Name: subcutaneous neurotrophin 4 infusion using osmotic pumps or direct muscular injection enhances aging rat laryngeal muscles
Uploaded: 2017-06-13T15:00:00.000+00:00
Duration: 5 min 50 s
Description: 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

Bu çalışma, Ulusal Sağırlık ve Diğer İletişim Bozuklukları Enstitüsünün (R21DC010806&#39;dan CAM ve JCS&#39;ye ve R01DC011285&#39;den CAM&#39;e) hibeleriyle desteklendi.

Bu protokol için 6 ve 30 aylıkken erkek Fischer 344-Brown Norveç sıçanları kullanın. Sıçanlar Ulusal Yaşlanma Enstitüsü&#39;nden kemirgen koloni elde edildi. Bu çalışma için sıçanlar kullandık, çünkü sıçan larenksinin yapısı, havayolu koruması ve türe özgü seslendirme işlevini yerine getiren işlevsel olarak insandakine benzerdi. Bu çalışma, PHS İnsan Gücü Bakımı ve Laboratuar Hayvanları Kullanımı, Laboratuar Hayvanlarının Bakımı ve Kullanımı için NIH Rehberi ve Hayvan Refahı Yasası (7 USC ve ark.); Hayvan kullanımı protokolü, Kentucky Üniversitesi&#39;nin Kurumsal Hayvan Bakımı ve Kullanım Komitesi (IACUC) tarafından onaylanmıştır. 1. Sıçan Anestezi <ol><li> Ketamin hidroklorür (dissosiyatif anestetik) ve ksilazin hidroklorür (sedatif ve analjezik) tamponlu salinde karıştırılarak anestezi hazırlayın. Nihai çözeltideki ketamin ve ksilazinin konsantrasyonları kg başına 100 mg / 8 mgVücut ağırlığı. </li><li> 25 G iğneli bir şırınga kullanarak intraperitoneal uygulama vasıtasıyla anestezi fareye enjekte edilir. </li><li> Parmağın ayak parmağını veya ayağı forsepsle tutarak sıçanın yeterince anestezi edildiğini belirleyin. Eğer sıçan sıkışmaya tepki vermezse, ameliyat başlayabilir. Sıçan ayak parmaklarına refleks veya kasılmalar ile tepki verirse, 1-2 dakika bekleyin ve sıkma testini tekrarlayın. Sıçan tekrar tepki gösteriyorsa, sıçanı yeni bir hayvanla değiştirin ve adım 1.2&#39;den başlayarak işlemi yineleyin. </li><li> Sıçanın hareketsiz kaldıktan sonra sıçan gözlerine oftalmik merhem uygulayın, böylece kornealar kurumaz. </li></ol> 2. Osmotik Pompa İmplantasyonu <ol><li> Sıçanın ventralini aseptik cerrahi alana yerleştirin. Bir preanestetik ilaç olarak meloksikam uygulayın. 25 G&#39;lik bir iğneli bir şırınga kullanarak intraperitoneal olarak 1-4 mg / kg vücut ağırlığı dozunda uygulayın. </li><li> Yaklaşık birimi kaldırmak için makas kullanınBoynunun arkasından 1 &quot;x 1&quot; kare kürk ve omuzlar arasında yaklaşık 1 &quot;kaudal alan olabildiğince cildin yakınına tıraş edin. </li><li> Dezenfekte etanol (70%) ile sırt ve boynu ıslatın. </li><li> Tıraştan sonra, boyun dorsal boyasını, alkolle bitirme iyod-alkol art arda 3 fırça ile fırçalayın. </li><li> Sıçanın vücut ısısını, 34 ° C&#39;ye ayarlanmış bir ısıtma yastığı üzerine yerleştirerek koruyun. </li><li> Aseptik olarak hazırlanmış ozmotik pompaları ya sistemik NTF4 tedavisi için 50 μL NTF4 veya tuzlu su ile doldurun ( Şekil 1 ). <ol><li> Deriden yaklaşık 2 cm genişliğinde yatay bir kesi yapmak için bir neşter kullanın, sadece kafa parçalarının arasından skapula arasında boşluk bırakın. Hemostatların ucunu yerleştirirken insizyonun arka kenarını bir elinizle forsepsle kaldırın ve posteri insizyona yavaşça itin. </li><li> Hemostatların ucu yaklaşık 2 cm sonraKesiye giden yolda, hemostatların üzerindeki kolları açın, insizyon bölgesine subkutan içi boş bir &quot;cep&quot; oluşturacak şekilde ipuçlarını genişletin. Bu, pompa için yerleştirme bölgesi olacaktır. </li></ol></li><li> NTF4&#39;ün herhangi bir etkileşimini ve cebin kesilme bölgesinin iyileşmesini en aza indirgemek için, pompa dağıtım portalı ucunu yerleştirdikten sonra yönlendirin. </li><li> 7-14 gün boyunca 50 μL NTF4 salin verin. 7 günlük grup, toplam 47.04 mg doz için 6.72 mg / gün NTF4 aldı. 14 günlük grup, 94.08 mg NTF4 25 toplam dozu için 6.72 mg / gün aldı. </li><li> Pompa yerleşimi için yapılan kesi kapatmak için 5-0 naylon dikiş ipliği, hemostatlar ve forseps kullanın. </li><li> Anesteziden kurtulduklarından sıçanları en az 30 dakika izleyin. İzlemenin tamamlanması için kriterler, hayvanın faal hale gelmesi, kafesin etrafında hareket edilmesi, içme suyunun alınması ve damat tutumu gibi diğer normal faaliyetlerin başlatılmasıdır. </li><li> Günlük hayvanları izleyinCerrahi alan iyileşmesi, normal yem ve su tüketimi ve idrar / dışkılardan geçme ve stres, ağrı veya diğer ameliyat sonrası komplikasyonların herhangi bir anormal davranış belirtileri gözlenerek ilk hafta için. </li><li> Eğer fare ağrı ya da sıkıntılı görünüyorsa, ağrıdan kurtulmak için her 24 saatte bir 5 mg / kg subkutanöz karprofen enjeksiyonu yaptırınız. </li><li> Bir enfeksiyon var gibi görünüyorsa, yaranın iyileşmesini sağlamak için bir veterinerle görüşün. </li><li> Hangi deney grubuna girildiğine bağlı olarak, ipliğin tahriş olmasını önlemek için ameliyattan 7-10 gün sonra 5-0 naylon dikişini çıkarın. </li></ol> 3. Direkt Enjeksiyonluk Sıçan Anestezisi <ol><li> Prosedürden önceki gece fareden yemek yemeyin. Bu, endoskopu veya enjeksiyon iğnesini bloke edecek yiyecek bulunmamasını sağlar. </li><li> Sıçanları tartın ve acepromaz 1-2 mg / kg vücut ağırlığı hazırlayın. Kas içine enjekte edin (IM konumuSol tiroksinoid kas). </li><li> Sıçanı, indüksiyon kutusuna yerleştirin. İndüksiyon kutusunda% 5 izofluran ve 1 Ç 2 ile anestezi uygulayın. </li><li> Sıçanı,% 2 izofluran ve 600 mL O 2 içeren burun konisine taşıyın. </li><li> Parmağın ayak parmağını veya ayağı forsepsle tutarak sıçanın yeterince anestezi edildiğini belirleyin. Sıçan tutamağa tepki vermezse, enjeksiyon protokolü başlayabilir. Sıçan ayak parmaklarına refleks veya kasılmalar ile tepki verirse, 1-2 dakika bekleyin ve sıkma testini tekrarlayın. Sıçan tekrar tepki gösteriyorsa, sıçanı yeni bir hayvanla değiştirin ve adım 3.4&#39;ten başlayarak yinelemeyi tekrarlayın. </li></ol> 4. Doğrudan Enjeksiyon ve Görselleştirme <ol><li> Enjeksiyondan önce 30 dakika boyunca 25 ° C&#39;ye ayarlanmış bir H20 b banyosunda NTF4 veya salin içeren aseptik olarak hazırlanmış 50-μL dozajlar yerleştirin. </li><li> Sıçanı bir pleksiglas platformda sırtüstü yatırın ve yatırın ( Şekil 2 ). SusSıçanı platformun üst kısmında bulunan bir kılavuz tel vasıtasıyla ön üst kesici uçlarından geri duran duruşta tutun. </li><li> 1.9 mm, 30 ° sinüs endoskopuna 50 mm, 30 gauge, 100 μL şırınga takın ( Şekil 3 ). NOT: Şırınga düzeneği kanülü sıkıca endoskopun dış duvarına tutan bir alet yardımıyla bağlanır. Endoskop vokal kıvrımların görselleştirilmesine ve şırınganın rehberliğine intraoral olarak izin verir. Kanül ucunun konumu, ucun endoskopik görünümü ( Şekil 4 ) aracılığıyla tam ve açık şekilde görülebilmesini sağlamak için her hayvandan önce ayarlanır. </li><li> Dili uzatmak ve yana doğru hareket ettirmek için lastik uçlu bir forseps kullanın. Daha sonra, oral açıklığı korumak için plastik bir spekulum takın. Kesikleri uçları kopararak pürüzsüz cilalanarak, 1.5 ila 2 cm&#39;lik bir uzunluğa kesilen 5 mL&#39;lik bir plastik şırınga varilinden spekulum yapın. </li><li> Işıklardaki ışıkları kapatM ve endoskopa bir halojen ışık kaynağı takın. İşlemi kaydetmek için video kaydediciyi açın. </li><li> Sıçanın ağzına yerleştirildiğinde cam ucu üzerinde yoğuşma gelişimini en aza indirgemek için endoskopun distal ucunu ılık suya birkaç saniye batırın. </li><li> Monitörden gelen görsel geribildirimi kullanarak, iğne dikkatli bir şekilde sol ses katmanının alanına yönlendirilir. </li><li> Vokal katın tümüne erişmek için solüsyonun solunum döngüsünün inspirasyon fazıyla birlikte enjeksiyon zamanı. Solunumun inspiratuar fazı boyunca vokal kat tam olarak açığa çıkar. <ol><li> Ses kıvrımı tamamen görünür hale geldiğinde, iğneyi vokal katın beyaz medyal kenarının yan tarafında bulunan sol tiroarenoid içine yerleştirin. İğne yerinde iken enjektörü şırınganın depresyondan geçirin. </li></ol></li><li> Endoskop ve video oynatıcıdaki halojen ışık kaynağını kapatın ve ro&#39;yu geri çevirinIşıklar. </li><li> Sıçanı evdeki kafese geri getirin ve ısıtma yastığına yerleştirin. </li><li> Isı pedinden çıkarmadan önce sıçanın iyileşmesine izin verin. Kafesdeki yiyecek ve suyu değiştirin. </li><li> Enjeksiyondan sonra sıçanları 7 gün boyunca izleyin ve ardından öksüzleştirin. Kriyoteksiyon için larideleri çıkarın 24 . </li></ol> Sıçanların Euthanizasyonu <ol><li> Sıçanları, ketamin hidroklorür ve ksilazin hidroklorür (100 mg / 8 mg / kg vücut ağırlığı enjekte edilen intraperitoneal enjeksiyon) ile anestezi altına alınız. </li><li> Medial torakotomiyi takiben kanama önleme yöntemiyle euthanize olun. </li></ol>

<ol>
	<li>Trupe, E. 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=Correlates+and+consequences+of+eating+dependency+in+institutionalized+elderly.">Correlates and consequences of eating dependency in institutionalized elderly.</a> J. Am. Geriatrics Society. 34 (3), 192-198 (1986).</li><li>Ward, P. H., Colton, R., McConnell, F., Malmgren, L., Kashima, H., Woodson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+of+the+voice+and+swallowing.">Aging of the voice and swallowing.</a> Otolaryngol. Head Neck Surg. 100 (4), 283-286 (1989).</li><li>Gay, E. G., Kronenfeld, J. J., Dettmann, F. 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+comparison+on+the+effect+of+regulation+on+health+care+for+the+older+American:+a+tale+of+two+states.">A comparison on the effect of regulation on health care for the older American: a tale of two states.</a> Gerontologist. 34 (6), 787-796 (1994).</li><li>Hagen, P., Lyons, G. D., Nuss, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysphonia+in+the+elderly:diagnosis+and+management+of+age-related+voice+changes.">Dysphonia in the elderly:diagnosis and management of age-related voice changes.</a> South. Med. J. 89 (2), 204-207 (1996).</li><li>Broniatowski, M., 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=Current+evaluation+and+treatment+of+patients+with+swallowing+disorders.">Current evaluation and treatment of patients with swallowing disorders.</a> Otolaryngol. Head Neck Surgery. 120 (4), 464-473 (1999).</li><li>Lundy, D. 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=Aspiration:+cause+and+implications.">Aspiration: cause and implications.</a> Otolaryngol. Head Neck Surg. 120 (4), 474-478 (1999).</li><li>Schindler, J. S., Kelly, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swallowing+disorders+in+the+elderly.">Swallowing disorders in the elderly.</a> Laryngoscope. 112 (4), 589-602 (2002).</li><li>Gambino, D. R., Malmgren, L. T., Gacek, R. R. <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+changes+in+the+neuromuscular+junctions+in+the+human+posterior+cricoarytenoid+muscles:+a+quantitative+study.">Age-related changes in the neuromuscular junctions in the human posterior cricoarytenoid muscles: a quantitative study.</a> Laryngoscope. 100 (3), 262-268 (1990).</li><li>Sinard, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aging+voice:+how+to+differentiate+disease+from+normal+changes.">The aging voice: how to differentiate disease from normal changes.</a> Geriatrics. 53 (7), 76-79 (1998).</li><li>Baker, K. K., Olson-Raming, L., Sapir, S., Luschei, E. S., Smith, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+vocal+loudness+in+young+and+old+adults.">Control of vocal loudness in young and old adults.</a> J. Speech Lang. Hear Res. 44 (2), 297-305 (2001).</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=Functional+and+morphological+evidence+of+age-related+denervation+in+rat+laryngeal+muscles.">Functional and morphological evidence of age-related denervation in rat laryngeal muscles.</a> J. Gerontol. A. Biol. Sci. Med. Sci. 64 (4), 435-442 (2009).</li><li>Lai, K., Ip, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsynaptic+signaling+of+new+players+at+the+neuromuscular+junction.">Postsynaptic signaling of new players at the neuromuscular junction.</a> J. Neurocytol. 32 (5-8), 727-741 (2003).</li><li>Huang, E. J., Reichardt, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins:+roles+in+neuronal+development+and+function.">Neurotrophins: roles in neuronal development and function.</a> Annu. Rev. Neurosci. 24 (1), 677-736 (2012).</li><li>Gonzalez, M., 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=Disruption+of+Trkb+mediated+signaling+induces+disassembly+of+postsynaptic+receptor+clusters+at+neuromuscular+junctions.">Disruption of Trkb mediated signaling induces disassembly of postsynaptic receptor clusters at neuromuscular junctions.</a> Neuron. 24 (3), 567-583 (1999).</li><li>Hirano, S., Kishimoto, Y., Suehiro, A., Kanemaru, S., Ito, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+aged+vocal+folds:+first+human+case+treated+with+fibroblast+growth+factor.">Regeneration of aged vocal folds: first human case treated with fibroblast growth factor.</a> Laryngoscope. 118 (12), 2254-2259 (2008).</li><li>Branski, R. 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=Effects+of+transforming+growth+factor-beta+1+on+human+vocal+fold+fibroblasts.">Effects of transforming growth factor-beta 1 on human vocal fold fibroblasts.</a> Ann. Oto. Rhinol. Laryngol. 118 (3), 218-226 (2009).</li><li>Kishimoto, Y., 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=Effects+of+exogenous+hepatocyte+growth+factor+on+vocal+fold+fibroblasts.">Effects of exogenous hepatocyte growth factor on vocal fold fibroblasts.</a> Ann. Otol. Rhinol. Laryngol. 118 (8), 606-611 (2009).</li><li>Belluardo, 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=Neuromuscular+junction+disassembly+and+muscle+fatigue+in+mice+lacking+neurotrophin-4.">Neuromuscular junction disassembly and muscle fatigue in mice lacking neurotrophin-4.</a> Mol. Cell. Neurosci. 18 (1), 56-57 (2001).</li><li>Johnson, A. M., Ciucci, M. R., Connor, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vocal+training+mitigates+age-related+changes+within+the+vocal+mechanisms+in+the+old+rat.">Vocal training mitigates age-related changes within the vocal mechanisms in the old rat.</a> J. Gerontol. A Biol. Sci. Med. Sci. 68 (12), 1458-1468 (2013).</li><li>Roscow, D. E., Parikh, P., Vivero, R. J., Casiano, R. R., Lundy, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+for+initial+dosing+of+botulinum+toxin+in+treatment+of+adductor+spasmodic+dysphonia.">Considerations for initial dosing of botulinum toxin in treatment of adductor spasmodic dysphonia.</a> Oto. HeadNeck Surg. 148 (6), 1003-1006 (2013).</li><li>Benninger, M. S., Hanick, A., Hicks, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cricothyroid+muscle+botulinum+toxin+injection+to+improve+airway+for+bilateral+recurrent+laryngeal+nerve+paralysis,+A+case+series.">Cricothyroid muscle botulinum toxin injection to improve airway for bilateral recurrent laryngeal nerve paralysis, A case series.</a> J. Voice. 30 (1), 96-99 (2016).</li><li>Coppoolse, J. M. 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>

Yazarların açıklayacak bir şeyi yok.

Larinks kasları yaşlanmanın olumsuz etkilerine karşı savunmasızdır. Önceki çalışmalar, kontraktil fonksi- yon ve miyozin izoform değişikliklerine ek olarak lif boyutu, toplam lif sayısı, rejeneratif yeteneği, NMJ boyutu ve miktarı değişikliklerini içeren yaşlanma laringeal kaslarında değişiklikler olduğunu göstermiştir 4 , 11 , 27 , 30 , ...

Larinks kasları hızla ve sürekli olarak kasılır ve yaşlanmanın olumsuz etkilerine karşı savunmasızdır. Bu sabit etkinliğin, 65 yaş üzerindeki 1 , 2 , 3 , 4 , 5 , 6 , 7 numaralı kişilerde görülen ses problemlerine veya disfajiye katkıda bulunduğu düşünülmektedir. Birkaç moleküler ve patofizyolojik mekanizma bu yaşla ilişkili disfonksiyona katkıda bulunmaktadır. Bu mekanizmalar laringeal mukozanın yeniden şekillendirilmesi, kas lifi atrofisi veya kaybı, vasküler kıvrımların eğilmesine ve glottis kapanmasının başarısız olmasına neden olan kas lifi rejenerasyonu veya atrofisinin eksikliği içerebilir 8,9,10,11. Şu anda, kanıtlanmış bir tıbbi tedavi yoktur.Bu kaslarda bu yaşa bağlı değişiklikleri kolayca önlemek veya rehabilite etmek. Nöromüsküler bulaşmanın etkinliğinin modülasyonu, nöromotor performansı büyük ölçüde etkiler. Nörotrofinin ailesi, sinir büyüme faktörü (NGF), beyin türevi sinir büyüme faktörü (BDNF), nörotrofin 3 (NTF3) ve NTF4 12 , 13&#39;ü içerir . Nörotrofinlerin sinaptik etkinliği 1 , 4&#39;ü modüle ettiği gösterilmiştir. Hepatosit büyüme faktörü, dönüştürücü büyüme faktörü beta ve fibroblast büyüme faktörü son zamanlarda insanlarda vokal kıvrım skarlanması 15-17 tedavisinde kullanılmıştır. NTF4 ayrıca NMJ etkinliğini düzenler; NTF4&#39;ten yoksun fareler , 11 , 18 , 19 numaralı NMJ&#39;leri parçalamıştır. Bu çalışmalar, tedavi edici etkilere yol açmaktadırLaringeal kas bozukluklarının yaşlanması ve büyüme faktörleri ile denervasyon. Vokal kıvrımların dokularına direkt enjeksiyon tedavileri insanlarda eşi benzeri görülmez. Örneğin, botulinum toksininin lokal enjeksiyonları şu anda spazmodik disfoni ve bilateral tekrarlayan laringeal sinir paralizisi gibi larenks kaslarını etkileyen nörolojik hareket bozuklukları için etkili bir tedavi olarak kullanılmaktadır 20,21. Hyaluronik asit hidrojel, vokal kat korkutma ve glotal yetmezliği tedavi etmek için kullanılan başka bir enjekte edilebilir 22 , 23 . Enjeksiyon larinkoplasti, çeşitli iletişim bozukluklarını tedavi etmek için kullanılabilir 24 . Bu doğrudan enjeksiyon yöntemleri vokal fonksiyonu iyileştirmek ve yaşlanan popülasyonlarda yutmak için söz verir.

<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

Yaşlılarda laringeal işlev bozukluğu, ses bozukluklarından disfajiye ve hava yolu koruyucu reflekslerde azalmaya neden olan önemli bir sakatlık nedenidir. Yaşa bağlı laringeal kas disfonksiyonunu hedef alan az sayıda, varsa, terapiler mevcuttur. Nörotrofinler, kas innervasyonu ve nöromüsküler kavşakların (NMJ&#39;lerin) farklılaşmasına katılırlar. Nörotrofinlerin nörotransmitter salınımını arttırarak nöromüsküler iletimi arttırdığı düşünülmektedir. Nöromusküler kavşaklar (NMJ&#39;ler) fonksiyonel denervasyon bulguları ile sıçan laringeal kas yaşlanmasında daha küçük ve daha az miktarda bulunur. İnsanın laringeal kas yaşlanmasında fonksiyon geliştirmek için NTF4&#39;ün ileride klinik kullanıma yönelik terapötik olarak etkilerini araştırdık. Burada, sıçan laringeal kasının yaşlanması NTF4 uygulamasına tepki olarak yeniden şekillenme yeteneğini araştırmak için sistemik uygulama ve doğrudan NTF4 enjeksiyonu için ayrıntılı protokolü sunuyoruz. Bu yöntemde, sıçanlar NTF4&#39;ü ya sistemik olarak oSmotik pompa veya vokal kıvrımlar yoluyla doğrudan enjeksiyon yoluyla uygulanabilir. Larinks kasları ayrıldı ve morfolojinin histolojik incelenmesi ve yaşla ilişkili denervasyon için kullanıldı.

Sıçanlar, 2 hafta ozmotik pompa infüzyonundan sonra veya NTF4&#39;ün doğrudan enjeksiyonundan 1 hafta sonra ötenazi yapıldı. Larenjler hasat edildi, kriyoprotektan (% 30 sükroz ve% 70 fosfat tamponlu salin) içine yerleştirildi ve daha sonra bir kriyostat ile 10 um&#39;lik genişlikte kesit olarak kesildi. Yaşlanma laringeal kasları, NTF4 25&#39;in uygulanmasından etkilenir. Genç ve yaşlı sıçanın yanısıra, tiroidenoid kasların enjekte edilen ...

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

Burada, sistemik olarak ve direkt olarak sıçan yaşlanmasına bağlı laringeal kasların yeniden şekillendirilmesi için nevrotropin 4&#39;ü (NTF4) tanımlamak için bir protokol sunuyoruz.

Osmotik Pompalar veya Doğrudan Kas Enjeksiyonu Kullanılarak Subkutanöz Neurotrophin 4 İnfüzyonu, Yaşlanan Farenin Larinks Kaslarını Arttırır

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

subcutaneous-neurotrophin-4-infusion-using-osmotic-pumps-or-direct

Research

JoVE Journal

Developmental Biology

8.4K Görüntüleme.  University of Kentucky.  Burada, sistemik olarak ve direkt olarak sıçan yaşlanmasına bağlı laringeal kasların yeniden şekillendirilmesi için nevrotropin 4'ü (NTF4) tanımlamak için bir protokol sunuyoruz. 

Video: 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.

Amyotrophic Lateral Sclerosis ve Travmatik Spinal Kord Yaralanması Servikal Ventral Horn Hedefleme intraspinal Hücre Transplantasyonu

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

Tıp

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.

Nöromüsküler Elektriksel Stimülasyon tarafından Kas Eğitimi A Mürin Modeli

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.

Subkutan Muskarinik Antagonistleri Yönetim ve Farelerde levator Auris longus&#39;un Triple-immün

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

Nörobilim

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;

Tibial Sinir transeksiyonu - Farelerde denervasyon bağlı İskelet Kas Atrofi için bir Standart Model

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.

Periferik Sinir Hasarı Sonrası Fonksiyonel Kurtarma Değerlendirmesinde Koku Ensheathing Hücre Nakli

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.

Motor End Plates boyunca İntramüsküler Enjeksiyonlar: Doğrudan Motor Nöronlar içine Servis Tarayıcıları Bir Minimal İnvaziv Yaklaşım

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.

Otolog kök hücre yakınındaki at sol tekrarlayan gırtlak sinir sinir Stimülatörü güdümlü enjeksiyon

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;

Vivo Elektromiyografide Kronik İçin İmplante Edilebilir Bir Sistem

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.

Nöromüsküler Analizler için Sıçan Vokal Kıvrımının Hazırlanması

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>Fare Levator Auris Longus Kasında Elektroporasyon ve Sinir Hasarı

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>Yazar Spotlight: Sinir Yaralanması Sonrası Nöromüsküler Kavşakta Kas Proteinlerinin Rejeneratif Rolleri

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.

Kranial Levator Auris Longus İskelet Kasının Kombine İn Vivo Elektroporasyonu ve Kısa Süreli Reinnervasyonu

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