이 작품은 NIH 보조금 EY019312과 EY021126 (VJK), 워싱턴 대학 안과학 교실 및 Visual 과학에 EY002687에 의해 지원되었다, 연구에 의해 실명을 방지 할 수 있습니다.

모든 실험 프로토콜은 실험 동물의 관리 및 사용에 대한 가이드에 따라했고, 워싱턴 대학의 기관 동물 연구위원회에 의해 승인되었다. 1. 관류 및 시편 홀더 설정 <ol><li> 실험의 날에 신선한 망막 관류에 대한 솔루션을 준비합니다. 증류수 및 탈 이온수를 사용합니다. 다음 세 가지 방법 중 하나를 사용합니다. <ol><li> 미디어와의 NaHCO3 1.9 G : &#39;에임스 1 병 (1ℓ) 솔루션&#39;을 에임스를 중탄산염이 함유 준비 </li><li> (MM)에 로크의 솔루션 준비 : 112.5 염화나트륨, 3.6의 KCl, 2.4의 MgCl 2, 1.2 CaCl2를, 10.0 HEPES를, 20.0 NaHCO3를, 3.0 나 숙시 네이트, 0.5 나 조미료, 0.02 EDTA, 10.0 포도당, 0.1 % MEM 비타민과 아미노산 산 </li><li> (MM)에 준비 HEPES 완충 벨소리 솔루션 : 133.3 염화나트륨, 3.3의 KCl, 2.0의 MgCl 2, 1.0 CaCl2를, 10.0 포도당, 0.01 EDTA, 12.0 HEPES, pH를1 M의 NaOH 5.8 ml의 ~ 7.5로 조정, 0.72 G / L Leibovitz 문화 매체 L-15을 추가합니다. 어떤 관류 매체를 사용하여 광 수용체 응답을 분리하는 100 μm의 BaCl 2 - 50 μm의 DL-AP4 50 - 20를 사용합니다. </li></ol></li><li> (MM)에 전극 (28)에 대한 솔루션을 준비 : 140.0 염화나트륨, 3.6의 KCl, CaCl2를, 3 HEPES, 0.01 EDTA 2.4의 MgCl 2, 1.2 및 7.4에 pH를 조정 - NaOH로 7.5. 전극 액은 수개월 동안 실온에서 저장 될 수있다. </li><li> 준비하고 시편 홀더를 테스트합니다. <ol><li> 시험편 홀더의 바닥 부의 돔 위에 블랙 / 회색 여과지를 붙인다 (도 1A 참조). 돔 플랫 탑의 가장자리 주위 신중 이성 분 5 분 에폭시 접착제를 확산. 필요한 경우, 해부 현미경으로 그것을 할. </li><li> 접착제가 거의 건조 (약 4 분)이 될 때까지 기다렸다가 평면 항목을 사용하여 돔에 여과지를 누릅니다. 접착제 종이 필터 실험 전에 적어도 하루.여과지를 여러 번 사용할 수 있지만, 레코딩 개월 후에 교체되어야한다. 대체 용지를 설치하기 전에 접착제 잔류 물에서 조심스럽게 지붕을 청소하는 70 % 에탄올을 사용합니다. </li><li> 전극 액은 공기 방울을 방지하고 전극 채널로 나사산 어댑터로 둘러싸인 펠렛 전극 (도 1A 및 B 참조)하려고 나사가 구비 된 전극 채널을 채운다. 네 개의 나사와 시편 홀더의 상단과 하단 부분을 연결하고 전극 용액으로 관류 라인을 입력합니다. </li><li> 멀티 미터 (그림 1B)를 사용하여 각 전극 쌍의 리드 사이의 저항과 전압을 측정합니다. 저항 100 Ωk 10 MV 아래의 전압 채널 거품 무료이며 전극이 좋은 상태에있는 경우 이하이어야한다. </li></ol></li><li> 유리 병에 관류 매체의 700 mL의 - 400 붓는다. 또 다른 300 ㎖의 망막 및 S 해부에 사용되는 별도냉장고에서 그것을 찢었다. 열교환 블록 관류 튜브를 설정하고 가열판에 예열 된 블록을 배치 (도 1d 참조). </li><li> (그림 1D 참조) (에임스 &#39;또는 로크를 사용하는 경우) 연결이 2 / O 2 가스를 공동가 캡 병 및 관류 튜빙을 묶습니다. 37 O C의 미디어와 병을 예열 및 가열 플레이트 또는 37으로 설정 물을 욕조에 빛 자극 장치의 상단에 배치 - 39 O를 C (17). <ol><li> 국무 중력 중심의 흐름을 시작 관류 미디어를 작성하여 관류 라인. <ol><li> LKC 시스템에서, 다음 실험 기간 동안 병에 관류 용액의 저하 수준에 의해 영향을받지 않고, 유량 조절기에 의해 조절되는 중요한 중력 유동을 제공하도록 자극 부 (17)의 위쪽에 용기를 배치했다. </li><li> Ocuscience 시스템에서, P를 배치시편 홀더 긴 관류 출력 라인 노이즈를 최소화하고 배치하기 위해 패러데이 케이지 안에 erfusion 병 용액의 중력 구동을 증가시키는 잘 시험편 홀더 (관류 병)의 레벨 이하 (단계 2.8 참조). 이러한 출력 라인을 방패 ERG 신호에 전자 노이즈의 커플 링을 방지하도록 상기 증폭기의 접지에 실드를 연결한다. </li></ol></li></ol></li><li> 유량 조절기를 사용하여 / 분을 5 ml - 3 관류를 조정합니다. 적절한 규제와 실린더에서 5 % CO 2 / 95 % O 2 가스를 연결하고 공기 돌을 통해 병에 미디어의 꾸준한 버블을 보장하기 위해 유량을 조절합니다. </li></ol> 2. 샘플 준비 <ol><li> 직선 블레이 딩 microscissors, 하나 또는 두 개의 45 O 핀셋, 면도날 및 필터 종이의 사각형 조각을 포함하여 깨끗하고 날카로운 해부 악기를 조립합니다. </li><li> 감기 perfu의 약 200 ml에 붓고큰 페트리 접시에 시온 용액 (돔 포함) 시험편 홀더의 전체 바닥 부분 용액에 침지 될 수 있도록. 돔의 망막을 장착 할 때이 단계가 중요하게된다 (단계 2.6 참조). 일부 솔루션 carbogen (5 % CO 95분의 2 %의 O 2)로 포화되도록 설계되어 있지만, 이는 해부 상업적 필수적이지 여기 기재된 실험을 수행하지 않았다. </li><li> 녹음하기 전에 12 시간 - 전형적인 실험, 어두운 / 라이트 사이클과 6을 어두운 - 적응 12/12 시간에 동물을 유지. (현미경 광원 앞에 예를 들어, 빨간색 필터를 사용) 희미한 붉은 빛 아래 자궁 전위 다음 이산화탄소 흡입 동물을 안락사 및 어두운 빨간색 또는 적외선 조명 아래 모든 다음 절차를 수행합니다. <ol><li> 핀셋을 사용하여 눈을 잡아 당겨 미디어에 넣어. 페이퍼 (예를 들어, 일부 정규 여과지)의 작은 조각에 한번에 하나의 눈으로 만들어 놓고핀셋 눈 (이 여기에 솔루션의 외부에서 수행되는) 잡고 오라 세라의 수준에 약 조명. </li></ol></li><li> 오라 세라 따라 절단 (또는 가까운 눈의 적도에) microscissors와 각막과 렌즈를 제거합니다. 큰 페트리 접시에 차가운 미디어에서 아이 컵을 놓고 다른 눈으로 동일한 절차를 반복합니다. </li><li> 가능한 손상으로 망막을 유지하기 위해 망막과 공막 사이에 위를 유지하여 시신경 향해 아이 컵의 상단에서 작은 절개를 잘라. 그립 개의 핀셋을 사용하여 망막 분리를 위해 서로 떨어져 핀셋 당겨 의해 절개 양측으로부터 공막. <ol><li> 시신경을 잘라 말단 표면에 물리적 인 접촉의 최소 금액으로 망막을 분리하려고합니다. RPE 주로 박리 과정에서 자동 망막 분리한다. 그것은을 수행하는 것보다 망막의 기계적 교란을 피하기 위해 더 중요해부 빠르게 망막은 일반적으로 응답 특성에 심각한 영향없이 용액 중에서 적어도 30 분 동안 항온 처리 할 수​​있다. </li></ol></li><li> 시편 홀더 (그림 1C)의 돔에 망막를 탑재합니다. 해부 망막으로 페트리 접시에 시료 홀더의 바닥 부 담가. 돔 상기 망막 위쪽 감광체 측 (절연 망막의 볼록면)을 밀어 망막 여과지에 부착되도록 시험편 홀더를 해제. 다른 망막에 대해이 절차를 반복합니다. </li><li> 전기 망막 사이의 크로스 토크뿐만 아니라 노이즈와 신호 입환을 방지하기 위해주의 깊게 홀더 플레이트를 건조. 시험편 홀더는 홀더의 하부 및 상부 부분 사이의 액 유출을 방지 할뿐만 아니라 개별 망막의 감광체 및 신경절 세포 양쪽의 전기적 분리를 돕기 위해 바닥 부 돔 주위에 O 링을 갖는다. (네 개의 나사와 홀더의 상단 부분을 연결도 1b 참조), 주사기와 바늘을 이용하여 관류 용액으로 관류 채널을 채운다. </li><li> 열교환 블록 옆 시험편 홀더를 전송하고 시험편 홀더 (도 1D)에 대한 입력 및 출력 관류 라인을 연결한다. (상부 전극 증폭기 접지 / 마이너스 핀에 연결) ERG 증폭기에 전극을 연결하는 어댑터를 사용하여 표본 홀더에 자극기 / 제어 유닛을 부착하거나 따라 자극 부에 가열 패드를 밀어 생체 내에서 그 위에 에르그 시스템이 사용됩니다. </li></ol> 3. 녹음 <ol><li> 생체 시스템의 소프트웨어를 사용하여 앰프와 자극 설정을 구성합니다. 300 Hz로 필터링 1 ~ 10 kHz와 로우 패스 사이의 값으로 인수 주파수를 설정합니다. 하이 패스 필터링을 사용하지 마십시오. 60 Hz에서 (또는 유럽에서 50 Hz에서) 필요한 경우 필터를 홈을 사용합니다. </li><li> 빛 자극과 재치없이 기록 기준시간 희미한 자극 (예., -35 dB의 0.3 MCD의 SM -2의 녹색 빛) 전극과 망막 샘플 사이 좋은 전기적 연결을 테스트합니다. 망막 온도 함수 안정된 상태에 도달하도록 데이터 수집을 시작하기 전에 20 분 - 10 기다린. <ol><li> 특정 실험에 따라 자극 파라미터를 선택하고 데이터 수집을 시작합니다. 예를 들면, 0dB까지 0.1 1,000까지 MCD의 SM에서 -40에서 녹색 빛을 사용 -2 암순응 응답 가족을 위해. 봉 배경 빛에 의해 억제 할 필요가 포토 픽 녹음에 약 2 밝은 로그 단위 3 깜박 사용 (3-30 CDM -2) 또는 프로브 플래시 (-2 = 1dB 약 1.3 카드뮴 SM을, 그림 3 참조). </li><li> 그러나 정확한 빛의 강도 값이 시스템 교정 및 실험 조건에 다소 의존 할 수 있음을 기억하십시오. 경험적으로,도 5에 의해 다운 스케일 강도 - 10 배는 생체에 비해 </eM&gt; 녹음 녹색 빛 (17)를 사용하여. (상업적 어댑터 시스템에 포함) 망막 위의 개구를 가진 블랙 커버 시험편 홀더에 광 산란을 감소시키고 시스템이 생체 내에서 상용의 Ganzfeld 구함으로써 두 망막의 균일하고 동일한 자극을 용이하게하는데 사용될 수있다. 참고 : 어두운 적응 망막에서 녹음 들어, 일반적인 실험 표백제 안료의 무시할 부분에 사용되는 테스트 깜박의 강도가되도록 망막 색소 상피 중심의 재생의 부족은 문제가되지 않습니다. 또한, 이전에 콘 안료 재생 여전히 뮐러 세포주기 (20)를 통하여 시각적 절연 망막에서 일어날 수 있음을 보여왔다. </li></ol></li><li> 실험은 몇 시간에 30 분부터 일반적으로 마지막으로 이러한 변화는 기술적 인 문제를 나타낼 수로, 실험 기간 동안베이스 라인 드리프트, 소음 및 응답의 안정성을 모니터링 할 수 있습니다. 참고 : 노이즈 증가 또는 감소D 응답 진폭은 공기 관류 또는 전극 채널 거품, 너무 낮거나 높은 온도, 용액 누출 / 유출 또는 망막의 변위를 표시 할 수있다. </li></ol> 4. 청소 <ol><li> , 관류 라인으로부터 시험편 홀더를 분리하여 열고 여과지로부터 망막 플러시. 전극을 제거하고 증류수 (에탄올을 사용하지 않는)로 씻어. 에탄올 및 / 또는 증류수 (관류 채널 포함) 시편 홀더를 청소합니다. 조심스럽게 세척 관류 관 70&gt;와 % 에탄올. </li><li> 증류수 (세제를 사용하지 마십시오)와 관류 병을 씻어. 에탄올과 같은 병을 세척하기 위해 사용될 수있다. </li></ol>

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세인트 루이스의 워싱턴 대학은 Xenotec, 사와 라이센스 계약을 가지고 있으며, 생체 어댑터의 판매 로열티를받을 수 있습니다.

<html> 우리는 여기서 생체 ERG 어댑터와 함께 생체 ERG 시스템 구성 요소를 사용하여 두 개의 격리 된 마우스의 망막 동시에 고품질 생체 ERG 녹화를 얻기위한 중요한 단계를 보여준다. 본 연구에서 우리는 동일한 용액 (어느 에임스 &#39;, 로크 또는 링거)과 동물 모두에서 망막 관류하지만 약물 테스트를 위해 상이한 솔루션 등 각 망막 관류하는 것도 가능하다. 고품질...

전위도 (ERG)은 빛에 의해 트리거 망막의 전기 활동을 기록 할 수있는 잘 확립 된 기법이다. ERG 신호는 망막의 세포 외 공간 저항에 흐르는 전류에 의한 방사 전압 변화 (감광체 바이폴라 셀의 축 방향)에 의해 주로 생성된다. 제 ERG 신호 은점 (1)의 표면으로부터 그렌에 의해 1865 년에 기록되었다. Einthoven 유쾌한 1908 2 B-, A-라는 세 가지 파도에 빛의 발병 ERG 응답, 및 C 파 지금 쌍극 세포, 감광체 주로 활동을 반영하는 것으로 알려져 있으며, 색소 상피를 분리 세포, 각각 3-8. 에르그 지역 (미소 전극과 (생체) 고립 그대로 망막에 걸쳐, 고립 된 눈 준비 (9)에서, 마취 동물 또는 (생체 내) 인간의 눈에서 3,10-15 또는에서 특정 망막 층을 기록 할 수 있습니다ERG) 4,16. 이들 중에서, 생체 ERG 현재 망막 기능을 평가하기 위해 가장 널리 사용되는 방법이다. 또한 진단 목적으로 이용 될 수 있거나 또는 동물이나 환자의 망막 질환의 진행을 따라 비 침습적 방법이다. 그러나 생체 내 에르그 녹음은 종종 안구 생리 소음 (예, 호흡과 심장 활동)에 의해 오염, 여러 중복 구성 요소와 복잡한 신호를 생성한다. 로컬 ERG는 망막의 특정 층에 걸쳐 신호를 기록하기 위해 사용될 수 있지만, 가장 침습적이고 다른 ERG 기록 구성과 비교하여 낮은 신호대 잡음비 (SNR)를 갖는다. 지역 망막 전위도 기술적으로 요구하고 고가의 장비 (예를 들어, 현미경과 미세 조작기)가 필요합니다. 그대로 고립 된 망막에서 Transretinal ERG (생체 ERG)는 안정적이고 HIG을 허용 생체 내 및 지역 에르그 방법 사이의 타협을 제공합니다동물이나 인간 (17)의 손상 망막에서 시간 SNR 녹음. 최근에,이 방법은 포유 동물, 영장류 및 인간 망막 18-20에 막대와 콘 감광체 기능을 연구하기 위해 성공적으로 사용되어왔다. 또한, 인해 생체 망막 색소 상피 세포의 부재로, ERG 신호의 포지티브 형 C 파 성분을 제거하고 눈에 띄는 네거티브 느린 PIII 성분은 생체 녹화 드러난다. 느린 PIII 성분은 망막 21-23에서 뮐러 아교 세포의 활성에서 발생하는 것으로 나타났다. 따라서, 생체 외 ERG 방법도 그대로 망막 뮐러 세포를 연구하기 위해 사용될 수있다. 여러 연구는 또한 생체 ERG 레코딩은 망막 (24) 주위되는 약물의 농도를 측정하고, 약 25 ~ 27의 안전성과 효능을 테스트하는 데 사용될 수 보여 주었다. 생체 시스템의 여러 상업이 가능하며,반드시 광범위한 전기 생리학 배경이없는 많은 실험실에서 사용. 대조적으로, 체외 장치 (17)는 최근까지 극소수 실험실은이 강력한 기술을 활용되는 결과로서 사용할 수 없었다. 그것은 망막의 생리와 병리에 대한 우리의 지식을 향상하고, 질병을 눈부신에 대한 새로운 치료법을 개발하기 위해 더 많은 실험실에 생체 에르그 녹음을 사용할 수 있도록하는 것이 도움이 될 것입니다. 우리는 여기에 간단하고 저렴한 생체 ERG 장치 (17)을 설명하고는 일단이 상기 조작 패널 및 원뿔 - 매개 신호를 기록하는 몇몇 시판 생체 ERG 시스템과 조합하여 사용될 수있는 방법을 보여준다 (A- 및 B- 파)와 함수 그대로 야생형 마우스 망막에서 뮐러 세포 (PIII을 느리게).

<table><tbody><tr><td>&lt;i&gt;In vivo&lt;/i&gt; ERG system</td><td>OcuScience</td><td>HMsERG</td><td>&lt;a href=&quot;http://www.ocuscience.us/id77.html&quot;&gt;www.ocuscience.us/id77.html&lt;/a&gt;</td></tr><tr><td>&lt;i&gt;In vivo&lt;/i&gt; ERG system</td><td>LKC Technologies</td><td>UTAS-E 3000</td><td>&lt;a href=&quot;http://www.lkc.com/products/UTAS/bigshot.html&quot;&gt;www.lkc.com/products/UTAS/bigshot.html&lt;/a&gt;</td></tr><tr><td>&lt;i&gt;Ex vivo&lt;/i&gt; adapter</td><td>OcuScience</td><td>Ex VIVO ERG adapter</td><td>&lt;a href=&quot;http://www.ocuscience.us/id107.html&quot;&gt;www.ocuscience.us/id107.html&lt;/a&gt;</td></tr><tr><td>Dissection microscope</td><td>North Central Instruments</td><td>Leica M80</td><td>&lt;a href=&quot;http://www.ncimicro.com/store/ListProduct.asp?bSub=T&amp;amp;idCategory=27&quot;&gt;May use any brand&lt;/a&gt;</td></tr><tr><td>IR emitter</td><td>Opto Diode Corp.</td><td>OD-50L</td><td>&lt;a href=&quot;http://www.optodiode.com/&quot;&gt;www.optodiode.com&lt;/a&gt;</td></tr><tr><td>Prowler Night Vision Scopes</td><td>B.E. Meyers Electro Optics</td><td>D4300-I</td><td>Military grade product.</td></tr><tr><td>Red filter</td><td>Rosco Laboratories</td><td>Roscolux #27 Medium Red</td><td>May be used instead of IR system</td></tr><tr><td>Red head light</td><td>OcuScience</td><td>ERGX011</td><td>&lt;a href=&quot;http://www.ocuscience.us/catalog/i29.html&quot;&gt;www.ocuscience.us/catalog/i29.html&lt;/a&gt;</td></tr><tr><td>Microscissors</td><td>WPI, Inc.</td><td>500086</td><td>&lt;a href=&quot;http://www.wpiinc.com/&quot;&gt;www.wpiinc.com/&lt;/a&gt;</td></tr><tr><td>Dumont tweezers #5</td><td>WPI, Inc.</td><td>14101</td><td></td></tr><tr><td>Razor blades</td><td>Electron Microscopy Sciences</td><td>72000</td><td>&lt;a href=&quot;http://www.emsdiasum.com/&quot;&gt;www.emsdiasum.com&lt;/a&gt;</td></tr><tr><td>Scale</td><td>Metler Toledo</td><td>AB54-S/FACT</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;May use any brand&lt;/a&gt;</td></tr><tr><td>pH meter and electrode</td><td>Beckman Coulter</td><td>pHI 350</td><td>May use any brand</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;May use any brand&lt;/a&gt;</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>60129</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;May use any brand&lt;/a&gt;</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>63020</td><td>1.0 M solution</td></tr><tr><td>CaCl2</td><td>Sigma-Aldrich</td><td>21114</td><td>1.0 M solution</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>431788</td><td>May use any brand</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td>May use any brand</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma-Aldrich</td><td>S6297</td><td>May use any brand</td></tr><tr><td>Ames medium</td><td>Sigma-Aldrich</td><td>A1420</td><td>May use any brand</td></tr><tr><td>BaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>B0750</td><td>May use any brand</td></tr><tr><td>DL-AP4</td><td>Tocris Bioscience</td><td>101</td><td>May use any brand</td></tr><tr><td>Succinic acid disodium salt</td><td>Sigma-Aldrich</td><td>224731</td><td>May use any brand</td></tr><tr><td>L-Glutamic acid</td><td>Sigma-Aldrich</td><td>G2834</td><td>May use any brand</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G7528</td><td>May use any brand</td></tr><tr><td>Leibovitz culture medium L-15</td><td>Sigma-Aldrich</td><td>L4386</td><td>May use any brand</td></tr><tr><td>MEM vitamins</td><td>Sigma-Aldrich</td><td>M6895</td><td></td></tr><tr><td>MEM amino acids</td><td>Sigma-Aldrich</td><td>M5550</td><td></td></tr><tr><td>Carbogen</td><td>Airgas</td><td>UN3156</td><td>5% CO&lt;sub&gt;2&lt;/sub&gt;</td></tr></tbody></table>

simultaneous ex vivo functional testing of two retinas by in vivo electroretinogram system

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

우리는 암순응 야생형에서 플래시 응답을 기록 실험 프로토콜 표준 관류 다른 해결책 (도 2)를 사용하여 상술 한도 1에 도시를 수행하여 (WT) C57BL / 6 마우스의 망막. 응답 파형과 반응 속도뿐만 아니라로드 광 수용체의 민감도는 에임스 &#39;와 로크의 미디어 (그림 2A와 B)에서 유사 나타났다. 한편, HEPES 완충 링거액 (NO 중탄산염 또는 5 % CO 95분의 2 %의 O <...

Watch this Scientific Journal Video about Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System at JoVE.com

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

동시<em&gt; 생체</em두 망막의&gt; 기능 테스트로<em&gt; 생체</em&gt; 전위도 시스템

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

simultaneous-ex-vivo-functional-testing-two-retinas-vivo

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

9.1K 조회수.  Washington University in St. Louis. Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

비디오: Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Multifocal Electroretinograms

A limitation of traditional full-field electroretinograms (ERG) for the diagnosis of retinopathy is lack of sensitivity. Generally, ERG results are normal unless more than approximately 20% of the retina is affected. In practical terms, a patient might be legally blind as a result of macular degeneration or other scotomas and still appear normal, according to traditional full field ERG. An important development in ERGs is the multifocal ERG (mfERG). Erich Sutter adapted the mathematical sequences called binary m-sequences enabling the isolation from a single electrical signal an electroretinogram representing less than each square millimeter of retina in response to a visual stimulus1.
Results that are generated by mfERG appear similar to those generated by flash ERG. In contrast to flash ERG, which best generates data appropriate for whole-eye disorders. The basic mfERG result is based on the calculated mathematical average of an approximation of the positive deflection component of traditional ERG response, known as the b-wave1. Multifocal ERG programs measure electrical activity from more than a hundred retinal areas per eye, in a few minutes. The enhanced spatial resolution enables scotomas and retinal dysfunction to be mapped and quantified.
In the protocol below, we describe the recording of mfERGs using a bipolar speculum contact lens.
Components of mfERG systems vary between manufacturers. For the presentation of visible stimulus, some suitable CRT monitors are available but most systems have adopted the use of flat-panel liquid crystal displays (LCD). The visual stimuli depicted here, were produced by a LCD microdisplay subtending 35 - 40 degrees horizontally and 30 - 35 degrees vertically of visual field, and calibrated to produce multifocal flash intensities of 2.7 cd s m-2. Amplification was 50K. Lower and upper bandpass limits were 10 and 300 Hz. The software packages used were VERIS versions 5 and 6.

The development of the multifocal electroretinogram (mfERG) is an important advance in the diagnosis and characterization of retinopathy. Multifocal electroretinograms are a mathematical average of an approximation of a b-wave. Software programs can derive ERGs from more than a hundred retinal areas in a few minutes per eye. Scotomas and retinal dysfunction can be mapped and quantified.

A limitation of traditional full-field electroretinograms (ERG) for the diagnosis of retinopathy is lack of sensitivity.  Generally, ERG results are ...

연구

의학

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light whereas cones operate continuously under rapidly changing bright light conditions. Absorption of light by rod- and cone-specific visual pigments in the outer segments of photoreceptors triggers a phototransduction cascade that eventually leads to closure of cyclic nucleotide-gated channels on the plasma membrane and cell hyperpolarization. This light-induced change in membrane current and potential can be registered as a photoresponse, by either classical suction electrode recording technique1,2 or by transretinal electroretinogram recordings (ERG) from isolated retinas with pharmacologically blocked postsynaptic response components3-5. The latter method allows drug-accessible long-lasting recordings from mouse photoreceptors and is particularly useful for obtaining stable photoresponses from the scarce and fragile mouse cones. In the case of cones, such experiments can be performed both in dark-adapted conditions and following intense illumination that bleaches essentially all visual pigment, to monitor the process of cone photosensitivity recovery during dark adaptation6,7. In this video, we will show how to perform rod- and M/L-cone-driven transretinal recordings from dark-adapted mouse retina. Rod recordings will be carried out using retina of wild type (C57Bl/6) mice. For simplicity, cone recordings will be obtained from genetically modified rod transducin &alpha;-subunit knockout (T&alpha;-/-) mice which lack rod signaling8.

We describe a relatively simple method of transretinal electroretinogram (ERG) recordings for obtaining rod and cone photoresponses from intact mouse retina. This approach takes advantage of the block of synaptic transmission from photoreceptors to isolate their light responses and record them using field electrodes placed across the isolated flat-mounted retina.

마우스 망막에서 Transretinal 에르그 레코딩 :로드 및 원뿔 Photoresponses

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light ...

신경과학

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

손상 망막의 특성 Electrophysiological GFP - 표현 세포 집단

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Direct-Coupled Electroretinogram (DC-ERG) for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a specialized monolayer of cells strategically located between the retina and the choriocapillaris that maintain the overall health and structural integrity of the photoreceptors. The RPE is polarized, exhibiting apically and basally located receptors or channels, and performs vectoral transport of water, ions, metabolites, and secretes several cytokines.
In vivo noninvasive measurements of RPE function can be made using direct-coupled ERGs (DC-ERGs). The methodology behind the DC-ERG was pioneered by Marmorstein, Peachey, and colleagues using a custom-built stimulation recording system and later demonstrated using a commercially available system. The DC-ERG technique uses glass capillaries filled with Hank&#8217;s buffered salt solution (HBSS) to measure the slower electrical responses of the RPE elicited from light-evoked concentration changes in the subretinal space due to photoreceptor activity. The prolonged light stimulus and length of the DC-ERG recording make it vulnerable to drift and noise resulting in a low yield of useable recordings. Here, we present a fast, reliable method for improving the stability of the recordings while reducing noise by using vacuum pressure to reduce/eliminate bubbles that result from outgassing of the HBSS and electrode holder. Additionally, power line artifacts are attenuated using a voltage regulator/power conditioner. We include the necessary light stimulation protocols for a commercially available ERG system as well as scripts for analysis of the DC-ERG components: c-wave, fast oscillation, light peak, and off response. Due to the improved ease of recordings and rapid analysis workflow, this simplified protocol is particularly useful in measuring age-related changes in RPE function, disease progression, and in the assessment of pharmacological intervention.

Direct-Coupled Electroretinogram DC-ERG for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium

Here, we present a method for recording light-evoked electrical responses of the retinal pigment epithelium (RPE) in mice using a technique known as DC-ERGs first described by Marmorstein, Peachey, and colleagues in the early 2000s.

마우스 망막 안료 상피의 빛 방출 전기 응답 기록을 위한 직접 결합 된 전기 전체노그램 (DC-ERG)

The retinal pigment epithelium (RPE) is a specialized monolayer of cells strategically located between the retina and the choriocapillaris that maintain ...

화학

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the retina. The ERG is a sensitive measure of changes in retinal function that are pan-retinal, but is less effective for detecting damage confined to a small area of retina. In the present work we describe how to record the &#8216;flash&#8217; ERG, which is the potential generated when the retina is exposed to a brief light flash. We describe methods of anaesthesia, mydriasis and corneal management during recording; how to keep the retina dark adapted; electrode materials and placement; the range and calibration of stimulus energy; recording parameters and the extraction of data. We also describe a method of inducing ischemia in one limb, and how to use the ERG to assess the effects of this remote-from-the-retina ischemia on retinal function after light damage. A two-flash protocol is described which allows isolation of the cone-driven component of the dark-adapted ERG, and thereby the separation of the rod and cone components. Because it can be recorded with techniques that are minimally invasive, the ERG has been widely used in studies of the physiology, pharmacology and toxicology of the retina. We describe one example of this usefulness, in which the ERG is used to assess the function of the light-damaged retina, with and without a neuroprotective intervention; preconditioning by remote ischemia.

The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

쥐 망막 및 원격 사지 허혈 양상의 보호 효과에 기능을 평가하기 위해 전위도를 사용하여

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the ...

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

전위도의 동시 녹화 및 마취 된 흰쥐의 비주얼 사건 관련 전위

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

경동공 이광자 마우스 망막의 생체 내 이미징

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in retinal diseases, and testing therapeutic interventions. The ex vivo electroretinogram (ERG) allows the quantification of contributions from individual cell types in the isolated retina by addition of specific pharmacological agents and evaluation of tissue-intrinsic changes independently of systemic influences. Retinal light responses can be measured using a specialized ex vivo ERG specimen holder and recording setup, modified from existing patch clamp or microelectrode array equipment. Particularly, the study of ON-bipolar cells, but also of photoreceptors, has been hampered by the slow but progressive decline of light responses in the ex vivo ERG over time. Increased perfusion speed and adjustment of the perfusate temperature improve ex vivo retinal function and maximize response amplitude and stability. The ex vivo ERG uniquely allows the study of individual retinal neuronal cell types. In addition, improvements to maximize response amplitudes and stability allow the investigation of light responses in retina samples from large animals, as well as human donor eyes, making the ex vivo ERG a valuable addition to the repertoire of techniques used to investigate retinal function.

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Modification of existing multielectrode array or patch clamp equipment makes the ex vivo electroretinogram more widely accessible. Improved methods to record and maintain ex vivo light responses facilitate the study of photoreceptor and ON-bipolar cell function in the healthy retina, animal models of eye diseases, and human donor retinas.

작고 큰 눈의 망막 기능을 연구하기 위해 생체 외 망막 전도의 설정 및 조건 최적화

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

망막 신경혈관 질환 연구를 위한 생체 외 모델로서의 성인 마우스 망막의 망막 외 이식편

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

Two-Photon In Vivo Imaging of the Mouse Retina

<a href='https://appjove.unicartagenaproxy.elogim.com/v/61970/transpupillary-two-photon-in-vivo-imaging-of-the-mouse-retina'>Source: Wang, Z., et al.&#160;Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp.&#160;(2021).</a>This video demonstrates a technique for in vivo retinal imaging using two-photon microscopy. Anesthetized mice are prepared for imaging by securing their heads, Applying eye lubricant, and placing a coverslip over the eye. Retinal ganglion cells (RGCs) expressing fluorescent proteins are first visualized using epifluorescence for alignment. Finally, two-photon imaging is employed, allowing for high-resolution visualization of RGCs.

Source: Wang, Z., et al.&#160;Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp.&#160;(2021).This video demonstrates a technique ...

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