Name: the fibular nerve injury method: a reliable assay to identify and test factors that repair neuromuscular junctions
Uploaded: 2016-08-11T15:00:00.000+00:00
Duration: 6 min 1 s
Description: Watch this Scientific Journal Video about The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions at JoVE.com

The authors thank members of the Valdez laboratory for intellectual input on experiments and comments on the manuscript.

모든 실험은 버지니아 공대 기관 동물 관리 및 사용위원회에 의해 승인 NIH 가이드 라인과 동물 프로토콜 하에서 수행 하였다. 수술 1. 준비 동물 <ol><li> 멸균 된 1 ml의 인슐린 주사기 케타민 (90 ㎎ / ㎏) 및 서혜부 피하 주사를 통해 자일 라진 (10 ㎎ / ㎏)의 혼합물로 생쥐를 마취. 캐리어 솔루션은 0.9 % 식염수, 17.4 ㎎ / ㎖ 케타민, 2.6 ㎎ / ㎖ 자일 라진의 혼합물을 포함한다. 을 적용하려면 약물 치료를 기다리는 동안 케이지 다시 동물을 놓습니다. 주 : 로딩 용량이 절차 동안 충분한 마취를 제공하지 않는 경우, 부하 용량의 추가 25 %가 주입 될 수있다. </li><li> 모니터 동물은 정상 호흡 요금과 각성 수준의 적절한 우울증를 확인하기 위해 주사를 게시 할 수 있습니다. 충분히 마취 할 때 어떤 반응을 유도하지해야 뒷발 핀치와 각성 수준을 확인합니다. 참고 :이 일반적으로 3-5 소요25g 30에 평균 젊은 성인 마우스 분. 동물은 여전히​​ 10 분 포스트 분사 후 응답하는 경우, 마취 로딩 도즈의 추가 25 %가 주입 될 수있다. </li><li> 건조 함을 방지하기 위해 동물의 눈에 바셀린 가벼운 미네랄 오일 안과 연고를 적용합니다. 깨끗하고 평평한 표면에 케이지와 장소에서 동물을 제거합니다. 사지의 좌우 측면을 노출, 전기 헤어 트리머를 사용하여 골반에 발에서 원하는 뒷다리를 면도. </li><li> 1 분 동안 면도 사이트에 화학 머리 제거를 적용합니다. 수동으로 실험실 와이프를 사용하여 머리를 제거합니다. 실험실 청소 면도 한 영역은 에탄올에 배어 와이프. </li></ol> 2. 수술 <ol><li> 오토 클레이브 또는 다른 적절한 방법을 통해 수술 도구를 소독. 80 % 에탄올 / H 2 0과 수술 부위 및 수술 보드를 청소합니다. proviodine으로 수술 부위를 소독. 수술 보드에 마우스를 놓고 사지 구속과 맞 춥니 다. 유지무릎 관절을 가진 해부학 적 자연 위치에서 대상 뒷다리가 약간 내부 또는 외부 회전없이 확장. </li><li> 수술 현미경으로 동물과 보드를 놓습니다. 표면 랜드 마크, 특히 뼈 무릎 관절과 전 경골근과 비복근 근육 사이의 능선의 촉진을 통해 적절한 절개 사이트에 동양. </li><li> 그립의 일반 집게를 사용하는 동안 메스 또는 스프링 가위를 사용하여 피부를 통해 약 3cm의 절개를합니다. 일반적인 비골 신경의 기본 과정에 수직 절개를합니다. </li><li> , 피상적 인 밴드를 통해 절개를 계속 이두근 대퇴골을 노출하고 광근의 근육을 lateralis. 연결 깊은 근막을 통해 절단하여 이러한 근육을 분리합니다. 1-2 센티미터 컷은 충분합니다. </li><li> 이두근을 철회하는 것은 꼬리 쪽 공통 비골 신경을 드러내는, 기계 견인기를 사용하여 근육 대퇴. </li><li> 그 간까지 근위 신경을 추적비복근 근육의 측면 머리의 건과 절 발견된다. 참고 : 노출 후퇴 피부와 근육의 추가 조작이 필요할 수 있습니다. 이 교차로는 신경 손상에 대한 안정 랜드 마크로서 사용된다. </li><li> 비복근 건의 측면 테두리에 병렬 방식으로 팁을 정렬, 미세 집게로 신경을 잡고. 5 초 동안 꾸준히, 열심히 압력을 적용하여 일반적인 비골 신경 크러시. </li><li> 수술 범위를 통해 육안 검사로 신경의 전체 호감을 확증. 그것은 부상의 사이트에서 반투명 나타납니다. 주변 축삭의 형광 단백질을 발현하는 마우스를 사용하는 경우, 형광 부상의 사이트에서 사라집니다. </li><li> 견인기를 제거하고 자신의 해부학 적 위치에 근육을 재편성. 6-0 실크 봉합사와 절개 사이트를 닫습니다. 1-3 간단한 중단 봉합 충분하다. 깨끗한 케이지에 가열 패드에 마우스를 회복 놓습니다. </li><li> 2 시간 포스트 오페라에 대한 모든 동물을 모니터링기 호흡과 마취에 대한 부작용에 ​​대해 확인합니다. 즉시 수술에서 회복 다음 피하 사타구니 주사를 통해 프레 노르 핀 0.05-0.10 ㎎ / ㎏의 초기 용량을 관리 할 수​​ 있습니다. 3 추가 투여 48 시간 동안 매 12 시간을 제공합니다. 전체 복구 후, 동물 보호 시설에 마우스를 반환합니다. </li></ol> 3. 분리 및 염색 신근의 심지 Longus (EDL) 근육 <ol><li> 이소 플루 란을 이용하여 동물을 희생. 흡수 labwipes로 포장 50 ML 튜브에 0.5 ml의에게 액체 이소 플루 란을 분배. 밀폐 2,500cm 3 실에서 동물과 캡핑 튜브를 놓습니다. 노출의 적어도 4 분이면 충분하다. 양측 눈꺼풀, 발가락 핀치, 꼬리 - 핀치 반사 손실에 대한 테스트는 각각의 동물 관류를 진행하기 전에 의식이 있는지 확인합니다. </li><li> 첫째로 transcardially 10 ㎖의 0.1M PBS 16 동물들을 관류하고 0.1M PBS (PH 7.4)의 4 % 파라 포름 알데히드의 25 ㎖. 헤파린 (30 대 / 20g 동물 체중) / ㎖) 10 단위 관류 결과를 향상 작은 모세관 침대에서 혈액 응고를 방지하기 위해 (PBS를 첨가 할 수있다. </li><li> 복부의 둘레 주위의 피부를 통해 횡 방향으로 절단 가위를 사용하여 뒷다리를 덮고있는 피부를 제거합니다. 뒷다리 사지와 발 집게를 사용하여 과거의 피부를 벗겨. </li><li> 잡고 집게로 박리에 의해 뒷다리의 표면 근막을 제거합니다. 주변 축삭에 형광 단백질을 발현하는 마우스를 사용하는 경우, 50 ㎖ 튜브에 4 % PFA에서 하룻밤 전체 쥐를 후 수정. PBS로 3 회 씻어. 주 : 고정 마우스는 4 ℃에서 PBS에 저장 될 수있다. 그렇지 않은 경우,이 단계를 건너 뛰고 후 고정 동물없이 3.6 단계로 진행합니다. </li><li> 가능한 한 그대로 근위 및 원위 힘줄을 유지해야되고, 마우스 뒷다리에서 EDL 근육 (18)을 해부하다. </li><li> 적어도 1 시간 동안 (1X PBS, 0.5 % 트리톤 X-100, 3 % BSA, 5 % 염소 혈청을 함유) 완충액 블로킹 EDL 근육 인큐베이션. </li><li및 synaptotagmin-2 (1 : 250) 3 일간 버퍼를 차단에 희석 항체 :&gt; 신경 미세 섬유 (1,000 일)를 포함 튜브 모터 축삭과 신경 터미널, 장소 근육을 시각화합니다. 근육을 1X PBS로 3 회 10 분마다 씻으십시오. 주 : 주변 축삭의 형광 단백질 (XFP)를 발현하는 마우스를 사용하는 경우이 단계를 건너 뜁니다. </li><li> 전체 NMJ의 신경 분포에 대한 양성 대조군으로 EDL 손상되지 않은 반대측을 얼룩. 음성 대조군 4 일 후 부상의 NMJ 완전히 denervated있는 시점에서 얻어진 EDL뿐만 아니라, 단지 차 항체로 염색 EDL을 포함해야한다. </li><li> 1 일 신경 미세 섬유 및 synaptotagmin-2를 검출하기 위해 적절한 찬란 태그 이차 항체와 근육을 품어. 근육을 1X PBS로 3 회 10 분마다 씻으십시오. 주의 :이 단계는 단계 3.10와 함께 수행 될 수있다. 주변 축삭의 형광 단백질을 발현하는 마우스를 사용하는 경우이 단계를 건너 뜁니다. </li><li> postsy을 시각화하는 방법NMJ의 naptic 지역은 5 μg의 / ㎖ 이상 2 시간 동안 버퍼를 차단에 희석 알렉사 - 555 복합 알파 - bungarotoxin와 근육을 품어. 근육을 1X PBS로 3 회 10 분마다 씻으십시오. </li><li> 양으로 대전 된 유리 슬라이드에 전체 근육을 마운트하려면, 슬라이드에 매체를 장착 기반 글리세린 몇 방울을 추가하고 coverslip에와 커버 슬라이드에 직접 근육을 놓습니다. 근육을 평평하게 슬라이드에 대해 커버 슬립을 누릅니다. 슬라이드 및 coverslip에 사용하는 실험실 와이프의 경계에서 설치 미디어를 만끽 해보세요. 커버 슬립 및 슬라이드 사이의 가장자리를 밀봉 매니큐어를 적용합니다. </li></ol> 4. 영상 및 데이터 분석 <ol><li> NMJs 화상 488, 555 및 633 nm의 광을 여기하고 20X 및 40X 목적으로 방출 된 광을 포착 갖춘 공 ​​초점 레이저 주사 현미경을 사용하여 EDL 근육의 구조를 분석한다. </li><li> 전체 NMJs 시각화 광 초의 최대 강도 투사 이미지를 만들TIONS는 떨어져 NMJ의 가장 높은 보이는 지역 가장 낮은에서 연장 1 μm의 2 간격. 시중에서 판매하는 이미징 소프트웨어를 사용하여 최대 강도 예측을 만듭니다. </li><li> 1) 완전히 denervated = 시냅스 사이트가 축삭과 AChRs 사이의 축삭과 접촉, 5 % 미만의 colocalization을 완전히없는 상태입니다으로 reinnervation의 속도를 확인하려면 NMJs을 분류합니다. 2) 부분적으로 신경 지배는 = 축삭은 부분적으로 축삭과 AChRs 사이의 postsynapse, 5~95%의 colocalization을 중첩. 3) 전체 신경 분포 = 사전 및 사후 시냅스 사이의 거의 완벽한 동격, 축삭과 AChRs 사이의보다 큰 95 % colocalization을. 결상면에 수직으로 놓여 또는 완전히 이미지에 가시화되지 NMJs를 제외합니다. 주 : 모든 실험에서, 적어도 3 동물 및 동물 당 50 NMJs을 조사 하였다. 결과는 0.05 미만의 P 값을 갖는 학생 t-test를 이용하여 의미있는 것으로 간주 하였다. </li><li> 연산자를 눈 멀게하려면 별도의 개인이 수행 할 수 있습니다수술 및 이미지 분석. 치료 그룹의 지식없이, 분석기는 NMJ 점수와 목적이 될 수 있습니다. 대안 적으로, 무작위로 이미지 및 소스 동물 모르게 분석을 위해 조작자에게 제공 될 수있다. </li></ol> 5. 정량적 PCR <ol><li> 이소 플루 란과 자궁 경부 전위를 사용하여 동물을 희생. 피부와 3.3 단계를 따라 다리 근육을 덮고 표면 근막을 제거합니다. 전 경골근과 3.4 단계에있어서, EDL 근육을 해부하다. </li><li> 플래시는 액체 질소에 비해 1.5 ML 튜브의 전체 경골근과 EDL 근육을 동결. 부분적으로 액체 질소에 잠긴 미리 냉장 박격포에 튜브와 장소에서 조직을 제거합니다. 막자 사발을 사용하여 미세한 분말로 갈아 냉동 근육. </li><li> 시판 RNA 추출 시약에 냉동 근육 분말을 용해시키고, 시판의 키트를 제조자의 난에있어서와 RNA 추출 및 게놈 DNA의 제거를 수행nstructions. </li><li> 제조업체의 지시에 따라 시판중인 역전사 효소 믹스 역전사를 수행한다. </li><li> qPCR에 적절한 하우스 키핑 유전자를 이용하여 시판되는 키트를 사용하여 수행 (물질의 표를 참조). (재료의 표 참조) PCR을 수행하기 위해 시중에서 판매하는 정량적 PCR 열 순환기를 사용합니다. </li><li> 58 ° C로 설정 소둔 온도. Taq 중합 효소 / SYBR 녹색 믹스 제조업체의 사양에 추가로 자전거 매개 변수를 조정합니다. 프라이머 특이성에 대한 테스트 및 이량 체 형성을 프라이머 95 ° C에 65 ° C에서 0.5 ° C 증가 증가로 구성된 열 자전거 타는 프로그램에서 최종 용융 곡선 단계를 포함합니다. </li><li> 제어 유전자로서 18S RNA를 사용 ΔΔCT 방법 21 - 2 의한 상대적인 mRNA의 발현 수준을 결정한다. </li></ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+cellular+interactions+in+peripheral+nerve+regeneration.">Molecular mechanisms of cellular interactions in peripheral nerve regeneration.</a> Curr Opin Neurol. 14 (5), 635-639 (2001).</li><li>Gaudet, A. D., Popovich, P. G., Ramer, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wallerian+degeneration:+gaining+perspective+on+inflammatory+events+after+peripheral+nerve+injury.">Wallerian degeneration: gaining perspective on inflammatory events after peripheral nerve injury.</a> J Neuroinflammation. 8, 110 (2011).</li><li>Chen, P., Piao, X., Bonaldo, P. <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+macrophages+in+Wallerian+degeneration+and+axonal+regeneration+after+peripheral+nerve+injury.">Role of macrophages in Wallerian degeneration and axonal regeneration after peripheral nerve injury.</a> Acta Neuropathol. 130 (5), 605-618 (2015).</li><li>Chen, Z. -. L., Yu, W. -. M., Strickland, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+regeneration.">Peripheral regeneration.</a> Annu Rev Neurosci. 30, 209-233 (2007).</li><li>Darabid, H., Perez-Gonzalez, A. P., Robitaille, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromuscular+synaptogenesis:+coordinating+partners+with+multiple+functions.">Neuromuscular synaptogenesis: coordinating partners with multiple functions.</a> Nat. Rev. Neurosci. 15 (11), 703-718 (2014).</li><li>Geuna, 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+sciatic+nerve+injury+model+in+pre-clinical+research.">The sciatic nerve injury model in pre-clinical research.</a> J. Neurosci. Methods. 243, 39-46 (2015).</li><li>Batt, J. A. E., Bain, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tibial+nerve+transection+-+a+standardized+model+for+denervation-induced+skeletal+muscle+atrophy+in+mice.">Tibial nerve transection - a standardized model for denervation-induced skeletal muscle atrophy in mice.</a> J. Vis. Exp. (81), e50657 (2013).</li><li>Savastano, L. E., Laurito, S. R., Fitt, M. R., Rasmussen, J. A., Gonzalez Polo, V., Patterson, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sciatic+nerve+injury:+a+simple+and+subtle+model+for+investigating+many+aspects+of+nervous+system+damage+and+recovery.">Sciatic nerve injury: a simple and subtle model for investigating many aspects of nervous system damage and recovery.</a> J. Neurosci. Methods. 227, 166-180 (2014).</li><li>Kang, H., Lichtman, 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=Motor+axon+regeneration+and+muscle+reinnervation+in+young+adult+and+aged+animals.">Motor axon regeneration and muscle reinnervation in young adult and aged animals.</a> J Neurosci. 33 (50), 19480-19491 (2013).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> J. Vis. Exp. (65), e3564 (2012).</li><li>Feng, G., Mellor, R. 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=Imaging+Neuronal+Subsets+in+Transgenic+Mice+Expressing+Multiple+Spectral+Variants+of+GFP.">Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP.</a> Neuron. 28 (1), 41-51 (2000).</li><li>Sanes, J. R., Lichtman, 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=Development+of+the+vertebrate+neuromuscular+junction.">Development of the vertebrate neuromuscular junction.</a> Annu Rev Neurosci. 22, 389-442 (1999).</li><li>Bowen, D. C., Park, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+and+regulation+of+MuSK+at+the+neuromuscular+junction.">Localization and regulation of MuSK at the neuromuscular junction.</a> Dev Biol. 199 (2), 309-319 (1998).</li><li>Gay, S., Jublanc, E., Bonnieu, A., Bacou, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myostatin+deficiency+is+associated+with+an+increase+in+number+of+total+axons+and+motor+axons+innervating+mouse+tibialis+anterior+muscle.">Myostatin deficiency is associated with an increase in number of total axons and motor axons innervating mouse tibialis anterior muscle.</a> Muscle Nerve. 45 (5), 698-704 (2012).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25 (4), 402-408 (2001).</li></ol>

The authors have nothing to disclose.

이 논문에서 제시하는 방법은 신경근 접합부 (NMJ)을 수리에 관여하는 메커니즘을 식별하는 고유 한 기회를 제공합니다. 이 무릎 근처 비복근 건 통과하여이 방법은 공통 비골 신경 분쇄 포함한다. 우리는 집게와 신경 압축의 5 초 후, 완전한 변성 부상 후 사일에 의해 주목을 보여준다. 젊은 성인 쥐에서 알파 - 모터 축삭은 십이일에 의해 손상되지 않은 쥐와는 구별 시냅스 사이트의 개혁에서 남중...

In young adult and healthy animals, the neuromuscular junction (NMJ) is a highly stable connection between the presynapse, the nerve ending of an &#945;-motor axon, and the postsynapse, the specialized region of an extrafusal muscle fiber where nicotinic acetylcholine receptors (AChRs) selectively aggregate1. The nearly perfect apposition of the pre- and post-synaptic apparatuses is necessary for proper neurotransmission, survival of &#945;-motor neurons and muscle fibers and motor function. Unfortunately, the function of the NMJ is adversely affected by aging, diseases such as amyotrophic lateral sclerosis (ALS), autoimmune diseases and injury to muscles and peripheral nerves2-5. These insults often result in degeneration of presynaptic nerve endings, leaving muscles denervated and significantly altering motor skills. For this reason, the identification of molecules that function to maintain and repair the NMJ has become a priority. Because peripheral nerves regenerate and reinnervate targets, peripheral nerve injury models have been used to identify molecular changes associated with regenerating NMJs. 
Peripheral nerve injury models often involve either completely cutting or crushing specific nerve branches6. Following a cut, the endoneurial tube has to be reformed, delaying axonal regeneration and reinnervation of target cells and tissues. The severity of this type of injury also causes axons to meander away from their original path, resulting in their failure to reach original targets. This is in contrast to nerves injured via crush where the endoneurium remains contiguous, providing a path for efficient and proper regrowth of regenerating axons. It also allows axons to find and reinnervate their original muscle fiber partners. Irrespective of injury model, there are a number of cellular and molecular changes that must occur for axons to regenerate and reinnervate targets. After an injury, the nerve segment proximal to the target is broken down and removed via a process termed Wallerian Degeneration7. This process involves reprogramming and de-differentiation of Schwann cells into non-myelinating cells that secrete regenerative factors, clear myelin, and recruit macrophages to the site of injury8. Macrophages in turn complete the clearance of myelin and axonal debris, which would otherwise impede growth of the regenerating axon9. In parallel, motor and sensory neurons activate mechanisms needed to promote regeneration of their severed axons. Once the regenerating axon reaches the target, it must transform from a growth cone to a nerve ending capable of properly transmitting (for motor axons) or receiving (for sensory axons) information10. In this regard, alpha-motor axons undergo a series of well-orchestrated changes that culminate in their growth cone differentiating into a fully functional presynaptic nerve ending that nearly perfectly opposes the post-synaptic site on the target muscle fiber11. 
The sciatic, tibial and accessory nerves have been the primary choices for studying axonal and NMJ regeneration12-14. However, there are a number of drawbacks when using these models to examine cellular and molecular changes associated with regenerating NMJs between animals and under different conditions. Firstly, the sciatic nerve supplies the majority of the muscles of the hind limb, with injury significantly limiting both movement and sensation. It is therefore not possible to use this method to study the impact of exercise alone or in combination with other factors. Additionally, the sciatic nerve is a rather thick structure and thus requires a large amount of compressive force to fully injure all axons. This in turn may result in complete transection of the more superficial axons while leaving the endoneurial tube of deeper lying axons intact, introducing significant variability in the rate and fidelity of regeneration among these axons. Complete transection of this nerve is even less desirable given that many axons will fail to reconnect with the same muscle fibers. Complicating matters, the sciatic nerve possesses intrinsic anatomic variability, both in the number and site of origin of its terminal nerve branches. It is therefore very difficult to lesion the same site. While the tibial nerve is smaller and more amenable to crush injuries, there is also no readily available landmark to serve as a lesion site for this nerve branch. 
The accessory nerve branch (part of cranial nerve XI) that supplies the sternocleidomastoid muscle has also been used to study regeneration of NMJs15. This nerve is particularly attractive because NMJs in the sternocleidomastoid muscle can be more readily imaged in live animals compared to NMJs in other muscles. But similar to the sciatic and tibial nerves, there is no specific landmark that can be used to injure this nerve in the same location, limiting it as a model for comparing regeneration of NMJs among individual animals of an experimental cohort. An inconsistent lesion site introduces variability in the rates of NMJ reinnervation. Due to these shortcomings, the procedure presented here utilizes the injury of a different peripheral nerve branch to examine regenerating NMJs. 
The common fibular nerve, also called the common peroneal nerve, contains many features that make it a reliable nerve to examine regeneration of NMJs between animals and across different treatments. The common fibular nerve has a predictable anatomic course as it runs over the tendon of the lateral head of the gastrocnemius muscle in the knee, the intersection serving as a stable landmark for lesions. The nerve is accessed through a small and minimally invasive incision near but anatomically segregated from the muscles of interest. The findings presented here demonstrate that regenerating motor axons begin to reform NMJs in the extensor digitorum longus (EDL) muscle 8 days after crushing the fibular nerve in 70 days old young adult female mice. Importantly, the pattern and rate of reinnervation is consistent among animals of the same age and sex and therefore provide a reliable injury model that will significantly hasten our understanding of the cellular and molecular changes required to maintain and repair NMJs.

<table><tbody><tr><td>Ketamine</td><td>VetOne</td><td>501072</td><td></td></tr><tr><td>Xylazine</td><td>Lloyd Inc.&amp;nbsp;</td><td>003437&amp;nbsp;</td><td></td></tr><tr><td>Buprenorphine</td><td>&amp;nbsp;Zoopharm</td><td>1Z-73000-150910&amp;nbsp;</td><td></td></tr><tr><td>Nair</td><td>Nair</td><td></td><td></td></tr><tr><td>Kim-wipes</td><td>Kimtech</td><td>34155</td><td></td></tr><tr><td>Electric Razor</td><td>Braintree Scientific</td><td>CLP-64800</td><td></td></tr><tr><td>80% EtOH/H&lt;sub&gt;2&lt;/sub&gt;0</td><td></td><td></td><td></td></tr><tr><td>10% Proviodine</td><td></td><td></td><td></td></tr><tr><td>1 ml Insulin Syringe</td><td></td><td></td><td></td></tr><tr><td>Spring Scissors</td><td>Vannas</td><td>91500-09</td><td></td></tr><tr><td>No. 15 scalpel</td><td>Braintree Scientific</td><td>SSS 15</td><td></td></tr><tr><td>#5 Forceps</td><td>Dumont</td><td>11252-00</td><td></td></tr><tr><td>6-0 silk suture on reverse cutting needle</td><td>&amp;nbsp;Suture Express</td><td>752B&amp;nbsp;</td><td></td></tr><tr><td>Rodent Heating Pad</td><td>Braintree Scientific</td><td>AP-R-18.5</td><td></td></tr><tr><td>Alexa 555 conjugated alpha-BTX</td><td>Molecular Probes</td><td>B35451</td><td></td></tr><tr><td>Vectashield</td><td>Vector Labs</td><td>H-1000</td><td></td></tr><tr><td>Olympus Stereo Zoom Microscope</td><td>Olympus</td><td>562037192</td><td></td></tr><tr><td>Zeiss 700 Confocal Microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Variable-flow peristaltic perfusion pump</td><td>Fisher Scientific</td><td>13-876-3</td><td></td></tr><tr><td>Aurum Total RNA Mini Kit</td><td>Bio-Rad</td><td>7326820</td><td></td></tr><tr><td>Bio-Rad iScript RT Supermix</td><td>Bio-Rad</td><td>1708840</td><td></td></tr><tr><td>SsoFast Evagreen Supermix</td><td>Bio-Rad</td><td>1725200</td><td></td></tr><tr><td>Bio-Rad CFX96</td><td>Bio-Rad</td><td>1855196</td><td></td></tr><tr><td>Puralube Vet ointment</td><td>Puralube</td><td>1621</td><td></td></tr><tr><td>Synaptotagmin-2 antibody</td><td>Antibodies-Online</td><td>ABIN401605</td><td></td></tr><tr><td>Neurofilament antibody</td><td>Antibodies-Online</td><td>ABIN2475842</td><td></td></tr></tbody></table>

the fibular nerve injury method: a reliable assay to identify and test factors that repair neuromuscular junctions

신경 근육 접합부 (NMJ)은 노화, 상해 및 질병으로 인한 해로운 구조적 및 기능적 변화를 겪는다. 따라서, 유지 보수 NMJs 관련된 세포 및 분자 변화를 이해하는 것이 필수적이다. 이를 위해, 우리는 신뢰성 있고 일관 생쥐 NMJs 재생 조사하는 방법을 개발 하였다. 이 무릎 근처 비복근 근육 힘줄의 측방 헤드를 통과 신경 손상이 방법은 공통 비골 신경 분쇄 포함한다. 칠십일 세 여자 마우스를 사용하여, 우리는 모터 축삭 7 일 후 호감 내에서 이전 시냅스 목표를 reinnervate하기 시작 것을 보여줍니다. 그들은 완전히 십이일에 의해 이전 시냅스 영역을 다시 선점. 이 부상 방법의 신뢰성을 확인하기 위해, 우리는 개별 칠십일 세 여성 쥐 사이 reinnervation 속도를 비교했다. 우리는 reinnervated 시냅스 사이트의 수는 7, 9에서 마우스와 유사한 것을 발견하고, 십이일 후 호감. 있는지 확인하려면이 상처 분석법 또한 근육 분자량 변화를 비교하기 위해 사용될 수 있고, 우리는 근육의 니코틴 성 수용체 (감마 ACHR) 및 근육 특정 키나아제 (MUSK)의 감마 서브 유닛의 수준을 조사 하였다. 감마 - ACHR 서브 유닛과 사향이 높은 다음 탈 신경을 상향 조절하고 NMJs의 reinnervation 다음 정상 수준으로 돌아되어 있습니다. 우리는이 유전자와 근육의 신경 분포 상태에 대한 성적 수준 사이의 밀접한 관계를 발견했다. 우리는이 방법은 NMJ 및 기타 시냅스 수리에 관련된 세포 및 분자 변화에 대한 우리의 이해를 가속화 할 것이라고 믿는다.

일반적인 비골 신경은 또한, 공통 비골 신경이라고는 다리 (그림 1A)의 전방 측면에 비골의 머리 주위 스윙 오금 포사 위의 좌골 신경에서 발생한다. 피상적 깊은 비골 신경으로 분기 함께 발과 발가락의 dorsiflexors을 공급 (전방 경골근, 신근 심지 longus 및 브레비스 및 신근 halluces longus 근육), 그리고 다리의 everters (peroneus 근육)이 그것. 신경이 또한 발과 다리의 ...

Watch this Scientific Journal Video about The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions at JoVE.com

The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions

We have developed a nerve injury method to reliably examine muscle reinnervation, and thus regeneration of neuromuscular junctions in mice. This technique involves injuring the common fibular nerve via a simple and highly reproducible surgery. Muscle reinnervation in then assessed by whole-mounting the extensor digitorum longus muscle.

비골 신경 손상 방법 : 신뢰할 수있는 분석은 확인하고 테스트 즉, 수리 신경 근육 학 접합 요인

The neuromuscular junction (NMJ) undergoes deleterious structural and functional changes as a result of aging, injury and disease. Thus, it is imperative ...

the-fibular-nerve-injury-method-reliable-assay-to-identify-test

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

15.6K 조회수.  Virginia Tech. We have developed a nerve injury method to reliably examine muscle reinnervation, and thus regeneration of neuromuscular junctions in mice. This technique involves injuring the common fibular nerve via a simple and highly reproducible surgery. Muscle reinnervation in then assessed by whole-mounting the extensor digitorum longus muscle. 

비디오: The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

전체 마운트 근육 분석에 의한 재현성 마우스 좌골 신경 크러시와 중생의 후속 평가

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

연구

신경과학

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 ...

생체공학

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

경피적 전기 신경 자극을 사용하여 신경 근육 기능의 평가

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Murine Flexor Tendon Injury and Repair Surgery

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers and general hand function. Injuries to the FTs are common, and satisfactory healing is often impaired due to excess scar tissue and adhesions between the tendon and surrounding tissue. However, little is known about the molecular and cellular components of FT repair. To that end, a murine model of FT repair that recapitulates many aspects of healing in humans, including impaired range of motion and decreased mechanical properties, has been developed and previously described. Here an in-depth demonstration of this surgical procedure is provided, involving transection and subsequent repair of the flexor digitorum longus (FDL) tendon in the murine hind paw.&#160;This technique can be used to conduct lineage analysis of different cell types, assess the effects of gene gain or loss-of-function, and to test the efficacy of pharmacological interventions in the healing process. However, there are two primary limitations to this model: i) the FDL tendon in the mid-portion of the murine hind paw, where the transection and repair occur, is not surrounded by a synovial sheath. Therefore this model does not account for the potential contribution of the sheath to the scar formation process. ii) To protect the integrity of the repair site, the FT is released at the myotendinous junction, decreasing the mechanical forces of the tendon, likely contributing to increased scar formation. Isolation of sufficient cells from the granulation tissue of the FT during the healing process for flow cytometric analysis has proved challenging; cytology centrifugation to concentrate these cells is an alternate method used, and allows for generation of cell preparations on which immunofluorescent labeling can be performed. With this method, quantification of cells or proteins of interest during FT healing becomes possible.

Flexor tendons in the hand are commonly injured, leading to impaired hand function. However, the scar-tissue healing response is not well characterized. A murine model of flexor tendon healing is demonstrated here. This model can enhance overall understanding of the healing process and assess therapeutic approaches to improve healing.

쥐의 굴근 힘줄 손상 및 복구 수술

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers ...

의학

Functional and Physiological Methods of Evaluating Median Nerve Regeneration in the Rat

The main goal of this investigation is to show how to create and repair different types of median nerve (MN) lesions in the rat. Moreover, different methods of simulating postoperative physiotherapy are presented. Multiple standardized strategies are used to assess motor and sensory recovery using an MN model of peripheral nerve lesion and repair, thus permitting easy comparison of the results. Several options are included for providing a postoperative physiotherapy-like environment to rats that have undergone MN injuries. Finally, the paper provides a method to evaluate the recovery of the MN using several noninvasive tests (i.e., grasping test, pin prick test, ladder rung walking test, rope climbing test, and walking track analysis), and physiological measurements (infrared thermography, electroneuromyography, flexion strength evaluation, and flexor carpi radialis muscle weight determination). Hence, this model seems particularly appropriate to replicate a clinical scenario, facilitating extrapolation of results to the human species.
Although the sciatic nerve is the most studied nerve in peripheral nerve research, analysis of the rat MN presents various advantages. For example, there is a reduced incidence of joint contractures and automutilation of the affected limb in MN lesion studies. Furthermore, the MN is not covered by muscle masses, making its dissection easier than that of the sciatic nerve. In addition, MN recovery is observed sooner, because the MN is shorter than the sciatic nerve. Also, the MN has a parallel path to the ulnar nerve in the arm. Hence, the ulnar nerve can be easily used as the nerve graft for repairing MN injuries. Finally, the MN in rats is located in the forelimb, akin to the human upper limb; in humans, the upper limb is the site of most peripheral nerve lesions.

Presented is a protocol to produce different types of median nerve (MN) lesions and repair in the rat. Additionally, the protocol shows how to evaluate the functional recovery of the nerve using several noninvasive behavioral tests and physiological measurements.

쥐에서 중간 신경 재생을 평가하는 기능적 및 생리적 방법

The main goal of this investigation is to show how to create and repair different types of median nerve (MN) lesions in the rat. Moreover, different ...

Maximum Isometric Tetanic Force Measurement of the Tibialis Anterior Muscle in the Rat

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve grafts. Due to their limited availability and associated donor side morbidity, many studies in the field of nerve regeneration focus on alternative techniques to bridge a segmental nerve gap. In order to investigate the outcomes of surgical or pharmacological experimental treatment options, the rat sciatic nerve model is often used as a bioassay. There are a variety of outcome measurements used in rat models to determine the extent of nerve regeneration. The maximum output force of the target muscle remains the most relevant outcome for clinical translation of experimental therapies. Isometric force measurement of tetanic muscle contraction has previously been described as a reproducible and valid technique for evaluating motor recovery after nerve injury or repair in both rat and rabbit models. In this video, we will provide a step-by-step instruction of this invaluable procedure for assessment of functional recovery of the tibialis anterior muscle in a rat sciatic nerve defect model using optimized parameters. We will describe the necessary pre-surgical preparations in addition to the surgical approach and dissection of the common peroneal nerve and tibialis anterior muscle tendon. The isometric tetanic force measurement technique will be detailed. Determining the optimal muscle length and stimulus pulse frequency is explained and measuring the maximum tetanic muscle contraction is demonstrated.

Evaluation of motor recovery remains the benchmark outcome measure in experimental peripheral nerve studies. The isometric tetanic force measurement of the tibialis anterior muscle in the rat is an invaluable tool to assess functional outcomes after reconstruction of sciatic nerve defects. The methods and nuances are detailed in this article.

쥐의 티비아리스 전방 근육의 최대 동면 성 파타닉 힘 측정

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve ...

Implantation and Control of Wireless, Battery-free Systems for Peripheral Nerve Interfacing

Peripheral nerve interfaces are frequently used in experimental neuroscience and regenerative medicine for a wide variety of applications. Such interfaces can be sensors, actuators, or both. Traditional methods of peripheral nerve interfacing must either tether to an external system or rely on battery power that limits the time frame for operation. With recent developments of wireless, battery-free, and fully implantable peripheral nerve interfaces, a new class of devices can offer capabilities that match or exceed those of their wired or battery-powered precursors. This paper describes methods to (i) surgically implant and (ii) wirelessly power and control this system in adult rats. The sciatic and phrenic nerve models were selected as examples to highlight the versatility of this approach. The paper shows how the peripheral nerve interface can evoke compound muscle action potentials (CMAPs), deliver a therapeutic electrical stimulation protocol, and incorporate a conduit for the repair of peripheral nerve injury. Such devices offer expanded treatment options for single-dose or repeated dose therapeutic stimulation and can be adapted to a variety of nerve locations.

This is a protocol for the surgical implantation and operation of a wirelessly powered interface for peripheral nerves. We demonstrate the utility of this approach with examples from nerve stimulators placed on either the rat sciatic or phrenic nerve.

말초 신경 인터페이스를 위한 배터리가 필요 없는 무선 시스템의 이식 및 제어

Peripheral nerve interfaces are frequently used in experimental neuroscience and regenerative medicine for a wide variety of applications. Such interfaces ...

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

<meta charset="utf8"></head><body>마우스의 신경병증성 통증에 대한 수정된 보존 신경 손상 수술 모델

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

비골 신경 손상 방법 : 신뢰할 수있는 분석은 확인하고 테스트 즉, 수리 신경 근육 학 접합 요인

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