Name: kidney regeneration in adult zebrafish by gentamicin induced injury
Uploaded: 2015-08-03T14:30:00.000+00:00
Duration: 6 min 25 s
Description: Watch this Scientific Journal Video about Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury at JoVE.com

This work was supported by NIH grant F32DK091998 to CNK; NIH grant RO1DK041071 and Harvard Stem Cell Institute grant D001229 to IAD. The authors thank Neil Hukriede for the lhx1ain situ probe.

참고 : 윤리 문 : 모든 실험 연구에서 동물 사용에 대한 매사 추세 츠 종합 병원의 지침에 따라 실시 하였다. 1. 사전 준비 <ol><li> 얼마나 많은 성인 제브라 피쉬 6-12개월 이전을 다치게 결정합니다. 계획은 필요한 것보다 10 내지 20 %의 더 많은 물고기를 손상한다. 변동성을 최소화하기 위해, 사용 연령은 대략 동일한 크기 및 작은 유전 적 다양성을 갖도록 동일한 탱크에서 함께 자란 형제 물고기 유사한. 여성 물고기 인해 복부의 더 큰 용량, 남성 물고기에 비해 주입하는 것이 훨씬 쉽다. 그러나 남성과 여성이 비슷한 결과를 제공합니다. </li><li> 처음 실험을 수행 할 때, 고기의 특정 균주에 적절한 겐타 마이신 투여를 결정 도즈 곡선을 플롯. 겐타 마이신의 순도가 일괄 배치에 따라 다릅니다 때문에 실험에 사용하기 전에 겐타 마이신의 각각의 새로운 배치를 테스트합니다. 적절한 투여 량은 expressi을 평가하여 결정되어야한다같은 lhx1a으로 부상 마커에. (- D 그림 1A 참조). </li><li> 겐타 마이신 솔루션을 준비합니다. 젠타 마이신 스톡 용액을 -20 ° C에 저장되고 나중에 사용하기 위해 해동 될 수있다. 예를 들어, 0.5 g의 무게 물고기 80 ㎎ / ㎏ 용량 (체중의 80 mg을 겐타 마이신이 / ㎏)을 위해, 편리한 작업 솔루션으로 인산 완충 식염수에 겐타 마이신의 2 ㎎ / ㎖을 준비합니다. 이 물고기에 복강 내 주사에 대한 겐타 마이신의 20 μl를 제공합니다. 주 : 일반적으로 80 ~ 120 ㎎ / ㎏ 적절할 수 40 밀리그램 / kg 정도로 낮은 양호한 결과이지만 용량을 달성한다. 겐타 마이신은 실험실 직원에게 위험 노출을 최소화하기 위해 솔루션에서 구입하실 수 있습니다. 참고 : 고용량의 겐타 마이신 독성이 될 수 있습니다. 장갑을 착용하고 분말을 계량 할 때 마스크. </li><li> 물고기를 마취에 대해 0.016 %의 tricaine의 물 100ml를 준비합니다. 사용까지 4 ℃에서 pH를 7 점으로 조정 멸균 한 MilliQ 물에 용해 tricaine (4g / 1 리터)의 25 배 재고를 준비합니다. 4m를 희석0.016 %의 작업 농도 물고기 물 100ml에 L. 참고 : 처리 할 때 Tricaine는 마취 및 피부 자극성, 장갑을 착용하십시오. </li><li> 물고기는 국자 모양으로 전구를 절단 물을 배수하기 위해 바닥에 2 슬롯을 절단 플라스틱 전송 피펫에서 특종합니다. 준비된 겐타 마이신 용액 (10 μL 계조와) 부하 1 ㎖ 주사기와는 30½ G 바늘을 연결합니다. 공기 방울을 제거합니다. 바늘의 각도 팁 멀리 직면하고 주사기 표시 앞으로 읽을 직면하고 있는지 확인하기 위해 주사기 바늘을 트위스트. 제어 주사를 들어, 멸균 PBS로 주사기와 바늘을 준비합니다. </li><li> 깨끗한 표면에 스케일을 준비하거나 물고기를 건조 및 사출을위한 물고기를 잡고위한 보트뿐만 아니라 종이 타월 무게. </li><li> 관찰 하룻밤 후 주입 물고기를 잡고위한 뚜껑 개인의 작은 반 리터의 용기를 준비합니다. 작은 투명 플라스틱 결합 케이지는이 목적을 위해 유용하다. 이들용기는 흰색해서는 안 - 물고기 창고 흰색 상피 캐스트 검은 벤치 위에 관찰 할 수 있도록 이상적으로 그들이 명확해야한다. 물고기가 편안하게 수영을 충분히 물고기 물 각 컨테이너를 채 웁니다. </li></ol> 겐타 마이신 2. 복강 내 주사 <ol><li> 물고기 물에 0.016 %의 tricaine 용액 물고기 마취. 느리게하는 아가미 환기율에 대해 더 이상 터치 응답하지 물고기를 기다립니다. </li><li> 아가미 또는 지느러미 부상을 방지하기 위해 꼬리를 향해 머리에서 이동, 물고기 국자를 사용하여 물고기를 특종. 옆으로 부드럽게 물고기를 돌려 과잉의 물을 흡수하고 특종 여분의 물을 흔들어 종이 타월에 물고기를 놓습니다. </li><li> 다시 물고기를 특종 및 제로화 규모에 무게 보트에 배치하고, 물고기의 무게. 가장 가까운 0.25 g의 라운드, 그리고 주입하는 겐타 마이신의 적절한 양을 계산합니다. 참고 : 0.5 g의 물고기 8에서 20 μL 주입을 사용0 ㎎ / ㎏ 투여. </li><li> 다시 물고기를 특종 마른 접힌 종이 타월에 놓습니다. 오른손을 사용하여 주입하는 경우, 왼쪽 손에 종이 타월을 잡고 배를 쉽게 접근 할 수와 생선의 머리는 왼쪽을 가리키는 놓습니다. </li><li> 배설강에 배의 피부, 전방에 45 ° 각도로 바늘과 주사기를 잡습니다. 피부 바로 아래에 바늘을 밀어 다음 각도를 줄이고 내부 장기를 피하고, 피부 아래 앞으로 바늘을 밀어 넣습니다. 다음 바늘을 철회, 어떤 액체가 바늘 주위에 배출되지 않는 확인하기 위해 일시​​ 정지, 플런저에게 적절한 양의 우울. 물고기가 지속적으로 유지하는 것은 문제가있는 경우, 몸통에 대한 중괄호 팔꿈치를 안정. 참고 : 겐타 마이신 솔루션을 취급 할 때는 장갑을 착용 할 것. </li><li> 개별 용기에 각각의 물고기를 삭제합니다. 물고기를 관찰하고 마취로부터의 회복을 보장합니다. 28.5 ° C에서 물고기를 밤새 보관하십시오. 주 : 생선 즉시 회복되지 않는 경우, 트랜스의 플라스틱을 사용그 아가미 그것을 부활을 통해 FER 피펫 물을 관개한다. </li><li> 겐타 마이신의 동일 투여 여러 물고기를 주입 할 때 같은 주사기와 바늘을 사용합니다. 적절한 생물 학적 날카로운 물건 용기에 사용 된 주사기와 바늘 폐기하십시오. </li></ol> 상해 3. 포스트 사출 관측 <ol><li> 다음 날 (1 일 게시물 부상), 어두운 화면에서의 용기에 주입 된 물고기를 놓습니다. 부상당한 물고기 배설 죽은 상피 조직의 화이트 캐스트는 볼 수 있어야합니다. (- H 그림 1E 참조). 더 캐스트,이 없으면 어느 물고기 겐타 마이신 주입 부상되지 않았다 (도즈 하나 너무 낮은, 또는 사출 과정에서 유출 젠타 마이신의 일부) 또는 물고기가 심하게 요관 배설강의 완전한 폐색을 초래 부상 괴사 된 조직으로. 참고 : 물고기는 보통 2-3일 내에 죽을 몸에서 캐스트를 취소 할 수 없습니다와 이상에 대한 따라서 사용할 수없는 경우분석. 어떤 캐스트를 볼 수없는 경우, 아가미의 움직임이 정지 된 후 적어도 10 분 동안 침지 tricaine 물에 물고기를 안락사. </li><li> 흰색 조직 캐스트를 볼 수있는 경우를 제외하고 물고기를 설정하고 다른 물고기를 계속 검사. 겐타 마이신의 적절한 용량은 사용할 수있는 물고기 80 ~ 90 %가 발생합니다. 부상당한 물고기를 풀 한 다음 원하는 경우 다른 치료 그룹으로 분할합니다. </li></ol> 복구 물고기 4. 케어 <ol><li> 깨끗한 물과 한적한 환경에서 물고기를 유지합니다. 더러운 물과 밀집는 감염과 의도하지 않은 죽음으로 이어질 것입니다. 가능하면 매일 물을 더 이상 6보다 물고기 / 물고기 물 500㎖를 주기적으로 변경하려고합니다. 참고 :이 공간의 보존 또는 조사 비싼 약물 치료를 사용하여 실험을 수행하고자하는 경우 최소 볼륨입니다. </li><li> 삼일이 부상을 게시 할 때까지 물고기를 먹이를하지 마십시오. 복구 물고기는 처음에는 식사를하지 않으며, 탱크의 모든 음식은 분해 박테리아 GR을 촉진owth. 삼일에 부상을 게시 시작, 하루에 한 번 소량의 먹이를. </li></ol> 부상 신장 5. 분석 <ol><li> lhx1a의 mRNA 발현에 대한 현장 하이브리드의 경우, 신장이 원하는 시점들에서 물고기에서 수확 할 수있다. 아가미의 움직임이 멈출 때까지 얼음에 tricaine 물에 적어도 10 분 물고기를 안락사, 다음 면도날로 머리를 제거하고 뱃속을 열고 집게와 내부 장기를 제거합니다. </li><li> 장소 (지느러미 본체 벽에 부착 된 색소 기관)의 신장을두고 하룻밤 PBS에 4 % 파라 포름 알데히드에서 물고기를 수정합니다. 신장 (15)을 해부 및 현장 하이브리드 표준을 계속합니다. <ol><li> 간략하게, 실온에서 1 시간 동안 프로 테이나 제 K에 PBST (10 μg의 / ㎖)으로 투과성이 PBST (인산염 완충 식염수, 0.5 % 트윈 20)에서 신장 씻는다. 4 % 파라 포름 알데히드와 후 수정 및 PBST로 다시 세척 하였다. </li><li> 그런 다음 68에서 하룻밤 신장을 사전 하이브리드혼성화 완충액 (50 % 포름 아미드, 5 배 SSC, 50 μg의 / ㎖ 헤파린, 500 μg의 / ㎖ tRNA는 0.1 % 트윈 20, pH를 6.0)에서 C °. </li><li> 혼성화 완충액 제닌 표지 프로브와 샘플을 인큐베이션 한 후 68 ° C에서 100 %, 75 %, 50 %, 25 % 혼성화 완충액으로 5 내지 10 분 동안 각각 세척 한 다음, 실온으로 옮겨 30 분으로 두 번 씻어 배 0.1 % 트윈 20와 SSC, 0.1 % 트윈 20와 0.2 배씩 SSC와 30 분 동안 두 번 씻는다. </li><li> MAB의 샘플 3 배 5 분 (0.1 M 말레 산, 0.15 M의 NaCl, pH를 7.5) 10 % 염소 혈청과 MAB 4 ℃에서 하룻밤 차단 평형. 그리고 안티 - 디그 옥시 제닌 AP 팹 1에서 하룻밤 샘플을 품어 : 5,000 MAB에. </li><li> MAB 1 시간 동안 샘플 5 배를 세척하고, NTMT 15 분 (0.1 M 트리스 pH9.5, 0.05 M의 MgCl 2, 0.01 M의 NaCl, 50 μL 트윈 20)에 대해 3 배를 평형. </li><li> 그런 다음 신호를 감지하는 NBT / BCIP와 신장을 취급합니다. 4 % 파라 포름 알데히드와 신장을 수정하고 레모 디메틸 포름 아미드 배양탈 물에 하룻밤 표백 NBT / BCIP는, 초과했습니다. 신장을 세척 한 후 커버 슬립에서 50 % 글리세롤과 PBST에서 촬영. </li></ol></li></ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+renal+failure+in+zebrafish:+a+novel+system+to+study+a+complex+disease.">Acute renal failure in zebrafish: a novel system to study a complex disease.</a> Am J Physiol Renal Physiol. 288, F923-F929 (2005).</li><li>Gerlach, G. F., Schrader, L. N., Wingert, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+the+adult+zebrafish+kidney.">Dissection of the adult zebrafish kidney.</a> J Vis Exp. , (2011).</li><li>Fogazzi, G. B., Cameron, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+microscopy+from+the+seventeenth+century+to+the+present+day.">Urinary microscopy from the seventeenth century to the present day.</a> Kidney Int. 50, 1058-1068 (1996).</li><li>Nachtrab, G., Czerwinski, M., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexually+dimorphic+fin+regeneration+in+zebrafish+controlled+by+androgen/GSK3+signaling.">Sexually dimorphic fin regeneration in zebrafish controlled by androgen/GSK3 signaling.</a> Curr Biol. 21, 1912-1917 (2011).</li><li>Zhou, W., Hildebrandt, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+podocyte+injury+and+proteinuria+in+transgenic+zebrafish.">Inducible podocyte injury and proteinuria in transgenic zebrafish.</a> J Am Soc Nephrol. 23, 1039-1047 (2012).</li><li>Huang, J., 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=A+zebrafish+model+of+conditional+targeted+podocyte+ablation+and+regeneration.">A zebrafish model of conditional targeted podocyte ablation and regeneration.</a> Kidney Int. 83, 1193-1200 (2013).</li></ol>

The authors have no competing financial interests to disclose.

제브라 피쉬는 성인 mesonephric 신장 (6)를 포함하여 성인 장기의 재생을 공부에 이상적입니다. 최근의 연구는 더 나은 네프론 재생 과정에서 발생하는 단계의 특성을 분자 마커 및 새로운 유전자 변형 기자 라인의 장점을 촬영하고 7,12 책임 어떤 세포 수 있습니다. 비뇨기 캐스트 관찰 인간 16 신장 질환을 진단 세기 이상 동안 사용되어왔다. 여기에서 우리는 겐타 마이신 주?...

신장은 신체로부터 유체 항상성, 혈압 조절과 폐기물의 여과에 필수적이다. 포유 동물이 부상 차별화 된 상피 세포 1-4 사용 후 기존 네프론을 복구 할 수 있지만, 그들은 예약 된 줄기 세포 5의 풀을 결여하는 것, 새로운 네프론 드 노보를 형성 할 수 없습니다. 포유류 달리 성어 부상 6,7에 물고기의 성장 및 응답을 지원하는 성인 일생 동안 새로운 네프론을 형성 할 수있다. 제브라 피쉬, 다니오 레 리오 (rerio)는, 장기 재생 8-10의 연구를위한 귀중한 모델 생물과 인간의 신장의 수리 공학 응용 프로그램에 강력한 통찰력을 제공 할 수있는 잠재력을 가지고있다. Tg는 (Lhx1a : EGFP) 유전자 (11) 그러나, 성인 zebrafish의 신장 (12)에 부상에서 R + 세포 lhx1a의 응답을 제어하는 메커니즘을 네프론의 선조 세포의 풀 라벨을 도시 한분명이 메인. 아미노 글리코 사이드 겐타 마이신은 인간 13 알려진 신 독성 및 ototoxic 효과로 널리 사용되는 항생제이다. 겐타 마이신의 복강 내 주사는 물고기 6 급성 신장 손상을 유도 설립 방법이다. 물고기 모방에서이 부상 관 상피 세포와 과다 복용 14 젠타 마이신 후 인간에서 발생하는 사구체의 흉터의 손실. 겐타 마이신 주사 제브라 피쉬에 부상을 유도하는 것은 생산 된 많은 새로운 네프론으로, 강한, 동기 재생 반응을 유도하고 동시에 형성, 증식과 분화의 단계를 통해 진행하는 편리한 방법입니다. 이 프로토콜은 특이점을 최소화하기 위해 비 침습적 영상 심사 과정을 이용하여 성인 zebrafish의 신장에 강력하고 재현 부상에 대한 우리의 방법을 자세히 설명합니다. 우리는 겐타 마이신과 부상 상피 신장 조직과 형식의 죽음에 이르게한다는 사실을 활용다음 mesonephric 덕트 및 배설강에서 대중에 축적 신 세뇨관 캐스트의 이온. 이러한 어류로 전달되고 물에 육안으로 관찰 할 수있다. 이것은 우리가 다음 추가 실험을 위해 풀링 할 수 있습니다 강력한 치명적 부상, 생선을 선별 할 수 있습니다. 그들은 실험의 종점보다 균일하고 효율적으로 데이터를 수집 및 분석에 이르게 도달하기 전에 손상되지 않은 다이 물고기 또는 물고기의 숫자를 최소화한다. 또한, 특별한 장치 나 시약은 교육 또는 교육 환경에서 사용하기 위해이 방법을 비용 효율적이고 적절하게, 필요하지 않습니다. 우리의 방법을 사용하여, 여기에 우리가 신장 재생에 겐타 마이신 투여 량의 증가 효과뿐만 아니라 온도 상승의 효과를 보여줍니다.

<table><tbody><tr><td>gentamicin sodium sulfate</td><td>Sigma</td><td>G1264</td><td>TOXIC, purity varies from batch to batch</td></tr><tr><td>plastic transfer pipets</td><td>Fisher</td><td>13-711-7M</td><td></td></tr><tr><td>1 ml Norm-Ject syringes</td><td>Electron Microscopy Sciences</td><td>72520</td><td>green plastic syringes, ordinary 1 ml syringes are OK, but harder to read accurately</td></tr><tr><td>30 G1/2 needles</td><td>Becton Dickinson</td><td>305106</td><td></td></tr><tr><td>ethyl 3-aminobenzoaate methanesulfonate salt (tricaine)</td><td>Sigma</td><td>A5040</td><td>IRRITANT</td></tr><tr><td>16% paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Make 4% in 1x PBS for working solution</td></tr></tbody></table>

kidney regeneration in adult zebrafish by gentamicin induced injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

젠타 마이신 부상은 물에서 신장 상피 캐스트의 관찰 (도 1)에 의해 시각적으로 확인할 수있다. 겐타 마이신의 다른 용량은 재생 신장 lhx1a + 세포 집합체 (- D 그림 1A)의 수를 증가의 결과로, 성인 야생형 TuAB의 제브라 피쉬를 손상하는 데 사용되었다. 화이트 캐스트 쉽게 손상 후 물 일일 (- H도 1E)에서 볼 수있다. 고용량 물고기 갖는 캐스트 (그림 1I...

Watch this Scientific Journal Video about Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury at JoVE.com

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

겐타 마이신 유도 부상으로 성인 Zebrafish의에서 신장 재생

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

kidney-regeneration-in-adult-zebrafish-by-gentamicin-induced-injury

Research

JoVE Journal

Developmental Biology

10.8K 조회수.  Massachusetts General Hospital. Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons. 

비디오: Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Zebrafish 개발에 급성 신부전 모델링을위한 정확한 세포 제거 방법

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

연구

발생학

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

Zebrafish의 성인 Telencephalon의 자상 부상 : 척추 동물의 뇌 신경 발생과 재생을 조사하는 방법

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

신경과학

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

급성 신장 상해를 공부하기 위해 Zebrafish 애벌레의 정맥 Microinjections

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

생물학

Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury model was developed in the adult optic tectum of zebrafish and medaka and comparative histological and molecular analyses were performed to elucidate the molecular mechanisms regulating the high regenerative capacity of this tissue across these fish species. Here a stab wound injury model is presented for the adult optic tectum using a needle and histological analyses for proliferation and differentiation of the neural stem cells (NSCs). A needle was manually inserted into the central region of the optic tectum, and then the fish were intracardially perfused, and their brains were dissected. These tissues were then cryosectioned and evaluated using immunostaining against the appropriate NSC proliferation and differentiation markers. This tectum injury model provides robust and reproducible results in both zebrafish and medaka, allowing for comparing NSC responses after injury. This method is available for small teleosts, including zebrafish, medaka, and African killifish, and enables us to compare their regenerative capacity and investigate unique molecular mechanisms.

A mechanical brain injury model in the adult zebrafish is described to investigate the molecular mechanisms regulating their high regenerative capacity. The method explains to create a stab wound injury in the optic tectum of multiple species of small fish to evaluate the regenerative responses using fluorescent immunostaining.

재생 능력의 비교 분석을 위해 Zebrafish와 Medaka를 사용한 성인 시신경의 찔린 상처 부상 모델

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

직접 성인 Zebrafish의 신장에 세포의 이식

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

성인 Zebrafish의 신장의 해부

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Zebrafish의 Pronephros의 레이저 절제는 신장 상피 조직이 재생된를 공부

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

성인 Zebrafish의의 측면 라인 재생을위한 분석

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

성인 Zebrafish의 신장에서 네프론의 구성 및 기능 분석

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

성인 얼룩말 피시에 있는 Cisplatin에 의해 유도된 급성 신장 상해 모형

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...