat sectioning. The cork is placed in a small amount of freezing compound on the specimen stub. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54515/54515fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54515/54515fig3.jpg" /> 
Figure 3. Time Course Analysis of Skeletal Muscle Regeneration. A. Experimental scheme of acute skeletal muscle injury. B-E. Representative pictures of Hematoxylin &#38; Eosin stained sections of cardiotoxin (CTX)-treated muscles at indicated days after injury. B. The black continuous line (on the left side of the picture), encloses a couple of necrotic fibers, likely invaded by inflammatory cells. The dashed line (right side of the picture) marks an area of infiltrating mononucleated cells. C. A single immature myofiber with centrally located nucleus is indicated by the black circle. D-E. Large regenerated eosinophilic myofibers, with centrally located nuclei are marked by black circles. Scale bars represent 100 mm. F. Average of centrally nucleated myofiber size values in TA muscle sections. Values are mean &#177; SEM, 5 mice/group. G. Myofiber cross-sectional area (CSA) distribution at 6, 15, and 30 days after CTX injection. Values are mean &#177; SEM, 5 mice/group. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54515/54515fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Cardiotoxin 주입에 의한 급성 골격 근육 재생 유도

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

induction-acute-skeletal-muscle-regeneration-cardiotoxin

Nanyang Technological University

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JoVE Journal

Developmental Biology

21.9K 조회수.  Institute of Genetics and Biophysics "A. Buzzati Traverso", CNR. This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

비디오: Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

신경근육학 전기적 자극에 의​​해 근육 트레이닝의 Murine 모델

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

연구

의학

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

근육 재생의 마우스 모델에서 유도 만능 줄기 세포 유래 메소안지오블라스트 와 같은 근생 성 전구의 이식

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

생체공학

Analyzing Satellite Cell Function During Skeletal Muscle Regeneration by Cardiotoxin Injury and Injection of Self-delivering siRNA In Vivo

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite cells. Satellite cells are characterized by the expression of the transcription factor Pax7 and their location underneath the basal lamina in the resting skeletal muscle. Upon injury, satellite cells get activated, undergo self-renewal or differentiation to either form new myofibers or to fuse with damaged ones. The functionality of satellite cells in vivo can be investigated using a cardiotoxin based injury model of skeletal muscle. To study the function of one gene during the regeneration of skeletal muscle, transgenic mouse models are mostly used. Here, we present an alternative method to transgenic mice, to investigate the gene function in satellite cells during regeneration, e.g., in cases where transgenic mice are not available. We combine the cardiotoxin mediated injury of a specific skeletal muscle with the injection of a self-delivering siRNA into the regenerating muscle which is then taken up by satellite cells among other cells. Thereby, we provide a method to analyze gene function in satellite cells during regeneration under physiological conditions without the need for transgenic mice.

We describe an in vivo method to investigate the functional loss of a specific gene in satellite cells by using a combination of cardiotoxin mediated injury of skeletal muscle and the injection of a self-delivering siRNA.

심장 독소 부상에 의한 골격 근육 재생 중 위성 세포 기능 분석 및 생체 내 자체 전달 siRNA 주입

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite ...

유전학

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

수축 성 활동 유도 된 쥐 골격 근육 Phenotypic 적응 연구 만성 자극에의 응용

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

발생학

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

수정 된 버그 스트롬 기술을 이용하여 인간의 골격 근육 생검 절차

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

의 응용 프로그램<em&gt; 생체</em&gt; 조직 평가를위한 쥐 앞정 전방의 기능 테스트는 골격근 수리 엔지니어링

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Minimally Invasive Muscle Embedding (MIME) - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new muscle fibers under the right conditions. Although SCs can be harvested from muscle tissue and cultured in vitro, the resulting myoblast cells are not very effective in promoting myogenesis when transplanted into host muscle. Surgically exposing the host muscle and grafting segments of donor muscle tissue, or the isolated muscle fibers with their SCs onto host muscle, promotes better myogenesis compared to myoblast transplantation. We have developed a novel technique that we call Minimally Invasive Muscle Embedding (MIME). MIME involves passing a surgical needle through the host muscle, drawing a piece of donor muscle tissue through the needle track, and then leaving the donor tissue embedded in the host muscle so that it may act as a source of SCs for the host muscle. Here we describe in detail the steps involved in performing MIME in an immunodeficient mouse model that expresses a green fluorescent protein (GFP) in all of its cells. Immunodeficiency in the host mouse reduces the risk of immune rejection of the donor tissue, and GFP expression enables easy identification of the host muscle fibers (GFP+) and donor-cell-derived muscle fibers (GFP-). Our pilot data suggest that MIME can be used to implant an extensor digitorum longus (EDL) muscle from a donor mouse into the tibialis anterior (TA) muscle of a host mouse. Our data also suggest that when a myotoxin (barium chloride, BaCl2) is injected into the host muscle after MIME, there is evidence of donor-cell-derived myogenesis in the host muscle, with approximately 5%, 26%, 26% and 43% of the fibers in a single host TA muscle showing no host contribution, minimal host contribution, moderate host contribution, and maximal host contribution, respectively.

Minimally Invasive Muscle Embedding MIME - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

We describe a novel experimental technique that we call Minimally Invasive Muscle Embedding (MIME), which is based on the evidence that skeletal muscle tissue contains viable myogenic cells that can facilitate donor-cell-mediated myogenesis when implanted into a host muscle.

최소한 침략 적 근육 (MIME) 포함-기증자 세포 중재 하는 Myogenesis를 촉진 하기 위하여 새로운 실험 기법

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

근육 위성 세포의 분리, 문화, 및 이식

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

생물학

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

근육 줄기 세포에서 Autophagy의 원위치 Immunofluorescent 얼룩

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

쥐 모델의 근육 부상을 치료하기 위한 Ding의 롤 조작을 기반으로 한 마사지기

Mimicking Ding's Roll Method on Notexin-Induced Muscle Injury in Rats

Ding's roll method is one of the most commonly used manipulations in traditional Chinese massage (Tuina) clinics and one of the most influential contemporary Tuina manipulations in China. It is based on the traditional rolling method commonly used in the one finger Zen genre and named Ding's roll method. Due to its anti-inflammatory and blood circulation-promoting effects, Ding's rolling method has sound therapeutic effects on myopathy. Because of the large area of force applied to human skin, Ding's roll method is challenging to perform on experimental animals with small skin areas, such as rats and rabbits. Additionally, the strength of Tuina applied to the human body differs from that applied to experimental animals, so it may happen that the strength is too high or too low to achieve the therapeutic effect of Tuina during the experiment. This experiment aims to create a simple massager suitable for rats based on Ding's rolling manipulation parameters (strength, frequency, Tuina duration). The device can standardize manipulation in animal experiments and reduce the variation in Tuina force applied to different animals due to subjective factors. A rat model of notexin-induced skeletal muscle injury was established, and plasma injury markers creatine kinase (CK) and fatty acid binding protein 3 (FABP3) were used to assess the therapeutic effect of Tuina on skeletal muscle injury. The results showed that this Tuina massager could reduce the levels of CK and FABP3 expression and slow down the degree of skeletal muscle injury. Therefore, the Tuina massager described here, mimicking Ding's roll method, contributes to standardizing Tuina manipulation in experimental research and is of great help for subsequent research on the molecular mechanism of Tuina for myopathy.

저자 스포트라이트: 자체 제작 마사지기를 사용하여 쥐에서 Tuina 마사지 수행 

This protocol describes a simple device that mimics Ding's roll method, establishes a rat model of skeletal muscle injury, and uses hematoxylin-eosin staining to observe the pathology of damaged tissue and enzyme-linked immunosorbent assay to detect changes in serum damage markers.

Cardiotoxin 주입에 의한 급성 골격 근육 재생 유도

요약

더 많은 비디오 탐색

이 비디오의 챕터

신경근육학 전기적 자극에 의해 근육 트레이닝의 Murine 모델

근육 재생의 마우스 모델에서 유도 만능 줄기 세포 유래 메소안지오블라스트 와 같은 근생 성 전구의 이식

심장 독소 부상에 의한 골격 근육 재생 중 위성 세포 기능 분석 및 생체 내 자체 전달 siRNA 주입

수축 성 활동 유도 된 쥐 골격 근육 Phenotypic 적응 연구 만성 자극에의 응용

수정 된 버그 스트롬 기술을 이용하여 인간의 골격 근육 생검 절차

의 응용 프로그램

최소한 침략 적 근육 (MIME) 포함-기증자 세포 중재 하는 Myogenesis를 촉진 하기 위하여 새로운 실험 기법

근육 위성 세포의 분리, 문화, 및 이식

근육 줄기 세포에서 Autophagy의 원위치 Immunofluorescent 얼룩

쥐의 Notexin-Induced Muscle Injury에 대한 Ding의 롤 방법 모방

Cardiotoxin 주입에 의한 급성 골격 근육 재생 유도

요약

더 많은 비디오 탐색

이 비디오의 챕터

신경근육학 전기적 자극에 의​​해 근육 트레이닝의 Murine 모델

근육 재생의 마우스 모델에서 유도 만능 줄기 세포 유래 메소안지오블라스트 와 같은 근생 성 전구의 이식

심장 독소 부상에 의한 골격 근육 재생 중 위성 세포 기능 분석 및 생체 내 자체 전달 siRNA 주입

수축 성 활동 유도 된 쥐 골격 근육 Phenotypic 적응 연구 만성 자극에의 응용

수정 된 버그 스트롬 기술을 이용하여 인간의 골격 근육 생검 절차

의 응용 프로그램

최소한 침략 적 근육 (MIME) 포함-기증자 세포 중재 하는 Myogenesis를 촉진 하기 위하여 새로운 실험 기법

근육 위성 세포의 분리, 문화, 및 이식

근육 줄기 세포에서 Autophagy의 원위치 Immunofluorescent 얼룩

쥐의 Notexin-Induced Muscle Injury에 대한 Ding의 롤 방법 모방

신경근육학 전기적 자극에 의해 근육 트레이닝의 Murine 모델