이 연구는 난청 및 기타 의사 소통 장애 (R21DC010806에서 CAM 및 JCS로, R01DC011285에서 CAM로)의 국립 연구소로부터의 보조금에 의해 지원되었습니다.

이 프로토콜에 대해 6 개월에서 30 개월 된 수컷 Fischer 344-Brown Norway 쥐를 사용하십시오. 랫트는 국립 늙은 쥐 서식지 연구소 (National Institute of Aging)의 설치류 식민지에서 얻은 것이다. 랫트 후두의 구조는기도 보호 및 종 특이 적 발성을 위해 기능적으로 작용하는 인간의 것과 유사하기 때문에 본 연구를 위해 쥐를 사용했다.이 연구는 실험실 동물의 인간애 관리 및 사용에 관한 PHS 정책에 따라 수행되었으며, 실험실 동물의 관리 및 사용을위한 NIH 안내서 및 동물 복지법 (7 USC 외); 동물 사용 프로토콜은 University of Kentucky의 IACUC (Institutional Animal Care and Use Committee)에 의해 승인되었습니다. 1. 쥐의 마취 <ol><li> 완충 식염수에 ketamine hydrochloride (해리 성 마취제)와 xylazine hydrochloride (진정제 및 진통제)를 혼합하여 마취약을 준비합니다. 최종 용액에서 ketamine과 xylazine의 농도는 kg 당 100 mg / 8 mg체중 각각. </li><li> 25G 바늘과 주사기를 사용하여 intraperitoneal 투여하여 쥐에 마취제를 주사. </li><li> 쥐가 포셉과 발가락이나 발을 꼬집어 의해 충분히 anesthetized 있는지 확인하십시오. 쥐가 꼬집음에 반응하지 않으면 수술을 시작할 수 있습니다. 쥐가 반사 또는 근육 수축으로 발가락 핀치에 반응하면 1 ~ 2 분을 기다렸다가 핀치 테스트를 반복하십시오. 쥐가 다시 반응하면 쥐를 새 동물로 바꾸고 1.2 단계부터 시작하여 절차를 반복하십시오. </li><li> 쥐가 움직이지 않으면 각막이 마르지 않도록 쥐의 눈에 안 연고를 바릅니다. </li></ol> 2. 삼투압 펌프 이식 <ol><li> 쥐를 무균 수술 부위에 복강으로 놓습니다. Preanesthetic 약물로 meloxicam를 관리하십시오. 25G 주사 바늘이 달린 주사기를 사용하여 체중 1kg 당 1-4mg의 용량으로 복강 내 투여. </li><li> 클리퍼를 사용하여 근사치 제거목 뒤에서 모피 1 &quot;x 1&quot;정사각형, 어깨 사이에 약 1 &quot;꼬리 공간. 가능하면 피부 가까이에 면도하십시오. </li><li> 에탄올 소독 (70 %)으로 목과 뒷부분을 적시십시오. </li><li> 면도 후, 알코올로 요오드 - 알코올 마무리를 연속적으로 3 번 문지르면 목의 등면을 문지릅니다. </li><li> 34 ° C로 설정된 가열 패드 위에 놓아 쥐의 체온을 유지하십시오. </li><li> 전신 NTF4 처리를 위해 50 μL의 NTF4 또는 식염수로 무균 처리 된 삼투 펌프를 채 웁니다 ( 그림 1 ). <ol><li> 메스를 사용하여 피부를 가로 지르는 약 2cm의 수평 절개를하고, 견갑골 사이의 공간으로 단지 두개의 절개를하십시오. 한쪽 손으로 겸자로 절개의 후부 가장자리를 들어 올리면서 hemostats의 팁을 삽입하고 절개 부분을 부드럽게 밀어 넣습니다. </li><li> 지혈 기 끝이 약 2 cm c목덜미를 절개 부위에 놓고 지혈대의 손잡이를 열어 절개 부위의 피하에 중공 &quot;포켓&quot;을 형성하도록 팁을 확장시킵니다. 이것은 펌프의 배치 사이트가됩니다. </li></ol></li><li> NTF4의 상호 작용과 포켓 절개 부위의 치유를 최소화하기 위해 삽입시 펌프 전달 포털 끝을 먼저 배향하십시오. </li><li> NTF4 생리 식염수 50 μL를 7 14 일 동안 전달하십시오. 7 일군은 6.72 mg / day의 NTF4를 투여 받았고 총 용량은 47.04 mg이었다. 14 일군은 6.72 mg / 일을 투여 받았고 총 용량은 94.08 mg NTF4 25 이었다. </li><li> 5-0 나일론 봉합사, hemostats 및 포셉 펌프 배치를 위해 만들어진 절개를 닫습니다. </li><li> 마취에서 회복되면서 최소 30 분 동안 쥐를 관찰하십시오. 모니터링 완료 기준은 동물이 활성화되고, 새장 주위로 이동하고, 식수를 공급하고, 정리와 같은 기타 정상적인 활동을 시작하는 것을 포함합니다. </li><li> 매일 동물 관찰수술 부위의 치유, 정상적인 사료 및 물 소비, 소변 / 대변 통과, 스트레스, 통증 또는 기타 수술후 합병증의 비정상적인 행동 징후를 관찰하여 첫 주. </li><li> 쥐가 통증이나 고통을 겪고있는 것으로 나타나면 통증 완화를 위해 최대 24 시간마다 carprofen을 5 mg / kg 피하 주사하여 5 일 동안 투여하십시오. </li><li> 감염이있는 경우 수의사와상의하여 상처가 적절하게 치료되도록하십시오. </li><li> 어떤 실험 그룹에 따라 쥐가에 스레드에서 자극을 방지하기 위해 수술 후 7-10 일 5-0 나일론 봉합을 제거합니다. </li></ol> 3. 직접 주입을위한 쥐의 마취 <ol><li> 수술 전날 밤에 쥐에게서 음식물을 보류하십시오. 이렇게하면 내시경이나 주사 바늘을 막을 음식이 없습니다. </li><li> 쥐를 체중을 줄이고 아세 롤마 민 1 ~ 2 mg / kg 체중을 준비하십시오. 근육 주사로 주사하십시오 (IM 위치는thyroarytenoid 근육을 떠났다. </li><li> 유도 상자에 쥐를 놓으십시오. 5 % isoflurane과 1 LO 2로 유도 상자에서 마취를 유도하십시오. </li><li> 2 % isoflurane과 600mL O 2 를 사용하여 쥐 원추로 쥐를 이동하십시오. </li><li> 쥐가 포셉과 발가락이나 발을 꼬집어 의해 충분히 anesthetized 있는지 확인하십시오. 쥐가 핀치에 반응하지 않으면 주사 프로토콜을 시작할 수 있습니다. 쥐가 반사 또는 근육 수축으로 발가락 핀치에 반응하면 1 ~ 2 분을 기다렸다가 핀치 테스트를 반복하십시오. 쥐가 다시 반응하면 쥐를 새 동물로 바꾸고 단계 3.4부터 시작하여 절차를 반복하십시오. </li></ol> 4. 직접 주입 및 시각화 <ol><li> NTF4 또는 생리 식염수가 포함 된 무균 적으로 준비한 50 μL 용량을 주사하기 전에 30 분 동안 25 ° C로 설정된 H2O 배스에 놓습니다. </li><li> 플렉시 유리 플랫폼 ( 그림 2 )에서 쥐를 앙와위 자세로 올려 놓습니다. Sus플랫폼의 꼭대기를 가로 지르는 가이드 와이어를 통해 정면 상단 절치에서 젖혀지는 자세로 쥐를 지키십시오. </li><li> 50 mm, 30 게이지, 100 μL 주사기를 1.9 mm, 30 ° sinus 내시경에 부착하십시오 ( 그림 3 ). 참고 : 주사기 어셈블리는 내시경의 외벽에 캐뉼라를 단단히 고정하는 지그를 통해 부착됩니다. 내시경은 성대의 시각화 및 구강 내 주사기 안내를 허용합니다. 각 동물에 앞서 캐 뉼러 팁의 위치를 ​​조정하여 팁이 내시경보기를 통해 완벽하고 명확하게 보이는지 확인합니다 ( 그림 4 ). </li><li> 혀를 연장하고 그것을 옆으로 움직이려면 고무로 깎은 한 쌍의 집게를 사용하십시오. 그런 다음 구강 개통을 유지하기 위해 플라스틱 견본을 삽입하십시오. 1.5 ~ 2 cm 길이로 자른 5 mL 플라스틱 시린지 배럴에서 검체를 만들고, 절단 된 가장자리는 떨림을 제거하고 부드럽게 연마합니다. </li><li> 루아의 조명을 끄십시오.할로겐 광원을 내시경에 부착하십시오. 비디오 레코더를 켜서 절차를 캡처하십시오. </li><li> 쥐의 입에 넣을 때 유리 팁에 결로 현상을 최소화하기 위해 내시경의 말단을 따뜻한 물에 몇 초 동안 담그십시오. </li><li> 모니터의 시각적 피드백을 사용하여 바늘을 왼쪽 성대 영역으로 조심스럽게 당깁니다. </li><li> 성대에 완전히 접근하기 위해 동물 호흡주기의 흡기 단계로 용액 주입을 시간을 재십시오. 호흡의 흡기 단계에서 성대가 완전히 노출됩니다. <ol><li> 성대가 완전히 보이면, 바늘을 음성 골격의 흰색 중앙 가장자리 옆쪽에있는 왼쪽 thyroarytenoid에 삽입하십시오. 주사 바늘을 제 위치에 놓고 주사기의 우울증을 통해 주사액을 전달하십시오. </li></ol></li><li> 내시경과 비디오 플레이어의 할로겐 광원을 끄고 ro를 다시 켭니다.옴 조명. </li><li> 쥐를 집에 넣고 가열 패드에 놓습니다. </li><li> 발열 패드에서 제거하기 전에 쥐가 회복되도록하십시오. 새장에서 음식과 물을 교체하십시오. </li><li> 주입 후 7 일 동안 래트를 모니터링 한 다음 안락사하십시오. 절제를위한 후두 제거 24 . </li></ol> 5. 쥐의 안락사 <ol><li> 쥐를 케타민 하이드로 클로라이드 및 크 실라 진 하이드로 클로라이드 (체중 kg 당 100mg / 8mg 복강 내 주사)로 마취시킨다. </li><li> 내측 thoracotomy 다음 exsanguination에 의해 안락사. </li></ol>

<ol>
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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=An+in+vivo+study+of+composite+microgels+based+on+hyaluronic+acid+and+gelatin+for+the+reconstruction+of+surgically+injured+rat+vocal+folds.">An in vivo study of composite microgels based on hyaluronic acid and gelatin for the reconstruction of surgically injured rat vocal folds.</a> J. Speech Lang Hear Res. 57 (2), S658-S673 (2014).</li><li>Herteg&#229;rd, 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=Cross-linked+hyaluronan+used+as+augmentation+substance+for+treatment+of+glottal+insufficiency:+Safety+aspects+and+vocal+fold+function.">Cross-linked hyaluronan used as augmentation substance for treatment of glottal insufficiency: Safety aspects and vocal fold function.</a> Laryngoscope. 112 (12), 2211-2219 (2002).</li><li>Walker, W. F., Homberger, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anatomy and dissection of the rat. , (1997).</li><li>Ohno, T., Hirano, S., Rousseau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+changes+in+the+expression+and+deposition+of+vocal+fold+collagen+and+hyaluronan.">Age-associated changes in the expression and deposition of vocal fold collagen and hyaluronan.</a> Ann. Oto. Rhinol. Laryngol. 25 (1), 192-197 (2009).</li><li>Stemple, J. 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=Enhancement+of+aging+rat+laryngeal+muscles+with+endogenous+growth+factor+treatment.">Enhancement of aging rat laryngeal muscles with endogenous growth factor treatment.</a> Physiol. Rep. 4 (10), e12798 (2016).</li><li>McMullen, C. A., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+dysfunction+and+altered+metabolic+profile+of+the+aging+rat+thyroarytenoid+muscle.">Contractile dysfunction and altered metabolic profile of the aging rat thyroarytenoid muscle.</a> J. Appl. Phys. 100 (2), 602-608 (2006).</li><li>Engle, W. K., Cunningham, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+examination+of+muscle+tissue.+An+improved+trichrome+method+for+fresh-frozen+biopsy+section.">Rapid examination of muscle tissue. An improved trichrome method for fresh-frozen biopsy section.</a> Neurology. (13), 919-923 (1963).</li><li>Sheehan, D. C., Hrapchack, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+and+practice+of+histotechnology.">Theory and practice of histotechnology.</a> Battelle. , (1980).</li><li>Kulakowski, S. A., Parker, S. D., Personius, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+TrkB+expression+results+in+precocious+age-like+changes+in+neuromuscular+structure,+neurotransmission,+and+muscle+function.">Reduced TrkB expression results in precocious age-like changes in neuromuscular structure, neurotransmission, and muscle function.</a> J. Appl. Phys. 111 (3), 844-852 (2011).</li><li>Nishida, N., 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=Age-related+change+in+rat+intrinsic+laryngeal+muscles:+analysis+of+muscle+fibers,+muscle+fiber+proteins+and+subneural+apparatuses.">Age-related change in rat intrinsic laryngeal muscles: analysis of muscle fibers, muscle fiber proteins and subneural apparatuses.</a> Eur Arch Otorhinolaryngol. 270 (3), 975-984 (2013).</li></ol>

저자는 공개 할 것이 없습니다.

후두 근육은 노화의 부작용에 취약합니다. 이전의 연구들은 수축 기능과 myosin isoform 변이의 변화뿐만 아니라 섬유 크기, 총 섬유 수, 재생 능력, NMJ 크기 및 양 변화에 대한 후두 근육의 변화를 보여주었습니다 4 , 11 , 27 , 30 , 31 . 노화 후두 근육은 신경 영양소의 적용으로 바?...

후두 근육은 신속하고 지속적으로 수축하며 노화의 부작용에 취약합니다. 이 지속적인 활동은 65 세 이상의 1 , 2 , 3 , 4 , 5 , 6 , 7 명에서 관찰되는 목소리 문제 또는 연하 장애에 기여하는 것으로 생각됩니다. 몇 가지 분자 및 pathophysiologic 메커니즘이 나이 관련 기능 장애에 기여한다. 이러한 메커니즘은 후두 점막의 재건, 근육 섬유 위축 또는 손실, 성대의 굽힘과 성문 폐쇄의 원인이되는 근섬유 재생 또는 위축의 결핍을 포함 할 수 있습니다 8 , 9 , 10 , 11 . 현재로서는 입증 된 의학 요법이 없습니다.이러한 근육의 연령 관련 변화를 철저히 예방하거나 재활시킵니다. 신경근 전달의 효과를 조절하면 신경 모터의 성능에 큰 영향을 줄 수 있습니다. 뉴로 트로 핀의 계열은 신경 성장 인자 (NGF), 뇌 파생 신경 성장 인자 (BDNF), 뉴로 트로 핀 3 (NTF3) 및 NTF4 12 , 13을 포함 한다. 뉴로 트로 핀은 시냅스 효능을 조절하는 것으로 나타났습니다. 간세포 성장 인자, 형질 전환 성장 인자 베타 및 섬유 아세포 성장 인자는 최근 성대 흉터 치료를 위해 인간에서 사용되어왔다. NTF4는 또한 NMJ 효과를 조절합니다. NTF4가 결핍 된 마우스는 해체 된 NMJs 11 , 18 , 19를 보여준다. 이 연구는 치료자의 유망한 효과를 가져옵니다고령화 된 후두 근육 장애 및 성장 인자로 인한 노화. 성대 주름의 조직에 대한 직접 주사 치료제는 인간에게 전례가 없습니다. 예를 들어, 보툴리누스 독소의 국소 주사는 경련성 발성 장애 및 양측 재발 성 후두 신경 마비와 같은 후두 근육에 영향을 미치는 신경 학적 운동 장애에 대한 효과적인 치료법으로 현재 사용되고 있습니다 20 , 21 . Hyaluronic acid hydrogel은 보컬 폴드 징후와 성문 기능 부전을 치료하는 데 사용되는 또 다른 주사제입니다 22 , 23 . 주사 후두 성형술은 다양한 의사 소통 장애 치료에 사용될 수 있습니다 24 . 이러한 직접적인 주사 방법은 노령 인구에서 보컬 기능과 삼킴을 향상시킬 수 있습니다.

<table><tbody><tr><td>Neurotrophin 4</td><td>Pepro Tech</td><td>450-04</td><td>200 ng in 50 &amp;mu;L</td></tr><tr><td>Alzet Osmotic Pump</td><td>DURECT Corporation</td><td>2001D</td><td></td></tr><tr><td>30 &amp;deg; endoscope</td><td>Stoltz</td><td>61029D</td><td></td></tr><tr><td>50 mm 30 gauge 100-&amp;mu;L syringe</td><td>Hamilton</td><td>84850 and 201812</td><td></td></tr><tr><td>saline (sodium chloride solution)</td><td>Sigma-Aldrich</td><td>S8776</td><td></td></tr><tr><td>ketamine hydrochloride</td><td>Henry Schein</td><td>56344</td><td></td></tr><tr><td>xylazine hydrochloride</td><td>Henry Schein</td><td>33198</td><td></td></tr><tr><td>25 G 5/8 needle</td><td>Becton-Dickinson</td><td>305901</td><td></td></tr><tr><td>1 mL syringe</td><td>Becton-Dickinson</td><td>309659</td><td></td></tr><tr><td>ophthalmic ointment</td><td>Henry Schein</td><td>8897</td><td></td></tr><tr><td>clippers</td><td>Oster</td><td>44-018</td><td></td></tr><tr><td>ethanol</td><td>Decon</td><td>2716</td><td></td></tr><tr><td>iodine (Betadine)</td><td>Purdue Pharma L.P.</td><td>606404</td><td></td></tr><tr><td>heating pad</td><td>Sunbeam</td><td>731-5</td><td></td></tr><tr><td>5-0 nylon suture thread</td><td>AD Surgical</td><td>PMN-518R6</td><td></td></tr><tr><td>crile hemostat</td><td>Fine Science Tools</td><td>13005-14</td><td></td></tr><tr><td>delicate suture tying forceps</td><td>Fine Science Tools</td><td>11063-07</td><td></td></tr><tr><td>meloxicam</td><td>Henry Schein</td><td>49756</td><td></td></tr><tr><td>carprofen</td><td>Merritt Veterinary Supplies</td><td>148700</td><td></td></tr><tr><td>antibiotic ointment</td><td>Henry Schein</td><td>57110</td><td></td></tr><tr><td>acepromizine Aceproject</td><td>Henry Schein</td><td>3845</td><td></td></tr><tr><td>isoflurane Isothesia</td><td>Henry Schein</td><td>50033</td><td></td></tr><tr><td>induction box (anesthetizing box)</td><td>Harvard Apparatus</td><td>50-0116</td><td></td></tr><tr><td>oxygen compressed tank</td><td>Scott Gross</td><td>UN1072</td><td></td></tr><tr><td>plexiglas platform</td><td>Small Parts Inc (Amazon)</td><td></td><td></td></tr><tr><td>rubber tipped forceps</td><td>Fine science tools rubber</td><td>11075-00</td><td></td></tr><tr><td>liquid rubber for forceps above</td><td>Lowe&#039;s</td><td>42518</td><td></td></tr><tr><td>plastic spectula (BD syringe cut to length)</td><td>Becton-Dickinson</td><td>309659</td><td></td></tr><tr><td>halogen light source rhino-laryngeal stroboscope</td><td>Kay-Pentax</td><td>RLS 9100 B</td><td></td></tr><tr><td>video recorder</td><td>Kay-Pentax</td><td></td><td></td></tr><tr><td>sucrose</td><td>Sigma-Aldrich</td><td>S0389-500G</td><td></td></tr><tr><td>phosphate buffered saline</td><td>Sigma-Aldrich</td><td>P4417-100TAB</td><td></td></tr><tr><td>cryostat Mictotom HM525</td><td>Thermo Scientific</td><td>HM 525</td><td></td></tr><tr><td>Gill 1 hematoxylin</td><td>VWR</td><td>10143-142</td><td></td></tr><tr><td>Shandon eosin-Y alcoholic</td><td>Thermo Fisher Scientific</td><td>6766007</td><td></td></tr><tr><td>anti-sodium channel Nav1.5 antibody produced in rabbit</td><td>Sigma-Aldrich</td><td>S0819</td><td></td></tr><tr><td>Texas red-X phalloidin</td><td>Sigma-Aldrich</td><td>T7471</td><td></td></tr><tr><td>alpha- bungarotoxin alexa fluor 488 conjugate</td><td>Thermo Fisher Scientific</td><td>B-13422</td><td></td></tr><tr><td>Small animal anaesthesia machine</td><td>Smiths Medical</td><td>CDS 9000</td><td></td></tr></tbody></table>

subcutaneous neurotrophin 4 infusion using osmotic pumps or direct muscular injection enhances aging rat laryngeal muscles

노인에서 후두 장애는 음성 장애에서부터 연하 장애로,기도 보호 반사 신경 장애로 이어집니다. 연령 관련 후두 근육 기능 장애를 표적으로하는 치료법은 거의 없습니다. 뉴로 트로 핀 (neurotrophin)은 근육 신경 분포 및 신경근 접합부 (NMUs)의 분화에 관여합니다. 뉴로 트로 핀은 신경 전달 물질 방출을 증가시킴으로써 신경근 전달을 향상시키는 것으로 생각됩니다. neuromuscular junctions (NMJs)는 노화 된 쥐의 후두 근육에서 작고 덜 풍부 해지고, 기능적 소실의 증거가 있습니다. 우리는 노화 후두 근육의 기능을 향상시키는 치료제로서 미래의 임상 적 사용을위한 NTF4의 효과를 탐구했다. 여기서는 NTF4 적용에 대한 반응으로 쥐의 두드러기 후두 근육이 재 형성되는 능력을 조사하기 위해 NTF4의 전신 투여 및 직접 주사에 대한 상세한 프로토콜을 제공합니다. 이 방법에서는 쥐가 NTF4를 전신적 으로 o자극적 인 펌프 또는 성대를 통한 직접 주입에 의한 것입니다. 후속 근육을 절개하여 형태학 및 연령 관련 소실에 대한 조직 학적 검사에 사용 하였다.

2 주 동안 삼투압 펌프를 주입하거나 NTF4를 직접 주입 한 후 1 주일 동안 랫트를 안락사시켰다. 후두를 수확하고 동결 방지제 (30 % 자당 및 70 % 인산 완충 생리 식염수)에 넣은 다음 저온 유지 장치를 사용하여 10-μm 폭으로 연속 절편을 만들었습니다. 나이가 들어간 후두 근육은 NTF4 투여로 영향을받습니다 25 . 젊고 오래된 쥐 이외에, 우리는 thyroarytenoid 근?...

Watch this Scientific Journal Video about Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles at JoVE.com

Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles

여기에서 우리는 쥐 노화 후두 근육을 재구성하기 위해 전신적으로 그리고 직접적으로 뉴로 트로 핀 4 (NTF4)의 사용을 기술하는 프로토콜을 제시합니다.

삼투압 펌프 또는 직접적인 근육 주사를 이용한 피하 뉴로트로핀 4 주입으로 노화 후두 근육 강화

Laryngeal dysfunction in the elderly is a major cause of disability, from voice disorders to dysphagia and loss of airway protective reflexes. Few, if ...

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8.3K 조회수.  University of Kentucky.  여기에서 우리는 쥐 노화 후두 근육을 재구성하기 위해 전신적으로 그리고 직접적으로 뉴로 트로 핀 4 (NTF4)의 사용을 기술하는 프로토콜을 제시합니다. 

비디오: Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles

Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1 and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2.
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4. Transgenic mice 4,5 and rats 6 carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R) have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8.
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9.
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A mice and rats, as well as spinal cord injured rats and mice 11.

Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.

루게릭병과 외상 척수 상해에서 자궁암 복부 호른를 타겟팅하는 Intraspinal 세포 이식

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) ...

연구

의학

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 ...

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo pharmacological studies of the signals essential for the formation and maintenance of mammalian NMJs. However, drug penetration into these muscles after subcutaneous or intramuscular administration is often incomplete or uneven and many NMJs can remain unaffected. Although systemic administration with devices such as mini-pumps can improve the spatiotemporal effects, the invasive nature of this approach can cause confounding inflammatory responses and/or direct muscle damage. Moreover, complete analysis of the NMJs in a hind limb muscle is challenging because it requires time-consuming serial sectioning and extensive immunostaining. 
The mouse LAL is a thin, flat sheet of muscle located superficially on the dorsum of the neck. It is a fast-twitch muscle that functions to move the pinna. It contains rostral and caudal portions that originate from the midline of the cranium and extend laterally to the cartilaginous portion of each pinna. The muscle is supplied by a branch of the facial nerve that projects caudally as it exits the stylomastoid foramen. We and others have found LAL to be a convenient preparation that offers advantages for the investigation of both short and long-term in vivo effects of drugs on NMJs and muscles. First, its superficial location facilitates multiple local applications of drugs under light anesthesia. Second, its thinness (2-3 layers of muscle fibers) permits visualization and analysis of almost all the NMJs within the muscle. Third, the ease of dissecting it with its nerve intact together with the pattern of its innervation permits supplementary electrophysiological analysis in vitro9,5. Last, and perhaps most importantly, a small applied volume (&tilde;50&mu;l) easily covers the entire muscle surface, provides a uniform and prolonged exposure of all its NMJs to the drug and eliminates the need for a systemic approach1,8.

We describe procedures for repeated administration of inhibitors of muscarinic signaling to the levator auris longus (LAL) muscle of young adult mice and for subsequent immunostaining of its neuromuscular junctions (NMJs) in wholemounts. The LAL muscle has unique advantages for revealing in vivo pharmacological effects on NMJs.

피하 Muscarinic Antagonists의 관리 및 마우스에 Levator Auris의 Longus 근육의 트리플 - Immunostaining

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

신경과학

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

경골 신경 절개 - 마우스의 탈 신경에 의한 골격근의 위축에 대한 표준 모델

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

말초 신경 손상 후 기능 회복을 평가하기 위해 후각 Ensheathing 세포의 이식

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the development of several animal models of these neuromuscular disorders has provided the scientific community with different therapeutic scenarios aimed at delaying or reversing the progression of these conditions. By taking advantage of the retrograde machinery of neurons, one of these approaches has been to target skeletal muscles in order to shuttle therapeutic genes into corresponding spinal cord motor neurons. Although once promising, the success of such gene delivery approach has been hampered by the sub-optimal number of transduced motor neurons it has so far shown to yield. Motor end plates (MEPs) are highly specialized regions on the skeletal musculature that are in direct synaptic contact to the spinal cord &#945; motor neurons. In this regard, it is important to note that, so far, the efforts to retrogradely transfer genes into motor neurons were made without reference to the location of the MEP region in the targeted muscles. Here, we describe a simple protocol 1) to reveal the exact location of the MEPs on the surface of skeletal muscles and 2) to use this information to guide the intramuscular delivery and subsequent optimal retrograde transport of retrograde tracers into motor neurons. We hope to utilize the results from these tracing experiments in further studies into investigating retrograde transport of therapeutic genes to spinal cord motor neurons through the targeting of MEPs.

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

모터 엔드 플레이트 함께 근육 주사 : 직접 모터 뉴런에 셔틀 트레이서에 대한 최소 침습 접근

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator.
Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 107 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage.
This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.

Here, we present a protocol to inject autologous muscle-derived stem cells in the proximity to the recurrent laryngeal nerve with the help of an electrical nerve stimulator. This novel technique may become useful for the treatment of equine recurrent laryngeal neuropathy.

말 왼쪽된 회귀 후 두 신경 근처 헌 혈 줄기 세포의 신경 자극을 유도 주사

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent ...

An Implantable System For Chronic In Vivo Electromyography

Electromyography (EMG) measures the muscle response to electrical stimulation or spontaneous activity of motor units and plays an important role in assessing neuromuscular function. Chronic recording of EMG activity reflecting a muscle&#8217;s reinnervation status after nerve injury has been limited, due to the invasive nature of traditional EMG recording techniques. In this regard, an implantable system is designed for long-term, in vivo EMG recording and nerve stimulation. It has been applied and tested in a study on reinnervation of laryngeal muscles. This system consists of 1) two bipolar electrode nerve cuffs and leads for stimulating each of two nerves: the recurrent laryngeal nerve (RLN) and internal branch of the superior laryngeal nerve (SLN); 2) two EMG recording electrodes and leads for each of the two laryngeal muscles: posterior cricoarytenoid (PCA) muscle and thyroarytenoid-lateral cricoarytenoid (TA-LCA) muscle complex; and 3) a skin receptacle interfacing all implanted lead terminals to an external recording preamplifier and stimulator using a connection cable. The wire leads are Teflon-coated, multi-filament, type 316 stainless steel. They are coiled and can stretch during body movement of the awake animal to prevent lead breakage and electrode migration. This system is implanted during an aseptic surgery. Afterwards, baseline EMG recordings are performed before the RLN is transected in the second surgery to study muscle reinnervation. Throughout the study, multiple physiological sessions are conducted in the anesthetized animal to obtain evoked and spontaneous EMG activity that reflects the reinnervation status of laryngeal muscles. The system is compact, free of infection over the course of the study, and highly durable. This implantable system can provide a reliable platform for research in which long-term recording or nerve stimulation is required in an anesthetized or freely moving animal.&#160;

Presented here is a protocol for the manufacturing of an implantable system for in vivo chronological recording of evoked and spontaneous electromyographic potentials. The system is applied to the investigation of reinnervation of laryngeal muscles following nerve injury. &#160;&#160;

만성 생체 내 근전도를 위한 이식형 시스템

Electromyography (EMG) measures the muscle response to electrical stimulation or spontaneous activity of motor units and plays an important role in ...

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures for rat laryngeal dissection, flash-freezing, and cryosectioning of the vocal folds. This study describes how to cryosection vocal folds in both longitudinal and cross-sectional planes. A novelty of this protocol is the laryngeal tracking during cryosectioning that ensures accurate identification of the intrinsic laryngeal muscles and reduces the chance of tissue loss. Figures demonstrate the progressive cryosectioning in both planes. Twenty-nine rat hemi-larynges were cryosectioned and tracked from the emergence of the thyroid cartilage to the appearance of the first section that included the full vocal fold. The full vocal fold was visualized for all animals in both planes. There was high variability in the distance from the appearance of the thyroid cartilage to the appearance of the full vocal fold in both planes. Weight was not correlated to depth of laryngeal landmarks, suggesting individual variability and other factors related to tissue preparation may be responsible for the high variability in the appearance of landmarks during sectioning. This study details a methodology and presents morphological data for preparing the rat vocal fold for histochemical neuromuscular investigation. Due to high individual variability, laryngeal landmarks should be closely tracked during cryosectioning to prevent oversectioning tissue and tissue loss. The use of a consistent methodology, including adequate tissue preparation and awareness of landmarks within the rat larynx, will assist with consistent results across studies and aid new researchers interested in using the rat vocal fold as a model to investigate laryngeal neuromuscular mechanisms.

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

신경 근육 분석을위한 쥐 보컬 폴드의 준비

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...

<meta charset="utf8"></head><body>Electroporation and nerve injury in mouse levator auris longus muscle(마우스 거근 오리스 장근의 전기천공법과 신경 손상)

Combined In Vivo Electroporation and Short&#45;Term Reinnervation of the Cranial Levator Auris Longus Skeletal Muscle

The neuromuscular junction (NMJ) is the peripheral synapse controlling the contraction of skeletal muscle fibers to allow the coordinated movement of many organisms. At the NMJ, a presynaptic motor axon terminal contacts a muscle postsynaptic domain and is covered by terminal Schwann cells. The integrity and function of the NMJ is compromised under several conditions, including aging, neuromuscular diseases, and traumatic injuries. To analyze the potential contribution of muscle-derived proteins to NMJ maintenance and regeneration, an in vivo gene transfer strategy has been combined with the denervation of the cranial levator auris longus (LAL) muscle after mechanical nerve injury. Previous findings showed that the forced expression of control fluorescent proteins does not alter NMJ organization or neurotransmission. This procedure aims to describe a detailed method of in vivo electroporation of the LAL muscle followed by transection or crushing of the specific branch of the facial nerve innervating cranial muscles, leading to NMJ denervation and reinnervation, respectively. The combination of these experimental strategies in the convenient LAL muscle constitutes an efficient method to study the potential contribution of muscle protein overexpression or silencing in the context of short-term NMJ reinnervation.

<meta charset="utf8"></head><body>저자 스포트라이트: 신경근 접합부에서 신경 손상 후 근육 단백질의 재생 역할

Here, we present a protocol that combines in vivo electroporation and denervation of the cranial levator auris longus (LAL) muscle. This procedure enables the study of the potential role of muscle-derived proteins in the regeneration of the neuromuscular synapse.

In Vivo Electroporation과 Cranial Levator Auris Longus Skeletal Muscle의 단기 신경 재생이 결합되었습니다.

The neuromuscular junction (NMJ) is the peripheral synapse controlling the contraction of skeletal muscle fibers to allow the coordinated movement of many ...

삼투압 펌프 또는 직접적인 근육 주사를 이용한 피하 뉴로트로핀 4 주입으로 노화 후두 근육 강화

요약

더 많은 비디오 탐색

이 비디오의 챕터

루게릭병과 외상 척수 상해에서 자궁암 복부 호른를 타겟팅하는 Intraspinal 세포 이식

신경근육학 전기적 자극에 의해 근육 트레이닝의 Murine 모델

피하 Muscarinic Antagonists의 관리 및 마우스에 Levator Auris의 Longus 근육의 트리플 - Immunostaining

경골 신경 절개 - 마우스의 탈 신경에 의한 골격근의 위축에 대한 표준 모델

말초 신경 손상 후 기능 회복을 평가하기 위해 후각 Ensheathing 세포의 이식

모터 엔드 플레이트 함께 근육 주사 : 직접 모터 뉴런에 셔틀 트레이서에 대한 최소 침습 접근

말 왼쪽된 회귀 후 두 신경 근처 헌 혈 줄기 세포의 신경 자극을 유도 주사

만성 생체 내 근전도를 위한 이식형 시스템

신경 근육 분석을위한 쥐 보컬 폴드의 준비

In Vivo Electroporation과 Cranial Levator Auris Longus Skeletal Muscle의 단기 신경 재생이 결합되었습니다.

삼투압 펌프 또는 직접적인 근육 주사를 이용한 피하 뉴로트로핀 4 주입으로 노화 후두 근육 강화

요약

더 많은 비디오 탐색

이 비디오의 챕터

루게릭병과 외상 척수 상해에서 자궁암 복부 호른를 타겟팅하는 Intraspinal 세포 이식

신경근육학 전기적 자극에 의​​해 근육 트레이닝의 Murine 모델

피하 Muscarinic Antagonists의 관리 및 마우스에 Levator Auris의 Longus 근육의 트리플 - Immunostaining

경골 신경 절개 - 마우스의 탈 신경에 의한 골격근의 위축에 대한 표준 모델

말초 신경 손상 후 기능 회복을 평가하기 위해 후각 Ensheathing 세포의 이식

모터 엔드 플레이트 함께 근육 주사 : 직접 모터 뉴런에 셔틀 트레이서에 대한 최소 침습 접근

말 왼쪽된 회귀 후 두 신경 근처 헌 혈 줄기 세포의 신경 자극을 유도 주사

만성 생체 내 근전도를 위한 이식형 시스템

신경 근육 분석을위한 쥐 보컬 폴드의 준비

In Vivo Electroporation과 Cranial Levator Auris Longus Skeletal Muscle의 단기 신경 재생이 결합되었습니다.

신경근육학 전기적 자극에 의해 근육 트레이닝의 Murine 모델