This work is funded by NIH RO1 NS 40433 (K.J.J.).

실행 모든 절차는 의학 기관 동물 관리 및 사용위원회의 인디애나 대학의 승인을 건강 지침의 국립 연구소 따릅니다. 1. 수술 기법 <ol><li> 멸균 장갑, 악기, 그리고 NIH 가이드 라인 (25)에 따라 멸균 수술 필드를 사용하여이 절차를 수행하는 동안 무균 기술을 유지한다. 를 오토 클레이브에 의해 수술을 시작하기 전에 도구를 소독 (전체 목록은 특정 시약 / 장비의 표 참조). 작업 중에 도구를 소독 유리 구슬 살균기를 사용합니다. </li></ol> 2. 마취 및 준비 <ol><li> 수의학 이소 플루 란 기화기 시스템을 이용하여 0.9 L / 분의 산소 및 2.5 %의 이소 플루 란 혼합물로 마취 상자에 마우스를 마취. 마우스가 상자에서 제거하기 전에 몸의 위치 변화에 응답하지 않습니다 있는지 확인하십시오. </li><li> 양해 각서 (MOU)에 안과 연고를 적용그 자체의 눈이 마르지을 보호 할 수 있습니다. </li><li> 코 콘에있는 상자에서 가스 흐름을 전환합니다. 콘 내부의 코와 입으로 수술 패드와 흡수 벤치 종이로 덮여 가열 패드의 왼쪽에 정면으로 마우스를 놓습니다. 지속적으로 마우스의 호흡 리듬과 속도를 모니터링하고 필요에 따라 이소 플루 란 레벨을 조정 - 마취의 적절한 수준을 유지하기 위해 (2.5 사이의 3 % 이소 플루 란), 총 진정 작용을 확인하기 위해 발가락 핀치 반사를 사용합니다. </li></ol> 3. 수술 적 접근 <ol><li> 맞추고 수술 필드 입체경을 초점을 맞 춥니 다. 노즈콘을 조정하고이 시야의 가장자리에 위치하도록 아래로하는 테이프. </li><li> 마우스의 왼쪽에 누워, 절개 될 것이다 귀 뒤 영역을 노출 노즈콘을 우측 귀의 가장자리를 테이프. 후방 귀의 정맥 귀 가로로 이동하는 확인하십시오. 참고 t의 정확한 위치그 동물 귀의 테이핑 신속 안면 신경을 찾기 위해 매우 중요하다. </li><li> 에 70 % 에탄올 귀 뒤에 모피 적시고 면도기 또는 메스 블레이드를 사용하여 수술 부위를 면도. 사전 습윤 모피하는이 해부학 적 위치에 쉽게 면도 있습니다. </li><li> 70 % 에탄올 다음과 같은 베타 딘 수술 스크럽 (7.5 % 포비돈 요오드)와 같은 요오드 용액,로 피부를 청소합니다. 철저하게 지역을 소독이 청소를 두 번 더 반복합니다. </li><li> , 절개를 귀 돌기에 지역의 후방에 꼬리 쪽 귀에서 후방 귀의 정맥을 추적해서 결정해야합니다. 돌기에 3mm 후방 - 봄 가위를 사용하여 4mm의 절개 (2)를 확인합니다. </li><li> 무딘 절개를 이용하여 피하 지방 및 근막을 절개하다. 혈관 또는 근육 조직이 쉽게 손상 될 수 있기 때문에 가위로 직접 절단하지 마십시오. </li><li> 출혈이 발생하는 경우, 멸균 면봉으로 수술 부위에 압력을 적용적어도 30 초. 유체 상당한 손실이 발생하면, 최대 25 또는 제 27 G 바늘을 사용하여 멸균 0.9 % 식염수 용액 0.5 ml를 복강 내 주사를 마우스. </li><li> 안면 신경의 위치를​​, 몇 가지 주요 랜드 마크, 척추 부신경, 외이도를 사용하여 (아래에 설명) digastric 근육을 전방. 안면 신경의 가지가 가시화 될 때까지 이러한 랜드 마크 주위를 해부하다. 이 공개 될 때 신경은 크게 백색 고체 구조로 표시됩니다 및 근막 층이 기본 구조를 준수합니다. <ol><li> 피하 지방과 근막이 해부 된 이후, 승모근의 근육에 분포하는 두개골의 꼬리 부분에서 이동 척추 액세서리 신경을 찾을 수 있습니다. 안면 신경은 척추 부신경에 깊은이다. </li><li> 진주 흰색 보이는 안면 신경에 주동이 볼 수있는 연골 귀 운하를 찾을 수 있습니다. </li><li> 의 상단과 C에 놓여 전방 digastric 근육의 근육 배꼽 찾기안면 신경에 audal. </li></ol></li><li> 안면 신경의 가지 주요 가시화되면 stylomastoid 난원에서 기원을 찾을 등쪽 그들을 추적. 개방형 수술 부위를 보유하고있는 신경의 경로를 따라 봄 가위 팁을 발전 연 다음, 새로 고급 영역을 유지하기 위해 등쪽 집게를 이동 뾰족한 뒤몽 포셉 # 45분의 5를 사용. </li><li> 이 시점에서 광대뼈, 구강과 안면 신경의 트렁크, 변연 하악 가지를 시각화. 주 : 시간 ​​분기 난원에 가까운 발견 될 것이다. 상부 및 하부에 한계 하악 신경 가지에 가까운 턱, 따라서 그 신경 지점이 수준에서 표시되지 않습니다. <ol><li> 신경 절개를 수행하는 경우, 미세 팁 포셉으로 부드럽게 신경을 안정시키고 스프링 가위로 신경을 잘라. 뇌간의 신경을 avulsing 방지하기 위해 집게와 신경에 너무 많은 견인을 적용하지 마십시오. 푸시멀리 서로, 또는 절단 및 말초 신경의 일부를 제거에서 젊고 아름다운 여자가 재 연결이 발생하지 않도록합니다. </li><li> 호감 부상을 수행하는 경우, 모든 축삭을 절단 첫 번째 호감 사이트에 수직 인 제 2 각도로이 호감을 반복 일정한 압력을 사용하여 30 초 동안 신경을 압축 뒤몽 #에게 45분의 5 집게를 사용합니다. 그렇지 않으면 부상 동물 사이에 일관성이 없습니다, 30 초 호감 동안 압력의 변수 금액을 적용하지 마십시오. </li></ol></li></ol> 4. 결산 및 복구 <ol><li> 기본 구조를 통해 지방과 근육 위치를 조정합니다. </li><li> 절개의 가장자리를 대략적인 7.5 mm 상처 클립을 사용하여 상처를 닫는다. 봉합 또는 접착제는 또한 상처 폐쇄에 대한 허용됩니다. 수술후 진통제는이 시점에 제공 될 수있다. </li><li> 마우스의 귀에서 테이프를 제거합니다. 이소 플루 란 흐름을 끄고 마우스가 1 분 30 초 동안 순수한 산소를 흡입 할 수 있습니다. PL마취에서 회복 할 수없는 침구 빈 케이지에서 마우스 에이스. </li><li> 마우스가 복구 될 때, 안면 마비의 확증 흔적이 동작을 검사합니다. 수염이 마비 다시 뺨을 향해 각도됩니다, 코가 이탈되고, 눈은 공기의 퍼프에 대한 응답으로 깜박하지 않습니다. </li><li> 주택의 동물 공동 수술 후 그들은 여성의 경우. 그들은 더 공격적 강제 감염에 이르게 자신의 cagemate의 상처 클립을 제거하는 경향이 있기 때문에 공동으로 남성 쥐를 수용하지 마십시오. 필요한 경우,이 시점에서 수술후 진통제를 제공한다. </li><li> 어떤 감염 또는 기타 합병증은 수술 후 발생하지 않도록 수술 후 며칠 동안 하루에 한 번 마우스를 모니터링합니다. 그들은 자신에 타락하지 않은 경우 수술 10 일 - 상처 클립 (7)를 제거합니다. </li><li> 눈 깜빡임이 재 할 때까지, 매일 각막 합병증을 방지하기 위해 영향을받는 눈에 눈 연고를 윤활 적용적용 또는 안락사까지. </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the Care and Use of Laboratory Animals: Eighth Edition. , (2011).</li><li>Serpe, C. J., Kohm, A. P., Huppenbauer, C. B., Sanders, V., Jones, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exacerbation+of+Facial+Motoneuron+Loss+after+facial+nerve+transection+in+severe+combined+immunodeficient+(scid)+mice.">Exacerbation of Facial Motoneuron Loss after facial nerve transection in severe combined immunodeficient (scid) mice.</a> Neuroscience. 19, (1999).</li><li>Mesnard-Hoaglin, N. A., 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=SOD1(G93A)+transgenic+mouse+CD4(+)+T+cells+mediate+neuroprotection+after+facial+nerve+axotomy+when+removed+from+a+suppressive+peripheral+microenvironment.">SOD1(G93A) transgenic mouse CD4(+) T cells mediate neuroprotection after facial nerve axotomy when removed from a suppressive peripheral microenvironment.</a> Brain, Behavior, And Immunity. 40, 55-60 (2014).</li><li>Wang, 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=Establishment+and+assessment+of+the+perinatal+mouse+facial+nerve+axotomy+model+via+a+subauricular+incision+approach.">Establishment and assessment of the perinatal mouse facial nerve axotomy model via a subauricular incision approach.</a> Experimental Biology And Medicine. 237, 1249-1255 (2012).</li><li>Sharma, N., Moeller, C. W., Marzo, S. J., Jones, K. J., Foecking, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+treatments+enhance+recovery+following+facial+nerve+crush.">Combinatorial treatments enhance recovery following facial nerve crush.</a> The Laryngoscope. 120, 1523-1530 (2010).</li><li>Lieberman, D. M., Jan, T. A., Ahmad, S. O., Most, S. P. <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+corticosteroids+on+functional+recovery+and+neuron+survival+after+facial+nerve+injury+in+mice.">Effects of corticosteroids on functional recovery and neuron survival after facial nerve injury in mice.</a> Archives of Facial Plastic Surgery. 13, 117-124 (2011).</li><li>Serpe, C. J., Coers, S., Sanders, V. M., Jones, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++T,+but+not+CD8++or+B,+lymphocytes+mediate+facial+motoneuron+survival+after+facial+nerve+transection.">CD4+ T, but not CD8+ or B, lymphocytes mediate facial motoneuron survival after facial nerve transection.</a> Brain, Behavior, And Immunity. 17, 393-402 (2003).</li><li>Haulcomb, M. 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=Axotomy-induced+target+disconnection+promotes+an+additional+death+mechanism+involved+in+motoneuron+degeneration+in+ALS+transgenic+mice.">Axotomy-induced target disconnection promotes an additional death mechanism involved in motoneuron degeneration in ALS transgenic mice.</a> The Journal of Comparative Neurology. , (2014).</li><li>Bauder, A. R., Ferguson, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible+mouse+sciatic+nerve+crush+and+subsequent+assessment+of+regeneration+by+whole+mount+muscle+analysis.">Reproducible mouse sciatic nerve crush and subsequent assessment of regeneration by whole mount muscle analysis.</a> Journal of Visualized Experiments : JoVE. (60), (2012).</li><li>Richner, M., Bjerrum, O. J., Nykjaer, A., Vaegter, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spared+nerve+injury+(SNI)+model+of+induced+mechanical+allodynia+in+mice.">The spared nerve injury (SNI) model of induced mechanical allodynia in mice.</a> Journal of Visualized Experiments : JoVE. (54), (2011).</li></ol>

The authors have nothing to disclose.

The critical step for this protocol is positioning the mouse properly before surgery is begun. If the mouse is not lying flat on its side, the ear is not taped at the correct angle, or the incision is made in an incorrect location, then finding the facial nerve becomes much more difficult. When this technique is mastered, surgeries will take only minutes per mouse. 
Either sutures, glue, or wound clips can be used to close the wound. Wound clips are preferred because of the small size of incision and rapidity with which they can be applied. We have determined that if wound clips are used and the mice are housed together, female mice fare better than male mice because males housed together often remove their cagemate&#8217;s wound clips, leading to infection of the surgical site.
Modifications can be made to the injury, such as changing the location along the nerve to induce injury. For example, the injury could be administered along one of the branches of the facial nerve28, or the skull could be opened to deliver an intracranial injury29. In addition, a variety of treatments can be implemented, such as applying drugs to the nerve stump14, injecting medicines systemically30, or even performing cell transfers31. Motoneuron diseases such as amyotrophic lateral sclerosis can also be studied using the facial nerve axotomy model32. Ongoing applications of this technique involve studying nerve reconnection strategies after transection and the application of pharmaceutical agents to accelerate nerve regeneration after injury. 
One of the most useful aspects of this surgery is that it is easy to confirm successful axotomy by monitoring the mouse&#8217;s vibrissae orientation and movements as well as the eye blink reflex. 
The paralysis does not affect the animal&#8217;s vital functions and overall is well tolerated in the mouse. 
A major advantage of the facial nerve injury model is that the isolated study of motoneurons is possible. However, because this is a cranial nerve injury, results found using this method may not translate to spinal, autonomic, or sensory nerve injury models. To study these other nerve injury types, a sciatic33 or tibial34 nerve injury model is recommended. The drawback of these models is that there is crosstalk from the contralateral neurons in the spinal cord that can confound results. There is no crosstalk between the facial motor nuclei, so the contralateral facial motor nucleus can serve as a paired internal control. The facial nerve axotomy model presents many advantages for studying nerve injury and regeneration.

많은 말초 신경 손상 모델은 존재하지만, 운동 신경원의 연구를 위해 눈에 띄는 사람은 얼굴 신경 axotomy 모델입니다. 또한 뇌신경 VII로 알려진 안면 신경은 뇌교에서 유래와 얼굴 표정 1,2의 근육을 innervates. 이 수술 프로토콜에서 안면 신경은 stylomastoid 난원에서의 출구에 노출 자르거나 분쇄 중 하나입니다. 신경 손상의 중증도 순차 축삭 다발 감싸는 결합 조직층 인 축삭 endoneurium, 신경 외피 및 epineurium의 intactness에 기초하여 부상 차별화 랜드 (3) 분류, 다음 분류 될 수있다. 압박 손상 (axonotmesis)에서 축삭이 절단되지만, 신경 외피와 epineurium가 유지됩니다. 그대로 신경초가 축삭은 4,5를 자라게하는 내 도관 역할을하기 때문에 안면 신경 호감에서 완전한 기능 회복은 약 11 일 만에 발생합니다. 에다른 한편으로는, 절단 손상 (neurotmesis)에서 축삭 3 결합 조직층이 절단되고, 전체 말초 신경 근육 신경 분포를 복원 자라게한다. epineurium의 외과 재 연결이 자주 신경 절개 부상과 인간의 환자에서 수행하지만 복구 결과는 드물게 최적입니다. 또한이 연구는 신경 목표로 자라게 못하고 어떤 치료법이 개선 회생 프로세스를 가속화하기 위해 이용 될 수있는 이유 이해할 필요가있다. 안면 신경 axotomy 모델을 사용하여 신경 손상을 공부에 많은 이점이있다. 먼저, 안면 신경 axotomy 절차는 빠르고 간단하며 높은 재현성이고; 및 안면 근육의 마비 얻어진 중요한 기능에 영향을주지 않고 잘 동물에 의해 허용된다. 이것은 운동 신경원 토륨 비교적 균질 집단에 상주하기 때문에 단순화된다 운동 신경 세포체 공부 뇌 신경 손상 모델이므로뇌교에서 전자 얼굴 모터 핵. 일곱 subnuclei 근육의 특정 그룹을 지배하는 것이 각각의 특정 있기 때문에 인구는 얼굴 모터 핵 내에서 subnuclear 패턴에 따라 차이가 않기 때문에 axotomy에 대응 subnuclear 차이가 결과 파 -2,6,7- 영향을 미칠 수 있습니다. 안면 신경 손상 모델의 주요 이점은 신경 신경 분포가 매우 대칭 및 안면 모터 핵 8 사이의 크로스 토크가 없기 때문에 unaxotomized 사이드 페어링 내부 제어로서 역할을 할 수 있다는 것이다. 이 수술 방법을 사용하는 또 다른 장점은 직접 CNS 외상 또는 혈액 뇌 장벽 (9)의 파괴의 부족이다. 과도한 출혈 및 감염 등의 합병증이 절차와 드물다. 다양한 분석은 신경 손상에 대한 생리적 반응을 평가하기 위해 수행 될 수있다. 눈 깜빡임과 수염 활동의 회복은 행동으로 사용할 수 있습니다기능 회복에 10, 11의 측정. vibrissae 활동의 비디오 녹화는 현재 안면 신경의 신경 분포 (12, 13)의 복구를 검출하기위한 가장 강력한 방법이다. 안락사 후, 뇌간 조직 학적 분석은 안면 모터 핵 내에 운동 신경 세포체에 수행 될 수있다. 얼굴 모터 핵 부상 2,6에 대한 응답의 차동 검사를 허용, 일곱 subnuclei, 특정 얼굴 근육에 각각의 특정으로 세분된다. 안면 운동 신경원은 세포 생존을 정량 계산 될 수 있고, 또는 바이오 마커는 면역 조직 화학 및 특정 세포 집단 (14)을 식별하는 데 사용될 수있다. 얼굴 모터 핵 정확하게 신경 손상 (15, 16)에 대한 세포 반응의 분자 분석을 위해 레이저 캡처를 사용하여 microdissected 할 수 있습니다. 안면 신경 axotomy의 영향은 운동 피질 (17, 18)에서 분석 될 수있다. 또한, 신경 Wallerian 변성을 연구하기 위해 해부 할 수있다 19축삭 재생 (20), 및 근육 신경 근육 접합부 (21)을 연구하기 위해 제거 할 수 있습니다. 안면 신경 근육 axotomy도 21 및 면역계 응답 (23), 중추 및 말초 첨부 신경교 세포 (22)를 연구 대상으로 사용될 수있다. 많은 안면 신경 axotomy 모델 (24)을 연구하여 이루어진 하였지만 신경 손상 환자에 대한 중요한 문제이고, 현재의 치료법은 최적의 결과를 생성하는 데 실패하기 때문에, 말초 신경 손상의 추가 연구가 요구된다. 이 모델은 신경 손상에 대한 생리적 반응을 검사하고 신경 재생 치료의 효과를 분석하기위한 강력한 도구입니다.

<table border="0" cellpadding="0" cellspacing="0" width="980">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Name of Material/ Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:176px;">Catalog Number</td>
			<td style="width:256px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Stereo Microscope</td>
			<td style="width:167px;">Leica</td>
			<td style="width:176px;">M60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Labeling tape</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:176px;">15-952</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:381px;">Vannas-T&#252;bingen Spring Scissors - Straight/Sharp/8.5cm/5mm cutting edge</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:176px;">15003-08</td>
			<td>Sterilize before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:381px;">Dumont #5/45 Forceps - Standard tips/Angled 45&#176;/Dumoxel/11cm</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:176px;">11251-35</td>
			<td>Sterilize before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Michel Suture Clips - 7.5mm x 1.75 mm</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:176px;">12040-01</td>
			<td>Described as &#34;wound clip&#34; in protocol, sterilize before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Hagenbarth Cross Action Wound Clip Applier 5&#34;</td>
			<td style="width:167px;">George Tiemann &#38; Co</td>
			<td style="width:176px;">160-910</td>
			<td>Used to apply wound clip, sterilize before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:381px;">Michel Suture Clip Applicator &#38; Remover - For 7.5 mm Clips</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:176px;">12029-12</td>
			<td>Used to remove wound clip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">0.9% Sodium Chloride Injection, USP</td>
			<td style="width:167px;">Hospira</td>
			<td style="width:176px;">0409-4888-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Betadine, 16 oz, with dispenser</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:176px;">19-027132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">70% Ethanol</td>
			<td style="width:167px;"></td>
			<td style="width:176px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Glass Bead Sterilizer</td>
			<td style="width:167px;"></td>
			<td style="width:176px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

facial nerve axotomy in mice: a model to study motoneuron response to injury

이 수술 프로토콜의 목표는 stylomastoid 난원에서의 출구에서 얼굴 근육을 innervates 안면 신경을, 노출 및 중 하나를 절단 또는 말초 신경 손상을 유도하기 위해 그것을 분쇄하는 것입니다. 이 수술의 장점은 이와 다른 신경 손상 모델에 비하여 비교적 가벼운 수술 결과 결과, 단순성, 재현성 및 후속 안면 마비에서 중요한 기능이나 이동성에 효과가 부족하다. 뇌 신경 손상 모델을 사용하는 주요 장점은이 운동 신경원 운동 신경 세포체 연구를 단순화 뇌교에서 안면 모터 핵 비교적 균질 한 집단에 존재한다는 것이다. 때문에 안면 신경의 신경 분포의 대칭성과 안면 모터 핵 사이의 크로스 토크의 부족, 동작 unaxotomized 사이드 페어링 내부 제어로서 일방적으로 수행 될 수있다. 분석의 다양한 엉덩이에 수술을 수행 할 수 있습니다이 문서의 범위를 벗어 그 세부 사항의 생리적 반응이야. 예를 들어, 근육 기능의 회복을위한 행동 reinnervation 마커로서 기능하거나, 운동 신경원은 세포 생존을 측정하기 위해 정량화 될 수있다. 또한, 운동 신경원 정확하게 분자 분석을위한 레이저 미세 절제를 사용하여 캡처 할 수 있습니다. 안면 신경 axotomy 침습 및 내약성이기 때문에, 유전자 변형 마우스의 다양한 상에 이용 될 수있다. 또한,이 모델은 수술 말초 신경 손상의 치료 효과를 분석하는데 사용될 수있다. 안면 신경 손상뿐만 아니라 운동 신경원을 조사하는 수단을 제공하지만, 중추 및 말초 미세 교세포, 면역계 및 목표 근육의 응답도. 안면 신경 손상 모델은 신경 손상과 재생을 연구하기위한 강력한 도구 역할을 널리 말초 신경 손상 모델이다.

안면 신경 axotomy이 수행 된 후, 운동 신경 손상 손실의 결과로 발생한다. 손상 후 운동 신경 생존은 모두 운동 신경 생존 데이터를 요약 성별, 수술시 동물의 연령, 운동 신경 카운트가 완료되는 평가시기 및 모란 그래버 검토 (24)과 진노와 야마다 리뷰 (22) 등 많은 변수에 따라 달라집니다. 일반적으로 운동 신경원의 약 86 %는 사후 axotomy 14,15,26 이십팔일에서 살아 남기. 운동 신경 손실의 속도론은 Serpe 등의 알에 설명되어 있습니다. 2000 년 그림 1은 여러 유전자 ​​변형 생쥐에서 운동 신경 생존의 변화를 보여줍니다. 유의 한 차이는 유전 적 변화는베이스 라인 계산에 영향을주지 않는 것을 나타내는, 제어 측의 안면 운동 신경 카운트에서 관찰되지 않습니다. 상당한 세포 손실은 마우스 모델에서 관찰된다 (도 1a, D 84 % ± 2.0) 야생형 마우스에서 운동 신경의 생존에 비해근 위축성 측삭 경화증 (SOD1 G93A, 68 % ± 1, 그림 1B, E)뿐만 아니라 면역 결핍 재결합 활성화 유전자 2 ​​녹아웃 마우스 (27) (RAG-2 - - /도 1C, F, 57 % ± 2.5) . 도 2는 얼굴 모터에인가 핵 레이저 캡처 미세 절제 기술을 보여준다. 얼굴 전체 모터 핵 (도 2A-C) 포착 될 수 있거나 subnuclei는 (도 2D-F) 개별적으로 수집 될 수있다. 보다 정밀하게 들어, 운동 신경원 개별적으로 포착 될 수 있고, 나머지 neuropil은 분석 (도 2G-I)에 대해 수집 될 수있다. 3 subnuclear 특히 ventromedial 비교 샘플 및 ventrolateral subnuclei로부터 추출한 RNA 재의 qPCR의 결과를 도시 한 도표. 네 개의 유전자 테스트, β II 튜 불린, 성장 단백질-43 (갭-43) 관련, hemopoie이완제 및 신경 발현 서열 -1- (HN1), 뇌 유래 신경 영양 인자 (BDNF)의 모든 신경 재생 응답과 연관 axotomy 16 이후 두 subnuclei 및 유전자 발현 프로파일 간의 흥미로운 차이가있다. <img alt="그림 1" src="/files/ftp_upload/52382/52382fig1highres.jpg" /> 그림 1. 대표 관상 얼굴 모터 핵 섹션은 thionin로 염색 일째와 28 일째 안면 신경의 절개 후 정량화. 얼굴 모터 핵 (A, D) (WT), (B, E) SOD1 G93A, 및 (C, F) RAG-에서 표시됩니다 2 - / - 마우스 (제어 측, axotomized 쪽). 스케일 바는 120 μm의 =. 이 수치는 27에서 수정되었습니다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52382/52382fig1large.jpg" target="_blank">일의 더 큰 버전을 보려면 여기를 클릭하십시오</a>그림입니다. <img alt="그림 2" src="/files/ftp_upload/52382/52382fig2highres.jpg" /> 얼굴 모터 핵 그림 2. 레이저 미세 절제. (A) 부분 axotomized 얼굴 핵의 레이저 미세 절제, 레이저 microdissected 조직의 (C) 컬렉션 axotomized 얼굴 핵, (B)의 Thionin 묻은 부분. subnuclei의 템플릿 (D)가 특히 ventromedial를 식별하고, 레이저 미세 절제 (E, F)에 대한 안면 subnuclei ventrolateral을 컴퓨터 화면 상에 중첩되었다. 안면 운동 신경원 레이저는 가시 핵과 핵소체 그들의 형태에 기초 microdissected하고, 화살표 (G)로 나타낸 FMN 전지 본체 단편, ​​레이저 별도로 microdissected 및 제거 처리 동안 FMN mRNA를 (* 운동 신경, G, H를 나타낸다) neuropil (S)에불가능한 명령어. 모든 FMN과 세포체 단편을 수집 한 후, 남은 핵 안면 티슈 neuropil 샘플 (I)로 microdissected 레이저였다. 스케일 바는 100 μm의 =. 이 수치가 16에서 수정되었습니다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52382/52382fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/52382/52382fig3highres.jpg" /> 그림 3. 프로 - 재생 및 프로 생존 mRNA 발현 특히 ventromedial에서 (VM) 및 ventrolateral (VL) 안면 신경 axotomy 다음 얼굴 모터 subnuclear 지역.에 가로로 VM 및 VL 얼굴 subnuclei 상대적으로 SEM ± mRNA 발현의 평균 퍼센트 작동이되지 않는 제어 subnuclei (AD). mRNA 발현의 경시 βII은 튜 불린 대한 손상 없음 (0), 3, 7, 14, 28 DPO (A를 포함하지 </강한&gt;), GAP-43 (B), HN1 (C), 및 BDNF (D). #은 P &lt;0.05에서 VM에 비해 VL의 유의 한 차이를 나타냅니다. 이 그림은 16에서 수정되었습니다.

Watch this Scientific Journal Video about Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury at JoVE.com

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

마우스의 얼굴 신경 Axotomy : 부상으로 운동 신경 응답을 연구하는 모델

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

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Nanyang Technological University

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JoVE Journal

Medicine

12.8K 조회수.  Indiana University School of Medicine. We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques. 

비디오: Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

마우스의 급성 폐 상해를 유발하는 압력 제어 환기

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

연구

의학

The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared nerve injury (SNI) model induces symptoms of neuropathic pain such as mechanical allodynia i.e. pain due to tactile stimuli that do not normally provoke a painful response [1]. 
The SNI mouse model involves ligation of two of the three branches of the sciatic nerve (the tibial nerve and the common peroneal nerve), while the sural nerve is left intact [2]. The lesion results in marked hypersensitivity in the lateral area of the paw, which is innervated by the spared sural nerve. The non-operated side of the mouse can be used as a control. The advantages of the SNI model are the robustness of the response and that it doesn&#x2019;t require expert microsurgical skills.
The threshold for mechanical pain response is determined by testing with von Frey filaments of increasing bending force, which are repetitively pressed against the lateral area of the paw [3], [4]. A positive pain reaction is defined as sudden paw withdrawal, flinching and/or paw licking induced by the filament. A positive response in three out of five repetitive stimuli is defined as the pain threshold. 
As demonstrated in the video protocol, C57BL/6 mice experience profound allodynia as early as the day following surgery and maintain this for several weeks.

The Spared Nerve Injury SNI Model of Induced Mechanical Allodynia in Mice

The Spared Nerve Injury animal model is described here as a mouse model of peripheral neuropathic pain following partial denervation of the sciatic nerve by lesioning the tibial and common peroneal nerve branches, leaving the remaining sural nerve intact. Behavioral modification resulting from mechanical allodynia is quantified by von Frey filaments.

마우스에서 유도된 기계 Allodynia의 겪었던 신경 상해 (스니) 모델

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

약시의 억제의 측정 및 치료

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

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. ...

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.

Respiratory failure is the leading cause of death following a cervical spinal cord injury. Having a reproducible, quantifiable, and reliable pre-clinical animal model of respiratory failure induced by a partial cervical injury will help to understand the subsequent respiratory and non-respiratory neuroplasticity and allow testing putative repair strategies.

후 병변 호흡 신경 가소성을 연구하기 위해 자궁 경부 척수 손상의 생쥐 모델

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed ...

Chronic Constriction Injury of the Rat's Infraorbital Nerve (IoN-CCI) to Study Trigeminal Neuropathic Pain

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral procedures to study trigeminal neuropathic pain in rats. To meet the specificity of trigeminal neuropathic pain syndromes, the infraorbital nerve (IoN) is subjected to a chronic constriction injury (CCI) by loosely ligating the nerve. An intra-orbital approach is presented here to expose and ligate the IoN in the orbital cavity. After IoN ligation, rats exhibit changes in spontaneous behavior and in response to von Frey hair stimulation that are indicative of persistent pain and mechanical allodynia. Two phases can be defined in the development of the behavioral changes. During the first week following IoN-CCI (phase 1), rats show an increased and asymmetric face grooming activity, i.e., with face wash strokes primarily directed to the nerve-injured IoN territory. A distinction is made between face grooming behavior that is part of a more general body grooming behavior, which remains largely unaffected by IoN-CCI, and face grooming that is neither preceded nor followed by body grooming, which is significantly increased after IoN-CCI. During this period, responsiveness to mechanical stimulation of the IoN territory is reduced. This hyporesponsiveness is abruptly replaced by an extreme hyperresponsiveness whereby even very weak stimulus intensities provoke nocifensive behavior (phase 2). The phenomenological similarities between these behavioral alterations and reported signs of facial pain (i.e., responses to noxious stimulation of the face) suggest the presence of dysesthesia/paresthesia and mechanical allodynia in the ligated IoN territory.

Chronic Constriction Injury of the Rat's Infraorbital Nerve IoN-CCI to Study Trigeminal Neuropathic Pain

This manuscript describes a rodent model to study trigeminal neuropathic pain. The methods described include surgical procedures to perform a chronic constriction injury of the rat&#8217;s infraorbital nerve and the post-surgical behavioral tests to measure the changes in spontaneous and evoked behavior that are indicative of persistent pain and allodynia.

삼차 신경 병증 성 통증을 연구하기 위해 쥐의 눈꺼풀의 신경 (이온 CCI)의 만성 수축 부상

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral ...

A Repetitive Concussive Head Injury Model in Mice

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of knowledge on the injury and its effects. To develop a better understanding of concussions, animals are often used because they provide a controlled, rigorous, and efficient model. Studies have adapted traditional animal models to perform mTBI to stimulate mild injury severity by changing the injury parameters. These models have been used because they can produce morphologically similar brain injuries to the clinical condition and provide a spectrum of injury severities. However, they are limited in their ability to present the identical features of injuries in patients. Using a traditional impact system, a repetitive concussive injury (rCHI) model can induce mild to moderate human-like concussion. The injury degree can be determined by measuring the period of loss of consciousness (LOC) with a sign of a transient termination of breathing. The rCHI model is beneficial to use for its accuracy and simplicity in determining mTBI effects and potential treatments.

Concussion presents the most common type of traumatic brain injury. Therefore, a repetitive concussive animal model, which replicates the important features of an injury in patients, may provide a means to study concussion in a rigorous, controlled, and efficient manner.

마우스에 반복적 인 충격 포 헤드 손상 모델

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

마우스에서 가속 죽상 경화증의 유도: "와이어 부상" 모델

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between experimental animals. Several contusive SCI models have been developed to produce injuries using different mechanisms. The severity of the SCI is based on the height that a given weight is dropped, the injury force, or the spinal cord displacement. In the current study, we introduce a novel mouse contusive SCI device, the Louisville Injury System Apparatus (LISA) impactor, which can create a displacement-based SCI with high injury velocity and accuracy. This system utilizes laser distance sensors combined with advanced software to produce graded and highly-reproducible injuries. We performed a contusive SCI at the 10th thoracic vertebral (T10) level in mice to demonstrate the step-by-step procedure. The model can also be applied to the cervical and lumbar spinal levels.

We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.

마우스의 조직 변위에 근거한 척수 손상 모델

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Myography 와이어를 사용 하 여 마우스 Mesenteric 동맥의 isometric 수축 측정

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...