This work is supported by an R21 grant from the National Institutes of Health and by a grant from Hearing Research, Incorporated.

참고 : NIH 윤리 지침과 캘리포니아 대학의 기관 동물 관리 및 사용위원회, 샌프란시스코의 승인 프로토콜 요구 사항을 준수 모든 절차와 동물 취급. 1. 수술에 대한 동물 준비 <ol><li> 깨끗하고 전용 공간에서 수술을 수행하십시오. 수술 전에 유리 비드 살균기로 살균, 모든 수술 도구를 압력솥. 참고 :이 프로토콜에서 출생 후의 일을 10 ~ 12 (P10-12) FVB 마우스를 사용합니다. 다양한 연령 및 생쥐 균주는 특정 프로젝트의 요구를 충족시키기 위해 사용될 수있다. 수포 뼈 어렵게 가져 오기 때문에 P40보다 오래된 마우스는 어려울 수 있습니다. </li><li> 케타민 하이드로 클로라이드 (100 ㎎ / kg), 자일 라진 하이드로 클로라이드 (10 ㎎ / kg), 및 아세 프로 마진 (/ kg 2 mg)의 혼합물을 복강 내 주사로 마우스를 마취. 동물이 더 이상 이러한 발가락 핀치와 같은 고통스러운 자극에 반응 후에 수술 준비를 시작합니다. 만약 necessarY는 원래 평면 마취제를 복원 마취제 칵테일 접종 (1/5 원래 투여) 투여. 참고 :이 나이에 관찰 된 사망률의 대부분이 마취제 과다 복용에 의해 발생하기 때문에이 단계에서주의하십시오. </li><li> 동물의 깜빡임을 억제, 마취 동안 촉촉한 눈을 유지하기 위해 보호 안과 연고와 동물의 눈을 감아 라. </li><li> 마우스가 완전히 깨어 때까지 마취 후 저체온증을 방지하기 위해, 절차 전반에 걸쳐 가열 패드에 확장 목 마우스를 놓습니다. </li><li> 클리퍼와 왼쪽 포스트 귀의 지역을 면도 및 수술 조작 전에 70 %의 에탄올과 포비돈 - 요오드 소독. </li></ol> 2. 수술 및 벡터 주입 <ol><li> 고막 수포를 노출 후 귀의 접근 방식을 사용합니다. 포스트 귀의 근육을 노출 작은 가위와 피하 조직을 절개. 지방 조직을 수축하는 토륨 후절개의 전자 후방 측은 측두골을 노출 수직 절개 좌우로 근육을 분리. 이 절개가 적절한 시각화 편안하게 작업 할 수있을만큼 긴 있는지 확인하십시오. </li><li> 25 G 바늘 고막 수포를 관통하고 달팽이관의 기저 차례에 액세스 할 수 있도록 다시 뼈를 박리함으로써 집게와 필요에 따라 구멍을 확장 한 다음 stapedial 동맥과 둥근 창 막을 시각화하기 위해 충분히 확대 (RWM) . </li><li> 보로 실리케이트 모세관 피펫 중앙에 부드럽게 RWM에 구멍. 이 시점에서 RWM를 통해 유체 유출을 준수; 이것은 정상입니다. 유출이 안정 될 때까지 기다립니다 (달팽이관에서 effluxed 유체는 멸균 여과지로 건조). 참고 : 잡고 RWM 천공이 가능한 한 작게되도록 유리 바늘을 전진 할 때주의하십시오. 사용되는 현미경에 따라, 피펫이 피펫을 사용하여 미세 조작기를 사용하거나 손으로 잡고홀더. </li><li> 같은 방법으로 패치 클램프 피펫을 준비 끌어 당기는 피펫을 사용하여 주입 피펫을 준비합니다. 팁 직경이 큰 (약 15 μm의 직경) 아웃 바이러스를 피펫 팅과 것은 쉽게 수행되도록 있는지 확인합니다. </li><li> 주입 피펫에 AAV1-VGLUT3, AAV1-GFP 또는 AAV2-GFP의 1 ~ 2 μL를 그립니다. 유출은 (5 ~ 10 분) 안정화, microinject 같은 구멍을 통해 스칼라 고실에이 고정 볼륨이 이전에 RWM에서 만든 후. </li><li> 근육의 작은 플러그 피펫을 잡아 당겨 후 신속하게 RW 틈새를 밀봉 둥근 창에서 누설을 방지하기 위해 근육에 배치 조직 접착제의 작은 방울로 고정합니다. 참고 : 아주 잘 바늘 달팽이관 -이 단계에서 어떤 외 림프 누출을 방지하기 위해 근육의 작은 플러그를 제거한 후이 구멍을 밀봉 청력을 보존하는 것이 중요합니다. 밀봉하지 않으면 완전히 RWM은 시간이 지남에 따라 청력 손실을 발생합니다. </li><li> 주입 후, 커버지방 조직과 청각 수포 구멍은, 정상 위치로 포스트 귀의 근육과 지방 조직을 반환하고 6-0 이하 흡수 크롬 봉합 층에 상처를 봉합. </li><li> 포비돈 - 요오드 상처를 소독. </li></ol> 3. 수술 후 케어 <ol><li> 따뜻한 새장에 마우스를 놓고 그들이 완전히 다시 어머니와 함께으로 이동 회복이 될 때까지 방치하지 않습니다. 이 어머니와 함께 다시 새끼를 착용하기 전에 케이지에서 남성을 제거하는 것이 좋습니다. </li><li> 염증과 통증을 관리하는 피하 카프로 펜 수술 후 통증, 3 일에 한 번씩 24 시간 (2 ㎎ / ㎏)을 관리합니다. 고통, 이상 체중 감소, 통증, 또는 감염의 흔적을 매일 동물을 모니터링합니다. 일반적으로 모든 마우스가 정상적으로 작동해야 수술 후 3 일째에 의해. 고통 또는 질병의 흔적은 3 차 하루 마우스에 표시하는 경우, t를 안락사 고려제도적 지침에 따라 그는 마우스. </li></ol> 청성 뇌간 반응 (ABR) 녹음을 사용하여 바이러스 성 납품 후 인공 와우 기능 4. 평가 참고 : 청성 뇌간 반응 (ABR)이 청각 두피 아래에 배치 전극을 통해 뇌에 지속적인 전기 활동에서 추출 기록 잠재력을 유발합니다. 동물 소리로 자극한다. 그 결과 기록은 청각 자극의 발병 후 첫 10 밀리의 청각 경로에서 연속 점의 전기적 활동을 반영 다섯 파도로 구성되어 있습니다. <ol><li> 아세 프로 마진이없는 것을 제외하고,이 프로토콜의 섹션 1.2에 기재된 바와 같이 케타민 하이드로 클로라이드 및 자일 라진 하이드로 클로라이드의 혼합물을 복강 내 주사에 의해 생쥐를 마취. </li><li> 마우스가 완전히 깨어 때까지 마취 후 저체온증을 방지하기 위해, 청력 검사를 통해 가열 패드에 마우스를 놓습니다. 참고 : 사용이 실험에서 다음과 같은 소리 자극 : (31 Hz에서 프리젠 테이션 속도 5 밀리 초 시간)을 클릭합니다. </li><li> 왼쪽 귀 (참조)의 날개 아래에 정점에 피하 주사 바늘 전극을 놓고 반대쪽 귀 아래 (접지)과 이전에 방음 실 (27, 28)에 설명 된대로 기록 ABRS를 시작합니다. 아래에서 최대 진폭 5dB 음압 레벨 간격으로 기록 ABR 파형. </li><li> ABR 임계 값 수술 후 빠르면 4 일 정도의 바이러스 배달 후 결정 참고 : 임계 값 파도에 대한 피크은 응답하는 가장 낮은 자극 수준으로 정의된다 I-V 분명히했다 및 육안 검사에 반복적으로 존재. </li><li> 나는 인공 와우 신경의 활동을 분석하는 웨이브를 측정한다. ABR 검출 파형을 산출 자극 최저 레벨은 임계 값으로 정의된다. </li></ol> 면역 형광을 사용하여 5. 달팽이 형질 전환 유전자의 단백질 발현 <ol><li> intrap의 과다 복용에 의해 생쥐를 마취케타민과 염산 자일 라진 하이드로 클로라이드의 혼합물을 주입 eritoneal는 아세 프로 마진이없는 것을 제외하고,이 프로토콜의 섹션 1.2에 기재된 바와 같이. </li><li> 동물이 더 이상 이러한 발가락 핀치와 같은 고통스러운 자극에 반응 후에 머리를 목을 벨. </li><li> 26 설명 된대로 전체 마운트 면역을 위해 밖으로 및 프로세스 cochleae을 해부하다. 안티 VGLUT3 및 안티 7A 미오신 항체를 사용하여 전체 인공 실장 면역 형광 상세한 프로토콜은 에이 킬 등., 2013 (29)에 기재되어있다. </li><li> 250 희석 : GFP 라벨의 경우, 1에서 토끼 방지 GFP 항체 4 ° C에서 하룻밤 인공 와우 전체 마운트를 품어. </li><li> PBS에서 4,000 : PBS와 10 분 동안 두 번 cochleae 헹구어 낸 후 CY2에 결합 염소 항 - 토끼 IgG를 2 시간 동안 배양 1 희석. </li><li> 10 분 동안 두 번 PBS와 cochleae을 씻어 15 분 동안 DAPI으로 그들을 품어. </li><li> 유리 슬라이드에 cochleae을 마운트하고 싶게을 관찰공 초점 면역 형광 현미경을 어. </li></ol>

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B., Petit, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+genetics+of+deafness+illuminates+auditory+physiology.">How the genetics of deafness illuminates auditory physiology.</a> Annu. Rev. Physiol. 73, 311-334 (2011).</li><li>Jero, J., Tseng, C. J., Mhatre, A. N., Lalwani, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+surgical+approach+appropriate+for+targeted+cochlear+gene+therapy+in+the+mouse.">A surgical approach appropriate for targeted cochlear gene therapy in the mouse.</a> Hearing Research. 151 (1-2), 106-114 (2001).</li><li>Seal, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensorineural+deafness+and+seizures+in+mice+lacking+vesicular+glutamate+transporter+3.">Sensorineural deafness and seizures in mice lacking vesicular glutamate transporter 3.</a> Neuron. 57 (2), 263-275 (2008).</li><li>Akil, O., 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=Restoration+of+hearing+in+the+VGLUT3+knockout+mouse+using+virally+mediated+gene+therapy.">Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy.</a> Neuron. 75 (2), 283-293 (2012).</li><li>Akil, O., 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=Progressive+deafness+and+altered+cochlear+innervation+in+knock-out+mice+lacking+prosaposin.">Progressive deafness and altered cochlear innervation in knock-out mice lacking prosaposin.</a> J. 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K., 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=Long-term+in+vivo+cochlear+transgene+expression+mediated+by+recombinant+adeno-associated+virus.">Long-term in vivo cochlear transgene expression mediated by recombinant adeno-associated virus.</a> Gene Ther. 5 (2), 277-281 (1998).</li><li>Raphael, Y., Frisancho, J. C., Roessler, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral-mediated+gene+transfer+into+guinea+pig+cochlear+cells+in+vivo.">Adenoviral-mediated gene transfer into guinea pig cochlear cells in vivo.</a> Neurosci. Lett. 207 (2), 137-141 (1996).</li><li>Weiss, M. A., Frisancho, J. C., Roessler, B. J., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+mediated+gene+transfer+in+the+cochlea.">Viral mediated gene transfer in the cochlea.</a> Int. J. Dev. Neurosci. 15 (4=5), 577-583 (1997).</li><li>Pettis, R. M., Han, J. J., Mhatre, A. N., Lalwani, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracochlear+infusion+of+recombinant+adeno+associated+virus:+Analysis+of+its+dissemination+to+near+and+distant+tissues.">Intracochlear infusion of recombinant adeno associated virus: Analysis of its dissemination to near and distant tissues.</a> Assoc. Res. Otolaryngol. Abstr. 21, 673 (1998).</li><li>Konish, i. 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No conflicts of interest declared.

본 연구에서는 상세히 복원하거나 유전자 결함에 의해 훼손되어 정상 청각 기능을 구출하기위한 목적으로, 인공 유전자 치료에 사용될 수있는 기술을 설명한다. 그것은 전형적 외상성이므로,이 방법은 인공 유전자 전달 분자 또는 기타 잠재적 인 치료 (30)에 안전하다. 인공 와우 치료를위한 다른 접근 방법은 복부 접근 (24), 마우스와 기니 돼지 cochleostomy (31, 32) 및 림프 낭...

유전자 치료는 긴 유전 청력 손실에 대한 잠재적 인 치료로 제안했지만,이 지역에서의 성공은 1 애매 남아있다. 현재까지 바이러스로 매개 방법론은 상대적으로 접근 할 수없는 달팽이관 내의 특정 세포 유형을 대상으로 이론적 능력 때문에 지배적했다. 모두 아데노 바이러스 (AV) 및 아데노 - 관련 바이러스 (AAV)는 인공 유전자 전달에 사용되어왔다. AAVs는 여러 이유로 달팽이관에서 유리하다. 그들은 복제 결핍 바이러스이고 효율적으로 신경 세포, 청력 손실의 원인의 수에 대한 중요한 목표를 포함한 다양한 종류의 세포로 형질 전환 분자를 전송할 수 있습니다. 셀에 AAV 항목은 특정 수용체 2에 의해 매개된다 세포 유형이되도록 형질 도입에 따라서, 특정 혈청 형의 선택은 호환되어야한다. AAVs 효과적으로 유모 세포를 형질 감염 3 및 TRA의 안정된 장기 발현 결과, 숙주 게놈 내로 통합 할nsgenic 단백질과 세포 4 표현형 변화. 이러한 모발 세포 재생 단기 애플리케이션에 반드시 유리한 반면, 장기적 발현 유전자 결함을 안정된 구조에 매우 중요하다. AAVs 임의의 인간 질병 또는 감염과 관련된 어떠한 5,6,7- 내이 신경 독성을 증명하지 않으므로, 손실 8 청력 상속 형태의 유전자 치료에 사용하기위한 이상적인 후보이다. 바이러스 벡터를 사용하여 포유류 내이 내로 외인성 유전 물질의 전사는 지난 10 년간 연구되어 손실 9 청력 유전자와 형태 모두 취득을 치료하기위한 유망한 기술로서 떠오르고있다. 달팽이관 잠재적 여러 가지 이유로 유전자 치료에 이상적인 대상이다 : 작은 부피 필요한 바이러스의 제한된 양을 필요 1); 다른 기관계 제한 부작용 2) 상대적 격리; 및 3)는 유체 충전식 챔버 바이러스 용이미로 10, 11,12,13,14, 15에 걸쳐 배달. 선천성 청각 장애의 마우스 모델은 체계적, 복제 방법으로 내이의 개발을 모니터링하는 연구의 많은 방법을 사용 할 수 있습니다. 마우스 cochleae의 작은 크기는 약간의 수술 어려움을 제시 않지만, 마우스가 다른 종 (16)에 비해 여러 가지 실험적인 장점 유전 청력 손실의 연구에 매우 중요한 모델 역할을합니다. 마우스 모델은 유전자 연관 분석, 상세한 형태 학적 관찰의 수집 및 병원성 시나리오를 시뮬레이션을 통해 특성의 범위의 평가를 허용; 같은, 그들은 바이러스로 매개 유전자 치료에 대한 좋은 후보입니다. 기술적 진보와 함께 생쥐에서의 광범위한 유전 연구는 실험실 가능한 17,18, 19, 20, 21에 걸쳐 재현 가능한 방식으로 유전자 변형 마우스를 생성하도록 만들었다. Furthermor모두 취득이 동물 모델 22, 23, 24에서 엄격한 테스트를 허용, 마우스의 청력 손실 표현형을 상속에 대한 전자, 다양한 모델이 존재한다. 따라서, 마우스 모델에서 바이러스로 매개 유전자 치료를 사용하여 청력을 보정하는 것은 인간의 질병에 대한 치료법에 대한 검색에 적절한 첫 번째 단계입니다. 우리는 이전에 수 포성 글루타메이트 트랜스 포터 3 (VGLUT3를) 부족 형질 전환 마우스 인해 IHC 리본 시냅스 (25)에서의 글루타메이트 방출의 부족으로 청각 장애인을 태어나는 것으로 나타났습니다. 이 돌연변이는 감각 유모 세포의 주요 퇴보로 이어질하지 않기 때문에, 이러한 돌연변이 생쥐는 잠재적으로 선천성 난청 인공 유전자 치료를 테스트하기에 훌륭한 모델입니다. 현재까지, 인공 유전자 치료 용 바이러스 전달 다수의 기술을 통해 라운드 cochleostomy 윈도우 확산 막, 둥근 막 윈도우 주입 및 전달을 포함하여 설명되었다. 강력한있다원점이라 장점들 및 이러한 방법 (9)의 각각의 단점. 여기에서 우리는 둥근 창 막 (RWM)를 통해 VGLUT3 KO 마우스 내 귀에 바이러스로 매개 유전자 전달을위한 수술 방법을보고한다. 포스트 귀의 RWM 주입 방법은 우수한 청각 보존과 최소 침습적이며, 비교적 빠르다. 우리가 이전에 게시 한 바와 같이,이 마우스 모델에서 청각 복원하기위한 노력의 일환으로, VGLUT3 유전자 (AAV1-VGLUT3)를 들고 AAV1 벡터는 출생 후 12 일에이 청각 장애 생쥐의 달팽이관에 도입되었다 (P! @), 결과 26 청력의 회복. 트랜스 유전자의 단백질 발현이 면역 형광 (IF)를 사용하여 확인하면서 VGLUT3 KO 마우스에서 보청기는 청각 뇌간 반응 (ABR)에 의해 확인되었다. 따라서이 방법론은 바이럴 - 매개 유전자 치료하고자 달리 난청 결과 유전자 결함을 보정 할 수 있다는 점을 입증 해 보여준다.

<table border="0" cellpadding="0" cellspacing="0" width="803">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:355px;">Name</td>
			<td style="width:177px;">Company</td>
			<td style="width:161px;">Catalog Number</td>
			<td style="width:111px;">Comments</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:355px;">Ketamine</td>
			<td style="width:177px;">Butler Schein</td>
			<td style="width:161px;"></td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:355px;">Xylazine</td>
			<td style="width:177px;">AnaSed</td>
			<td style="width:161px;"></td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:355px;">Acepromazine</td>
			<td style="width:177px;">Provided by UCSF LARC</td>
			<td style="width:161px;"></td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:355px;">Carprofen analgesia</td>
			<td style="width:177px;">Provided by UCSF LARC</td>
			<td style="width:161px;"></td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:355px;">Betadine</td>
			<td style="width:177px;">Betadine Puredue Pharma</td>
			<td style="width:161px;"></td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:355px;">dexamethasone ophthalmic ointment (TobraDex)</td>
			<td style="width:177px;">Alcon</td>
			<td style="width:161px;"></td>
			<td style="width:111px;"></td>
		</tr>
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			<td style="width:177px;">Braintree scientific, inc.</td>
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			<td height="24" style="height:24px;width:355px;">25G needle</td>
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			<td height="40" style="height:40px;width:355px;">Borosilicate capillary pipette</td>
			<td style="width:177px;">World precision instruments, inc.</td>
			<td style="width:161px;">1B100F-4</td>
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			<td height="24" style="height:24px;width:355px;">Suture PDS*plus Antibacterial</td>
			<td style="width:177px;">Ethicon</td>
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			<td height="24" style="height:24px;width:355px;">Tissue glue (Vetcode)</td>
			<td style="width:177px;">Butler Schein</td>
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			<td height="24" style="height:24px;width:355px;">Rabbit Anti-GFP antibody</td>
			<td style="width:177px;">Invitrogen</td>
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			<td height="20" style="height:20px;">Dissecting microscope&#160;&#160;&#160;&#160;&#160;</td>
			<td>Leica</td>
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			<td height="20" style="height:20px;">Flaming/ Brown Micropipette&#160;&#160;&#160;&#160;&#160;</td>
			<td>Sutter Instrument Co</td>
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			<td height="20" style="height:20px;">Puller Model P-97&#160;&#160;</td>
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			<td height="20" style="height:20px;">TDT BioSig III System&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>&#160;Tucker-Davis Technologies</td>
			<td></td>
			<td></td>
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surgical method for virally mediated gene delivery to the mouse inner ear through the round window membrane

감각 신경성 난청의 기능 회복을 달성하기 위해 사용되는 유전자 치료는 청력 손실에 기여하는 기본 분자 유전 메커니즘의 이해를 부여 할 것을 약속드립니다. 내이 (內耳)에 벡터의 도입은 기존의 구조에 손상을 최소화하면서 널리 달팽이관을 통해 에이전트를 배포하는 방법으로 수행해야합니다. 이 원고는 분자, 약물 및 마우스 출생 후의 일 10 라운드 창 막 (RWM)를 통해 이전에 바이러스 성 전달을 사용하여 마우스 와우 치료에 사용할 수있는 포스트 귀의 수술 방법을 설명합니다. 혈액 손실을 최소화하고 동물의 사망률을 피하면서이 수술 방법은 스칼라 고실에 신속하고 직접 전달할 수 있습니다. 이 기술은 필수적인 내부 및 중이의 구조뿐만 아니라 목 근육 완전히 청력을 유지하면서 무시할 또는 전혀 손상을 포함한다. 이 수술 기법, 소낭 glutam의 효능을 설명하기 위해3 녹아웃 (VGLUT3 KO) 마우스가 아데노 관련 바이러스 (AAV-1)를 사용하여 내 귀에 VGLUT3의 출산 후 청각 복구 선천성 청각 장애의 마우스 모델의 예로서 사용되는 수송을 먹었다.

기술적 특징과 인공 분자 치료 후 귀의 접근 방식의 유틸리티를 확인하려면 AAV1-VGLUT3, AAV1-GFP 및 AAV2-GFP는 RWM를 통해 P10-12 마우스 내이 (內耳)에 전달했다. 이 방법은 내부 유모 세포 내에서 성공적으로 유전자 발현을 보여줍니다 (IHC) (VGLUT3 그림 1과 GFP 그림 2와 GFP 그림 3A), 외부 유모 세포 (OHC) (GFP 그림 2)와 지원 세포 (GFP 그림 2와 그림 3A)</str...

Watch this Scientific Journal Video about Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane at JoVE.com

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

라운드 창 멤브레인을 통해 마우스 내부 귀에 바이러스로 매개 유전자 전달을위한 수술 방법

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

surgical-method-for-virally-mediated-gene-delivery-to-mouse-inner-ear

Research

JoVE Journal

Neuroscience

15.1K 조회수.  University of California, San Francisco. The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane. 

비디오: Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

휴일 두개골 윈도우를 사용하여 마우스 뇌 엑기스의 Intravital 현미경

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

연구

신경과학

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

척수 전기 생리학 II : 세포 흡입 전극 제작

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

분리 LacZ를 표현 유동세포계측법를 사용하여 마우스 내이 (内耳) 조직의 세포를

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

에 의해 개발 마우스 내이 (内耳)에 진 전송<em&gt; VIVO에서</em&gt; Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

마우스 뇌의 장기 이미징을위한 세련된와 철근 Thinned-해골 창

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

귀에 세포의 패치 클램프 녹음 절연 Zebrafish에서

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

이미징은 기본 양식 마우스 뉴런의 플라즈마 막에 수용체 삽입을 pHluorin 태그

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

결합 된 방법론을 활용하여 축삭 분기 및 신경 세포 형태 형성 동안 플라즈마 멤브레인 배달의 역할을 정의합니다

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

중추 신경계에 대상 물질 전달을위한 마우스 미세 주입 기술<i&gt;를 통해</i&gt; 내부 경동맥

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

마우스에 있는 Laminectomy 및 척수 창 이식

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...