저자는 설치류 모니터링, 처리 및 실험에서 부인 샤론 SPANA의 도움에 감사합니다. 박사 자금 지원은 비전에서 우수 시드니 대학과 호주 연구 센터에 의해 제공되고있다.

이 프로토콜은 시드니 대학의 동물 관리 가이드 라인을 따른다. 1. 만들기 전극 <ol><li> 양극 백금 와이어 직경 1-2mm의 짧은 (5cm) 길이에서 (각막 연락을 드릴 것 하나)를 구축합니다. 루프에 직경이 수 mm 그것을 패션. 앰프의 입력단에 도달하도록 충분한 시간, 통상의 리드에 연결이 루프 (도 4 참조). </li><li> AG / AgCl을 또한 컨벤션 리드에 연결된 직경 1-2mm를, 펠렛 사용 (동물의 입에 갈 것입니다) 음극를 구축합니다 (그림 4 참조). </li><li> (동물의 엉덩이에 들어갈 것) 기준 전극은, 또한 적당한 길이의 리드에 접속 깨끗한 피하 주사 바늘 (23 G)를 사용함에 따라 (도 4 참조). </li><li> 이상적으로, (양의 U를 세 개의 전극을 연결, 기기 제조업체가 제공하는 세 개의 핀 케이블을 사용(94); 각막, 부정 → 입, 앰프 엉덩이 → 참조). </li></ol> 가벼운 자극 및 망막 전위도 셋업 2. 연결 및 교정 <ol><li> 어두운 할 수있는 작은 기록 실험실을 작성 (또는 위치). 빨간색 만든 이상 - 더 - 벤치 빛 또는 붉은 머리 램프의 한쪽 또는 양쪽에 착용. </li><li> 1 룩스를 초과하지 않도록 설치하는 동안 쥐의 눈에 도달하는 붉은 빛의 조도를 확인하기 위해 럭스 미터를 사용합니다. 주 : 감광 필터는 램프 밝기를 감소시킬 수 있고, 램프의 광원은 특히 적색광을 방출한다. 광원이 낮은 (가시) 파장을 방출하는 경우 다크 적응이 손상됩니다. </li><li> (이 종종 불투명 테이프와 지속성을 필요) 기록 실험실을 입력 모든 미광을 봉쇄 이상 들어갈 정도로 큰, 그래서 희미 감광 필터를 (이이 장에서 구입하실 수 있습니다) 준비, 컴퓨터 화면 당신은에있을 것이다 실험실. 참고 : 길 잃은 빛과화면의 빛이 쥐 눈의 어두운 적응을 침해하기에 충분하다. </li><li> 데이터 수집 하드웨어에 앰프를 연결합니다. 앰프에 긍정적, 부정적 기준 리드를 연결합니다. 컴퓨터 및 LED Ganzfeld 전원부 안전하게 접지 전원에 연결되어 있는지 확인. 참고 : 일부 연구소는 건물 접지에 연결, 접지 포인트를 전문으로; 배수관은 효율적인 대안이다. </li><li> 연구 품질 복사계와 LED 광원을 보정합니다. 동물의 눈은 실험 기간 동안 위치 할 때의 위치에 미터의 센서를 고정합니다. </li><li> Ganzfeld LED가 깜박 사이의 플래시 에너지, 플래시 지속 시간, 플래시 반복과 시간에 단계적인 증가와 전체 필드 ERG 프로토콜을 실행하는 프로그램은, interstimuls 간격 (ISI) 설정을라고. 예를 들어 전체 필드 프로토콜은 표 1을 참조하십시오. 참고 : ERG 깜박 반복 희미 깜박에서 증가 풀 필드 b에단계 현명한 방식으로 오른쪽으로 깜박입니다. 트윈 플래시 프로그램은 전체 필드에서 프로토콜에 다음과로드와 콘의 답변 분리 할 수​​ 있습니다. </li></ol> 망막 전위도 실험에 3 일 이전 <ol><li> 어두운 기록하기 전에 12 시간 동안 흰쥐 적응. 이 미광이 제거 된 후, 기록 실험실에서 이렇게하는 것이 편리하다. </li></ol> 에르그 실험의 4 일 <ol><li> 동물이 기록하면서 부드럽게 가열 될 수 있도록 정렬합니다. 우리는 동물의 머리가 Ganzfeld에 항목에 정확한 지점에서 휴식 할 수 있도록 구축 된 빛 금속 플랫폼을 사용합니다. 이 플랫폼은 우리가 물을 욕조에 40 ℃로 예열 물 펌프 통해 붙박이 튜브를 가지고있다. 주 : 경험이 37 ° C에서 동물의 코어 온도를 유지 보여준다. </li><li> 어두운 조건에서 쥐의 무게를. 기록의 무게와 올바른 케타민 (60 ㎎ / ㎏)과 자일 라진을 구성 (5 ㎎ / ㎏) 투여 량. 쥐 세대를 억제TLY 및 복강 마취제를 주입. </li><li> 주입 시간이 있습니다. 동물이 의식이되면 (대개 5 분 이내)을 가볍게 반사 반응이 존재하는지, 한 발 패드를 곤란하게하여 마취의 깊이를 확인합니다. 그것은이 반사가 진행하기 전에, 없거나 약한 때까지 기다리는 것이 좋습니다. </li><li> 각막에 proxmethacaine의 단일 아트로핀의 드롭 및 다른를 적용합니다. </li><li> 검은 스레드의 10cm 길이를 잘라. 단순한 매듭으로 루프를 확인하고 눈의 적도 위에 루프 슬립. 살짝 조여; 효과는 최소한의 압력으로, 약간 앞으로 안구를 그릴 것입니다. 이 눈꺼풀 분명 각막을 유지합니다. </li><li> 적용 카보 머 눈은 각막 표면에 떨어진다. 확인 카보 머 각막 표면에 남아 있고 눈꺼풀이나 얼굴에 유출하지 않습니다. </li><li> 가열 플랫폼의 상단에 흡수 침구를 놓습니다. </li><li> Ganzfeld의 ​​구멍에 권장되는 장소에 머리를 침대에 위치 쥐. </li><li> 삽입 INT직장에 ernal 온도 프로브. 꼬리에 프로브 코드를 테이핑하여 위치에서 안전 온도 프로브. </li><li> 피하 뒤쪽 다리에 기준 전극 (23 G 바늘)를 삽입하고, 앰프에 연결합니다. </li><li> 안전하게 입에 음극 (AG / AgCl을 펠렛)을 놓습니다. 이 입 밖으로 미끄러지는 것을 방지하기 위해, 안정된 표면에 연결 리드를 부착합니다. </li><li> 각막의 중심을 통해 양극을 배치합니다. 미세 조작기를 사용하여 전극이 부드럽게 각막 접촉되도록. </li><li> 37.5 ° C - 확인 체온은 37.0이다. </li><li> 동물 올바른 위치에 전극이 제자리에되면, 불투명 한 재료로 전체 설치 (Ganzfeld과 동물) 드레이프 (어두운 적응을 유지하기 위해). 우리는 부드러운 검은 천을 사용하십시오. </li><li> 미리 수집 샘플링 5 밀리 초와 100-1000 밀리의 수집 시간이 2 kHz 샘플링 레이트로 설정 취득 소프트웨어. 내지 1,000으로 밴드 패스 필터를 설정할Hz에서 그 샘플링을 보장은 플래시 다음 ~ 250 밀리의 기간을 샘플링 트리거됩니다. </li><li> 기록 기준선을 확인합니다. 그것은 외부 소음이 없어야하지만, 일부 앰프 소음과 진동 호흡을 보여 주어야한다. </li><li> 베이스 라인은 외부 소음을 표시하는 경우, 문제 해결을 시작합니다. 대부분의 문제는 전극의 위치, 또는 접지에 미끄러지 관련이 있습니다. 녹음은 외부 소음의 자유 보장하기 위해 패러데이 케이지를 사용합니다. </li><li> 테스트 플래시, 0.4 로그 스코틀랜드 cd.sm -2를 실행합니다. 비슷한 ERG 파형 (2A)가 나타납니다 그림. 0.4 로그 스코틀랜드의 cd.sm -2 플래시 연구실 전형적인 반응에서 (: -474 ± 39 μV와 b 파 : 파 1,512 ± 160 μV, N = 11)입니다. </li><li> 어두운 동물 10 분 동안 재 - 적응 허용합니다. 이 기준을 다시 확인이 10 분을 사용하는 것이 편리하다. </li><li> 안정적인 신호의 다음과 같은 확인 녹음을 시작합니다. </li><li> 기록 세션의 끝에서, 그 본체를 확인 temperatURE 유지 하였다. 전극을 제거합니다. 각막에 카보 머 폴리머를 다시 적용합니다. 이 동물 하우징에 반환하기 전에, 완전히 모바일 및 활성화 될 때까지 동물이 열 패드를 복구 할 수 있습니다. </li></ol> 5. 원격 허혈 <ol><li> 깨어 또는 anesthetised 설치류 중 하나에서 원격 허혈을 수행합니다. </li><li> 동물 마취 경우 (위) 가열 플랫폼에 누워 무릎의 명확한 뒷다리의 상단 부분에 혈압계 커프 슬립. </li><li> 동물이 처리에 사용되는 경우, 마취없이이 절차를 수행 할 수있다; 이 두 사람이 필요합니다. 한 사람이 부드럽게 동물을 억제하고, 두 번째는 혈압계 커프를 적용하고 혈압계를 운영하고 있습니다. </li><li> 깨어 동물, 부드럽게 한 뒷다리 무료로, 동물을 포장 수건의 조각 ~ 15cm X 30-50cm를 사용합니다. 머리가 홀더의 팔과 몸통, 장소 사이에 자리 잡고으로, 왼쪽 팔뚝 (말)에 자사의 뒷면에있는 동물을 놓는다커프는 단지 설명했다. </li><li> 커프를 수축 및 공기압 밸브가 폐쇄되어 있는지. anesthetised 동물에서 160 mmHg로에 커프 펌프, 깨어 동물에서 180 mmHg로한다. 이 수축기 혈압 (일반적으로 140 mmHg로 160 mmHg로 각각)를 초과합니다. </li><li> 휴대용 펌프를 이용하여, 필요에 따라 이들의 압력을 유지한다. </li><li> 허혈 후 예정된 시간 (우리는 5 분 재관류로 구분 5 분의 2주기를 사용), 공기압 밸브를 풀어 커프 압을 수축. </li><li> 발바닥에 붙어 피부 온도 프로브 원격 허혈의 효과를 확인한다. 피부 온도는 일반적으로 5 분 이상, 32-30 ° C의에서 떨어지는 재관류에 복구합니다. </li></ol> 6. 가벼운 손상 <ol><li> 쥐가 어두운 적응 하룻밤 전에 가벼운 손상 과정에 있는지 확인하십시오. </li><li> (지체없이 우리의 실험에서) 사지 허혈 다음 적절한 시간에, 각 동물은 플렉시 글라스 박스, 위스콘신에 단독으로 배치됩니다바닥 기반의 용기에 번째 물과 음식. 참고 : 빛에 의한 손상은 흰둥이 동물에서 수행 할 수 있습니다. </li><li> 표준 시간 (보통 오전 9시)에서 미리 교정 1,000 룩스 흰색 빛에 전환하고 24 시간 동안이 상태를 유지한다. </li></ol> 7. 망막 전위도 데이터 추출 및 분석 <ol><li> ERG의 평균 웨이브 폼을 획득. 감산하여, 비 - 제로 기준에 대한 정확한 필요한 경우. </li><li> 기준선과 제 사이의 전압 차이로서, (높은 자극 강도에 중간에서 발표) 파의 진폭을 측정한다 (&lt;30 밀리 초 지연) 트로프 (도 1). </li><li> 일반적으로 80-100 밀리 (도 1)의 지연 시간에 발생하는 파장의 피크 파장은 다음의 양극 사이의 전압 차이와 같은 B 파 진폭을 측정한다. </li><li> 90 Hz의 전이 대역으로 60-235 Hz의 데이터를 필터링하는 푸리에 변환을 이용하여 진동을 격리 전위 <sup&gt; 7. 필요한 절연 진동 전위 신호는 필터링되지 않은 파형으로부터 감산 될 수있는 경우 파 물마루의 ID를 확인한다. </li><li> A-와 B 파 피크의 암시 적 시간 (대기 시간)도 유용한 측정 (그림 1)이 될 수 있습니다. 로드 응답을 분리하는 트윈 깜박을 사용합니다. 혼합 응답 (플래시 1)로드 응답을 분리하기 (그림 2)에서 콘 응답 (플래시 2)를 뺍니다. </li><li> 각각의 광 강도에게 파 및 b 파의 진폭 (후 처리 / 후 처리-기준)을 정상화 또는 치료 그룹에 대한 평균. 강도 - 반응 곡선은 플래시 에너지 대 그룹 진폭 및 에러 플롯. </li></ol>

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A., Bush, R. 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Investigative Ophthalmology &amp; Visual Science. 55 (4), 2088-2096 (2014).</li><li>Maccarone, R., Di Marco, S., Bisti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saffron+supplement+maintains+morphology+and+function+after+exposure+to+damaging+light+in+mammalian+retina.">Saffron supplement maintains morphology and function after exposure to damaging light in mammalian retina.</a> Investigative Ophthalmology &amp; Visual Science. 49 (3), 1254-1261 (2008).</li><li>Hood, D. C., Birch, D. 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조나단 스톤 CSCM Pty 주식 회사의 이사

위에서 설명한 어두운 적응 플래시 에르그 방법은 쥐의 망막 기능을 평가하기위한 신뢰할 수있는 방법이다. 파 및 b 파 모두 빛의 손상에 의해 감소​​되었다. 원격 허혈 양상은 파 및 b 파의 광 손상에 의한 감소를 완화. 망막 기능이 보존 원격 허혈 양상은 저산소증, 허혈 및 운동 8-10으로 보호 전처리의 다른 형태를 닮은 신경을 유도 것을 제안합니다. 녹화 설정, 광 자극의 파라​​미?...

ERG는 광에 응답하여 망막에 의해 발생하고, 눈의 각막 표면으로부터 기록 전위이다. 기록 조건을 신중하게 관리하는 경우, ERG 망막 기능을 평가하기 위해 다양한 방법으로 사용될 수있다. 여기서 우리는 &#39;플래시 ERG&#39;, 망막 Ganzfeld 배경에 제시된 간단한, 밝은 플래시에 노출되었을 때 발생하는 전위를 기록하는 방법을 설명했다. Ganzfeld는 균일하게 빛을 분산 빛의 플래시가 약 균일하게 전체 망막에 도달한다. 망막을 기록하기 전에 어두운 적응되며, 동물의 기록을 위해 준비 될 때 어두운 적응이 유지되는 경우, ERG 두로드와 콘 감광체에 의해 생성된다 얻어진. 어두운 적응 플래시 에르그는 두 가지 방법으로 분석 된 특성 파형을 가지고 있습니다. 먼저, ERG 파형의 초기와 후기 컴포넌트 구별하고 뉴런의 시퀀스와 관련되어왔다망막 알 활성화. 최초의 구성 요소는 짧은 대기 시간에 음의 가능성, 파 (그림 1)입니다. 이것은 양의 진행 가능성이 뒤에, B 파를했다. B 파의 상승 단계는 별도의 구성 요소 (진동 전위 또는 OPS) 간주되는 진동을 보여줍니다. 파장은 감광체에 의해 생성 된 것으로 간주되며, 무 축삭 세포에 의한 1 층의 내측 핵 세포 및 OPS 의한 B 표면파. 자극 강도에 기초하여, 매우 희미 깜박에 응답 암순응 임계 반응이 가능하다 불린다. 암순응 임계 응답은 망막 신경절 세포 2-4에서 생성하는 것으로 이해된다. 둘째, 플래시 ERG 빛 적응에 의해 분리 될 수 있고, 또는 두 플래시 프로토콜에 의해 일단이 상기 조작 패널과 원뿔 중심 성분으로, 후술. 포토 픽 조건 하에서, 콘 인구가 낮기 때문에, 전파는 쥐에서 검출되지이지만 OPS와 B 파장은5 취소합니다. 그의 망막 높은 콘 인구가 영장류에서, 일단이 상기 조작 패널과 원뿔 경로 모두 검출 파 (6)를 생성합니다. 종종 플래시 ERG로부터 추출 두 유용한 대책 감광체 인구가 위험한 점 밝은에 노출, 예를 들면, 감소도 2에 도시 된 전형적인 플래시 응답.와도 1에서와 같이 측정 A- 및 B- 파의 진폭, 아르 빛, 망막 전위도의 모든 구성 요소가 감소된다. 원격 허혈 (RIP)와 같은 신경 개입, A-와 B 파 (그림 3)의 진폭의 보존에 의해 검증 될 수있다. 요약하면, ERG의 분석은 건강 손상 가볍고 neuroprotected 망막 간의 비교를 가능하게한다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>PC computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Powerlab, 4 channel acquistion hardware</td>
			<td>AD Instruments</td>
			<td>PL 35044</td>
			<td>Acquistion of ERG</td>
		</tr>
		<tr>
			<td>Animal Bio Amp</td>
			<td>AD Instruments</td>
			<td>FE 136</td>
			<td>Amplifier for ERG</td>
		</tr>
		<tr>
			<td>Lab chart</td>
			<td>AD Instruments</td>
			<td></td>
			<td>Signal collection software</td>
		</tr>
		<tr>
			<td>Ganzfield</td>
			<td>Photometric solutions</td>
			<td>FS-250A</td>
			<td>Light stimulus</td>
		</tr>
		<tr>
			<td>Ganzfield operating system</td>
			<td>Photometric solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Research Radiometer</td>
			<td>International light technologies</td>
			<td>ILT-1700</td>
			<td>calibrate light series</td>
		</tr>
		<tr>
			<td>Lux meter</td>
			<td></td>
			<td>LX-1010B&#160;</td>
			<td>check red light illumanation</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lead wires</td>
			<td>AD Instruments</td>
			<td></td>
			<td>Connect postive, negative ground electrodes to amplifier</td>
		</tr>
		<tr>
			<td>Lead wires -aligator</td>
			<td>AD Instruments</td>
			<td></td>
			<td>ground ganzfield and acquistion hardware to computer</td>
		</tr>
		<tr>
			<td>Platinum wire 95%</td>
			<td>A&#38;E metals</td>
			<td></td>
			<td>postive electrode</td>
		</tr>
		<tr>
			<td>Mouth electrode Ag/AgCl Pellet</td>
			<td>SDR</td>
			<td>E205</td>
			<td>negative electode</td>
		</tr>
		<tr>
			<td>26 gauge needle</td>
			<td>BD</td>
			<td></td>
			<td>ground electode</td>
		</tr>
		<tr>
			<td>Water pump</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Homeothermic blanket system with flexible probe</td>
			<td>Harvard Appartus</td>
			<td>507222F</td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine 1% w/v</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>topical mydriasis</td>
		</tr>
		<tr>
			<td>Proxmethycaine 0.5% w/v</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>topical anaesthetic</td>
		</tr>
		<tr>
			<td>Visco tears eye drops</td>
			<td>Novartis</td>
			<td></td>
			<td>carbomer polymer</td>
		</tr>
		<tr>
			<td>Thread</td>
			<td></td>
			<td></td>
			<td>retract eye lid</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reusable adhesive</td>
			<td>Blu tac</td>
			<td></td>
			<td>Dim red headlamp. Affix electrodes</td>
		</tr>
		<tr>
			<td>Absorbent bedding</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamil - ketamine 100 mg/ml - 50 ml</td>
			<td>Troy Laboratories Pty Ltd</td>
			<td></td>
			<td>dissociative</td>
		</tr>
		<tr>
			<td>Xylium - Xylazine 100 mg/ml - 50 ml</td>
			<td>Troy Laboratories Pty Ltd</td>
			<td></td>
			<td>muscle relaxant</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

프로토콜은 생체 내 쥐의 망막 시각 기능을 측정 할 수있다. 전파, 광 수용체의 기능을 측정하고, B-파도 내측 망막 기능의 측정은,도 1에 주석 첨부된다. 도 2a에 도시 된 바와 같이 증가하는 빛 자극과로드 지배 ERG 신호 증가. 파장이 0.4 로그 스코틀랜드의 cd.sm -2 2.5 로그 스코틀랜드의 cd.sm에서 포화 될 때까지 파의 진폭이 증가 -2</s...

Watch this Scientific Journal Video about Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning at JoVE.com

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

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 ...

using-electroretinogram-to-assess-function-rodent-retina-protective

Research

JoVE Journal

Neuroscience

16.0K 조회수.  University of Sydney. 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.

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

Strategies for Study of Neuroprotection from Cold-preconditioning

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by development of effective, easy to administer and safe preconditioning treatments. We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. Low-level pro-inflammatory mediator signaling changes over time are essential for cold-preconditioning neuroprotection. This signaling is consistent with the basic tenets of physiological conditioning hormesis, which require that irritative stimuli reach a threshold magnitude with sufficient time for adaptation to the stimuli for protection to become evident.
Accordingly, delineation of the immune signaling involved in cold-preconditioning neuroprotection requires that biological systems and experimental manipulations plus technical capacities are highly reproducible and sensitive. Our approach is to use hippocampal slice cultures as an in vitro model that closely reflects their in vivo counterparts with multi-synaptic neural networks influenced by mature and quiescent macroglia / microglia. This glial state is particularly important for microglia since they are the principal source of cytokines, which are operative in the femtomolar range. Also, slice cultures can be maintained in vitro for several weeks, which is sufficient time to evoke activating stimuli and assess adaptive responses. Finally, environmental conditions can be accurately controlled using slice cultures so that cytokine signaling of cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects. Cytokine signaling system analyses require the use of sensitive and reproducible multiplexed techniques. We use quantitative PCR for TNF-&alpha; to screen for microglial activation followed by quantitative real-time qPCR array screening to assess tissue-wide cytokine changes. The latter is a most sensitive and reproducible means to measure multiple cytokine system signaling changes simultaneously. Significant changes are confirmed with targeted qPCR and then protein detection. We probe for tissue-based cytokine protein changes using multiplexed microsphere flow cytometric assays using Luminex technology. Cell-specific cytokine production is determined with double-label immunohistochemistry. Taken together, this brain tissue preparation and style of use, coupled to the suggested investigative strategies, may be an optimal approach for identifying potential targets for the development of novel therapeutics that could mimic the advantages of cold-preconditioning.

We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. We present strategies for such work that require biological systems, experimental manipulations plus technical capacities that are highly reproducible and sensitive.

콜드 preconditioning에서 Neuroprotection 연구를위한 전략

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

연구

신경과학

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

피부와 사지 본즈 Innervating 마우스 감각 신경 섬유의 조직 준비 및 Immunostaining

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

마우스 망막을 사용하여 중추 신경계의 혈관 재생의 평가

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

Zebrafish의 애벌레에서 비주얼 응답의 전위도 분석

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

CLARITY를 사용하여 마우스 척수에서 세로토닌 섬유 이미징 / CUBIC 기술

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

Electroporation에 의해 쥐 좌골 신경의 슈반 세포로 생체 내 유전자 전달에

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

가압된 설치류에 폭발을 이용한 Neurotrauma의 효과 중간 대뇌 동맥

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Sciatic 신경 호감 상해의 쥐 모형에 있는 기능 평가를 위한 3D 운동학 분석

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has examined the behavioral and neural effects of mindfulness meditation on episodic memory. Here we present a protocol that combines mindfulness meditation training, an episodic memory task, and EEG to examine how mindfulness meditation changes behavioral performance and the neural correlates of episodic memory. Subjects in a mindfulness meditation experimental group were compared to a waitlist control group. Subjects in the mindfulness meditation experimental group spent four weeks training and practicing mindfulness meditation. Mindfulness was measured before and after training using the Five Facet Mindfulness Questionnaire (FFMQ). Episodic memory was measured before and after training using a source recognition task. During the retrieval phase of the source recognition task, EEG was recorded. The results showed that mindfulness, source recognition behavioral performance, and EEG theta power in right frontal and left parietal channels increased following mindfulness meditation training. In addition, increases in mindfulness correlated with increases in theta power in right frontal channels. Therefore, results obtained from combining mindfulness meditation training, an episodic memory task, and EEG reveal the behavioral and neural effects of mindfulness meditation on episodic memory.

Here we present a protocol for combining mindfulness meditation training, an episodic memory task, and EEG to understand the behavioral and neural effects of mindfulness meditation on episodic memory.

행동과 EEG를 결합하여 마음 챙김 명상이 에피소드 기억에 미치는 영향 연구

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has ...